A polypeptide for alleviating or inhibiting ocular neovascularization and its use

Abstract

The present application belongs to the field of biomedical technology. More specifically, the present application relates to a polypeptide, a polypeptide composition and a pharmaceutical composition comprising the polypeptide, and the present application also relates to the use of the polypeptide and the polypeptide composition in the preparation of a pharmaceutical composition. Whether in vivo or in vitro, KV-7A exhibits a stronger therapeutic effect than KV-7, especially in the treatment of diseases related to corneal neovascularization. Therefore, the KV-7A of the present application has a wide range of application potential.

Description

Technical Field

[0001] The present application belongs to the field of biomedical technology. More specifically, the present application relates to a polypeptide, and a polypeptide composition and a pharmaceutical composition comprising the polypeptide. The present application also relates to the use of the polypeptide and the polypeptide composition in preparing a pharmaceutical composition. Background Art

[0002] The cornea is avascular in healthy individuals and is located in front of the iris and pupil. It is necessary for good vision and is also an important structure that protects the eye from infection and damage. However, in certain pathological conditions, new capillaries can grow within the cornea, forming corneal neovascularization (CNV).

[0003] A research report shows that about 1.4 million people develop corneal neovascularization-related diseases each year, of which 12%, or about 170,000 people, suffer from vision loss in the later stages of the disease. Vision loss has a huge economic impact on patients, their families, and society. For patients, blindness is prone to changes in social-emotional and self-cognition such as anxiety and depression, which may lead to social problems such as unemployment and marital problems. CNV is not only a risk factor for graft rejection after corneal transplantation, but also a major postoperative complication. Therefore, in the face of corneal neovascularization, a public health problem that threatens vision, is difficult to treat, and is becoming increasingly serious, finding a better drug or treatment to inhibit corneal neovascularization is crucial to maintaining good vision, improving quality of life, and enhancing social well-being.

[0004] Pterygium is a common ocular surface disease in clinic. The incidence of pterygium in people over 50 years old in southern my country is 33.01%. Pterygium is a multifactorial disease with invasive growth and recurrence tendency. The pathological process includes neovascularization, extracellular matrix remodeling and fibroblast proliferation, which manifests as subconjunctival benign fibrous connective tissue hyperplasia and blood vessels invading the corneal limbus, leading to visual obstruction, visual distortion, and even blindness.

[0005] The pathogenesis of pterygium is complex, and its occurrence and development involve multiple factors: genetic factors, environmental factors (ultraviolet rays, viral infection, etc.) and molecular factors (cytokines, growth factors such as VEGF, bFGF and TGFβ, and MMPs. However, the treatment of pterygium is relatively limited. Pterygium excision combined with conjunctival autotransplantation is the preferred method for treating primary and recurrent pterygium, but the recurrence rate after simple pterygium excision is as high as 30%, which brings great pain to patients. Therefore, finding drugs that can effectively inhibit factors such as VEGF, bFGF, and MMPs is the key to the early treatment of pterygium.

[0006] In the process of neovascularization, many angiogenic factors such as vascular endothelial growth factor (VEGF and its receptors), cytokines (CD146, CD31, etc.), matrix metalloproteinase family (MMP2, MMP9, MMP13, etc.), fibroblast growth factor (bFGF), inflammatory factors (IL-1β, IL-17, etc.), and tumor necrosis factor (TNFα) play an important role.

[0007] Human plasminogen Kringle5 (K5 for short) is the fifth Kringle domain of human plasminogen, containing 84 amino acids and a molecular weight of 14,000 Daltons. K5 is a ring structure formed by three disulfide bonds, and its structure and properties are stable. K5 can inhibit angiogenesis by inhibiting endothelial cell proliferation and migration, inhibiting pro-angiogenic factors such as VEGF and bFGF, and upregulating anti-angiogenic factors such as PEDF. K5 is considered to be an ideal anti-angiogenic drug. However, in the process of research and development of recombinant protein drugs, some controllable or uncontrollable factors often lead to the introduction of non-self sequences at both ends of the protein, such as His-tag, T7-tag, etc. This fusion protein sometimes affects biological activity and causes enhanced immunogenicity. In addition, since corneal neovascularization is located in the corneal stroma, protein inhibitors have a large molecular weight and are difficult to penetrate into the stroma to fully exert their effects. In addition, the production process is complex and the cost is high, so they have not been used in clinical practice.

[0008] The research group of the applicant compared the full-length sequences of plasminogen of 9 species, including humans, mice, common cattle, domestic brown rats, macaques, chimpanzees, zebrafish, jungle fowl and domestic dogs, by querying the NCBI database in the early stage, to understand which amino acid sequences are necessary to maintain activity. And further compared the homology of the Kringle domain between the above species, and found that there are conserved fragments (Asn-Tyr-Cys-Arg-Asn-Pro-Asp, NYCRNPD) in various species and genera, and named it KV-7. The applicant found that KV-7 can effectively inhibit the proliferation and migration of HUVECs, and can inhibit the angiogenesis of the chicken embryo chorioallantoic membrane, and applied for a patent (CN102250210B).

[0009] In order to further study and improve the anti-angiogenic effect of KV-7, the present application intends to provide a polypeptide with better effect than KV-7, which is of great significance for finding effective drugs to inhibit corneal neovascularization and the mechanism of angiogenesis and development. Summary of the invention

[0010] After research, the inventors of the present application found that after mutating KV-7, a polypeptide KV-7A with enhanced biological activity was obtained. Specifically, the inventors of the present application found that by mutating the 3rd amino acid of KV-7 (with reference to SEQ ID NO: 1) to alanine, the resulting polypeptide KV-7A has stronger biological activity than KV-7 (for example, treating pterygium, alleviating or inhibiting the formation of corneal neovascularization; alleviating or inhibiting the proliferation, migration and tube-forming ability of cells of corneal neovascularization; reducing or inhibiting the expression of VEGF (for example, VEGFA, VEGFC)). Whether in vivo or in vitro, KV-7A exhibits a stronger therapeutic effect than KV-7.

[0011] Therefore, in a first aspect, the present application provides a polypeptide, whose amino acid sequence has 1 amino acid mutation compared with the amino acid sequence shown in SEQ ID NO:1.

[0012] In certain embodiments, the polypeptide has a mutation at the position corresponding to the third position of the amino acid sequence of SEQ ID NO:1.

[0013] In certain embodiments, the polypeptide has the third amino acid residue of the amino acid sequence corresponding to SEQ ID NO: 1 mutated to alanine.

[0014] In certain embodiments, the polypeptide has the sequence shown in SEQ ID NO:2.

[0015] On the other hand, the present application provides a polypeptide composition, which comprises the polypeptide as described above, and one or more additional polypeptides.

[0016] In certain embodiments, the additional polypeptide is selected from human plasminogen Kringle1 or a conserved fragment thereof, human plasminogen Kringle2 or a conserved fragment thereof, human plasminogen Kringle3 or a conserved fragment thereof, human plasminogen Kringle4 or a conserved fragment thereof, human plasminogen Kringle5 or a conserved fragment thereof, KV-7, or any combination thereof.

[0017] In certain embodiments, the sequence of the additional polypeptide is selected from SEQ ID NO: 1, SEQ ID NO: 3, or any combination thereof.

[0018] In another aspect, the present application provides a nucleic acid molecule comprising or consisting of a nucleotide sequence encoding the polypeptide as described above.

[0019] In certain embodiments, the nucleotide sequence of the nucleic acid molecule is codon-optimized according to host cell preference.

[0020] Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, the nucleotide sequence encoding a polypeptide or polypeptide composition of the present invention may have a variety of different sequences. Therefore, unless otherwise indicated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and that encode the same amino acid sequence.

[0021] In another aspect, the present application provides a vector comprising the nucleic acid molecule as described above.

[0022] In certain embodiments, the vector is selected from a plasmid, a phagemid, an artificial chromosome (e.g., a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC) or a P1-derived artificial chromosome (PAC)), a phage (e.g., λ phage or M13 phage); and a viral vector (e.g., a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, a herpes virus vector).

[0023] In certain embodiments, the vector is used to express (eg, in vitro in a cell, or in vivo in a subject) a protein encoded by the nucleic acid molecule.

[0024] In certain embodiments, the vector is a vector for gene therapy (eg, a plasmid, a viral vector).

[0025] In another aspect, the present application provides a host cell comprising the isolated nucleic acid molecule or the vector as described above.

[0026] In certain embodiments, the host cell is selected from prokaryotic cells (eg, E. coli cells), and eukaryotic cells (eg, yeast cells, insect cells, plant cells).

[0027] In certain embodiments, the host cell is an animal cell (eg, a mammalian cell, such as a mouse cell, a human cell).

[0028] The vectors described above can be introduced into host cells by various suitable means, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, electroporation, TALEN method, ZFN method, non-viral vector-mediated transfection (such as liposome) or viral vector-mediated transfection (such as lentiviral infection, retroviral infection, adenoviral infection), and other physical, chemical or biological means for transfer into host cells, such as transposon technology, CRISPR-Cas9 and other technologies.

[0029] On the other hand, the present application provides a pharmaceutical composition, which contains an effective amount of the polypeptide as described above, or the polypeptide composition as described above, or the nucleic acid molecule as described above, or the vector as described above, or the host cell as described above; optionally, the pharmaceutical composition also contains a pharmaceutically acceptable carrier and / or excipient.

[0030] On the other hand, the present application provides a pharmaceutical composition as described above, wherein the pharmaceutical composition is in any one of the following dosage forms: ophthalmic preparations (e.g., eye drops, eye ointments, gels), ophthalmic injections (e.g., intraocular injections, fundus injections), and sustained-release agents.

[0031] In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0032] In certain embodiments, the pharmaceutically acceptable excipient is selected from a pH adjuster (e.g., hydrochloric acid, lactic acid, sulfuric acid, citric acid, sodium citrate, boric acid, sodium borate, triethanolamine, phosphates or mixtures thereof), a solubilizing agent, an osmotic pressure regulator (e.g., sodium chloride, glucose, mannitol, glycerol or propylene glycol or mixtures thereof), a viscosity modifier, an antioxidant, an antibacterial preservative (e.g., benzalkonium chloride, benzalkonium bromide, methylparaben, ethylparaben, propylparaben or mixtures thereof), a buffer, a suspending agent, a local anesthetic, a surfactant, a solubilizing agent, a wetting agent, an emulsifier, a filler, a gel matrix component (e.g., sodium hyaluronate, carbomer, poloxamer, chitosan, hydroxypropyl methylcellulose and mixtures thereof), or any combination thereof.

[0033] In certain embodiments, the pharmaceutical composition further comprises other active ingredients, for example, anti-inflammatory drugs (e.g., indomethacin, diclofenac sodium, ketorolac tromethamine, flurbiprofen sodium, pranoprofen), aminoglycoside antibacterial drugs (e.g., tobramycin, gentamicin, neomycin, amikacin, netilmicin, kanamycin), polyvinyl pyrrolidone.

[0034] In certain embodiments, the pharmaceutical composition has any one or more forms selected from the group consisting of: liquid, solid, semisolid, aerosol.

[0035] In certain embodiments, the pharmaceutical composition can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical composition of the present invention should be sterile and stable under production and storage conditions.

[0036] On the other hand, the present application provides the use of the polypeptide as described above, or the polypeptide composition as described above, or the nucleic acid molecule as described above, or the vector as described above, or the host cell as described above in the preparation of a pharmaceutical composition, wherein the pharmaceutical composition is used to alleviate or inhibit corneal neovascularization, or to treat symptoms caused by corneal neovascularization (e.g., decreased vision, blindness, corneal opacity), or to treat diseases related to ocular neovascularization.

[0037] Diseases caused by ocular neovascularization involve the cornea, iris, choroid and retina; the pathological changes such as exudation, hemorrhage and proliferation caused by it damage the structure and function of the eye and are an important cause of visual impairment. Most eye diseases have pathological processes such as inflammation, ischemia and hypoxia; therefore, eye diseases are closely related to ocular neovascularization. Among them, diseases related to ocular neovascularization include: diseases related to corneal neovascularization, diseases related to iris neovascularization, diseases related to choroidal neovascularization, and diseases related to retinal neovascularization.

[0038] In certain embodiments, the corneal neovascularization-related disease is selected from pterygium, corneal foreign body and trauma, corneal chemical injury and infectious, immune, dystrophic and exposure keratitis.

[0039] In certain embodiments, the retinal neovascularization-related disease is selected from retinal vein occlusion, diabetic retinopathy, idiopathic choroidal neovascular disease, retinopathy of prematurity, and neovascular age-related macular degeneration.

[0040] In certain embodiments, the pharmaceutical composition is used for:

[0041] (1) Alleviate or inhibit the formation of corneal neovascularization (e.g., the area of ​​corneal neovascularization, the length of corneal neovascularization);

[0042] (2) Alleviate or inhibit the proliferation, migration and / or tube-forming ability of corneal neovascularization cells;

[0043] (3) Reduce or inhibit the expression of VEGF (eg, VEGFA, VEGFC).

[0044] On the other hand, the present application provides a method for alleviating or inhibiting corneal neovascularization, which comprises administering a therapeutically effective amount of the polypeptide as described above, or the polypeptide composition as described above, or the nucleic acid molecule as described above, or the vector as described above, or the host cell as described above, or the pharmaceutical composition as described above to a subject in need thereof.

[0045] In certain embodiments, the methods are performed in vitro or in vivo.

[0046] On the other hand, the present application provides a method for treating symptoms caused by corneal neovascularization in a subject (e.g., decreased vision, blindness, corneal opacity), or treating a disease associated with corneal neovascularization (e.g., pterygium), comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide as described above, or a polypeptide composition as described above, or a nucleic acid molecule as described above, or a vector as described above, or a host cell as described above, or a pharmaceutical composition as described above.

[0047] In certain embodiments, the subject is a mammal.

[0048] In certain embodiments, the subject is a mouse or a human.

[0049] In certain embodiments, the method comprises administering to the subject a polypeptide expressing the present invention by dividing the dose into fractional doses, such as once, twice, three times or more.

[0050] In another aspect, the present application provides a method for expressing or producing the polypeptide as described above, the method comprising culturing the host cell as described above under conditions allowing protein expression, and optionally, recovering or purifying the expressed polypeptide.

[0051] In certain embodiments, the method comprises the following steps:

[0052] (1) constructing an expression vector comprising a nucleic acid sequence encoding the polypeptide as described above;

[0053] (2) introducing the expression vector into a host cell and culturing the host cell under conditions that allow protein expression;

[0054] (3) isolating and recovering the polypeptide from the cell culture of the host cell.

[0055] Definition of terms

[0056] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0057] As used herein, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0058] As used herein, the term "mutation" when used in the context of a protein / polypeptide refers to a mutation that has one or more amino acid differences (e.g., conservative amino acid substitutions) compared to the amino acid sequence of a reference protein / polypeptide and that retains the biological activity of the reference protein / polypeptide.

[0059] As used herein, the term "conservative amino acid substitution" means an amino acid substitution that does not adversely affect or alter the biological activity of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which amino acid residues are substituted with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0060] As used herein, the term "K5" stands for "human plasminogen Kringle5", which refers to the fifth Kringle domain of human plasminogen. It contains 84 amino acids and has a molecular weight of 14,000 Daltons. K5 is a ring structure formed by three disulfide bonds, and its structure and properties are stable. K5 can inhibit angiogenesis by inhibiting endothelial cell proliferation and migration, inhibiting pro-angiogenic factors such as VEGF and bFGF, and upregulating anti-angiogenic factors such as PEDF. In certain embodiments, K5 has an amino acid sequence as shown in SEQ ID NO:3.

[0061] Similarly, as used herein, the term "K4" stands for "human plasminogen Kringle 4" and refers to the fourth Kringle domain of human plasminogen. The term "K3" stands for "human plasminogen Kringle 3" and refers to the third Kringle domain of human plasminogen. The term "K2" stands for "human plasminogen Kringle 2" and refers to the second Kringle domain of human plasminogen. The term "K1" stands for "human plasminogen Kringle 1" and refers to the first Kringle domain of human plasminogen.

[0062] As used herein, the term "KV-7" refers to a polypeptide obtained in this way, by querying the NCBI database, comparing the full-length sequences of plasminogen from 9 species, including humans, mice, common cattle, domestic brown rats, macaques, chimpanzees, zebrafish, jungle fowl and domestic dogs, to understand which amino acid sequences are necessary for maintaining activity; and further comparing the homology of the Kringle domain between the above species, respectively, and finding that there are conserved fragments (i.e., Asn-Tyr-Cys-Arg-Asn-Pro-Asp, NYCRNPD) in various species and genera, and naming it KV-7. In certain embodiments, KV-7 has an amino acid sequence as shown in SEQ ID NO: 1.

[0063] According to the present application, the expression "corresponding to the amino acid sequence of SEQ ID NO: 1" refers to the amino acid position in the compared sequences that is at the equivalent position when the sequences are optimally aligned, i.e. when the sequences are aligned to obtain the highest percentage identity. Similarly, the expression "corresponding to the third position of SEQ ID NO: 1" refers to the amino acid position in the compared sequence that is at the equivalent position to the third position of SEQ ID NO: 1 when a sequence is optimally aligned with SEQ ID NO: 1, i.e. when a sequence is aligned with SEQ ID NO: 1 to obtain the highest percentage identity.

[0064] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0065] As used herein, the term "conserved fragment" refers to sequences with a high degree of similarity or identity, which can be nucleic acid sequences (such as RNA or DNA sequences) or protein sequences. The sequences of the conserved fragments from different species or the same organism are highly similar. By analyzing the genomes of organisms at different evolutionary stages, it is found that some sequences in the genomes of different organisms are surprisingly similar, and these similar sequences are so-called conserved fragments.

[0066] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0067] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.

[0068] As known to those skilled in the art, codons have degeneracy. That is, in the translation process of proteins, each amino acid may correspond to one or more codons, for example, up to 6 codons. Different species have great differences in the use of degenerate codons encoding a certain amino acid, and have different preferences. This preference phenomenon is called "codon preference". Therefore, as used herein, the term "codon preference" refers to the situation where a species prefers to use certain specific codons to encode amino acids. It is particularly advantageous in some cases to optimize the sequence of nucleic acid molecules according to codon preference, for example, it may help to improve the expression level of the protein encoded by the nucleic acid molecule. For example, when using Escherichia coli (or human cells) to express the polypeptide of the present application, it will be potentially advantageous to optimize the nucleic acid sequence encoding the polypeptide of the present application for the codon preference of Escherichia coli (or human cells).

[0069] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, preservatives. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, which can stabilize the desired activity of the active ingredient in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient includes a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0070] As used herein, the term "sustained release agent" refers to a preparation whose active ingredient is slowly released into the subject's body after administration (e.g., oral administration) to the subject and maintains a substantially stable level of therapeutically effective active ingredient over a relatively long period of time (e.g., about 8 hours to about 24 hours, or longer).

[0071] As used herein, the term "subject" refers to a mammal, such as a human, a cynomolgus monkey, or a mouse.

[0072] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain a desired effect. For example, a disease prevention effective amount refers to an amount sufficient to prevent, prevent, or delay the occurrence of the disease; a disease treatment effective amount refers to an amount sufficient to cure or at least partially prevent the disease and its complications in patients who already have the disease. Determining such an effective amount is well within the capabilities of those skilled in the art.

[0073] Advantageous Effects of the Invention

[0074] The polypeptide KV-7A of the present application has stronger biological activity than KV-7 and K5 (for example, relieving or inhibiting the formation of corneal neovascularization; relieving or inhibiting the proliferation, migration and / or tube-forming ability of cells of corneal neovascularization; reducing or inhibiting the expression of VEGF).

[0075] Whether in vivo or in vitro, KV-7A exhibits a stronger therapeutic effect than KV-7 (e.g., relieving or inhibiting corneal neovascularization and retinal neovascularization, treating symptoms caused by corneal neovascularization (e.g., decreased vision, blindness, corneal opacity) and symptoms caused by retinal neovascularization (e.g., varying degrees of decreased vision, visual distortion, color vision abnormalities, blindness, etc.), treating diseases related to corneal neovascularization (e.g., pterygium, corneal foreign bodies and trauma, corneal chemical injuries and infectious, immune, malnutrition and exposure keratitis, etc.)) and diseases related to retinal neovascularization (e.g., retinal vein occlusion, diabetic retinopathy, idiopathic choroidal neovascular disease, retinopathy of prematurity, neovascular age-related macular degeneration, etc.). Therefore, KV-7A has a wide range of application potential.

[0076] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 The blood vessel growth of the chick embryo chorioallantoic membrane in each group of Example 1 is shown, wherein: Figure 1 A is a 2x microscope photo of the chick embryo chorioallantoic membrane in different treatment groups; Figure 1 B is the statistical graph of the blood vessel area ratio of the chick embryo chorioallantoic membrane in each group (n≥5, **: P<0.01, ***: P<0.001).

[0078] Figure 2 The slit lamp photos of suture-induced corneal neovascularization in each group of Example 2 are shown, wherein: Figure 2 A is the slit lamp photograph of the eyeballs of rats in each group; Figure 2 B is a statistical chart of the relative ratio of corneal neovascularization area in each group; Figure 2 C is the statistical graph of the relative ratio of corneal neovascularization length in each group (**: P < 0.01; ***: P < 0.001).

[0079] Figure 3 The results of HE staining of corneal paraffin sections of CNV in each group of Example 3 are shown.

[0080] Figure 4 The CD146 immunofluorescence staining of corneal paraffin sections of each CNV group in Example 4 is shown.

[0081] Figure 5 The results of inhibiting the proliferation of HUVECs by KV-7A at different concentrations in Example 5 are shown; wherein, the difference significance of each group is compared with the model group (ie, the second column of bar graphs), ns: no statistically significant difference, *: P < 0.05, **: P < 0.01, ****: P < 0.0001.

[0082] Figure 6 The effect of different concentrations of KV-7A on HUVECs cell viability in Example 6 is shown; wherein, the difference significance of each group is compared with the control group (ie, the first column of bar graphs), ns: no statistically significant difference, ***: P < 0.005.

[0083] Figure 7 The effects of different concentrations of KV-7A on HCEs cell viability in Example 7 are shown; wherein, the differences in each group are compared with the control group (ie, the first column of bar graphs), ns: no statistically significant difference, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001.

[0084] Figure 8 The scratch test was performed to investigate the inhibition of HUVECs migration by KV-7A at different concentrations. Figure 8 A shows the migration of HUVECs in each group at 0, 6, and 12 hours after scratching, and the distance between the two white dashed lines is the width of the scratch; Figure 8 B is a statistical graph of the migration rate of each group at 6 and 12 hours after scratching (*: P < 0.05, **: P < 0.01, ****: P < 0.0001).

[0085] Fig. 9 The Transwell experiment was shown to investigate the inhibition of HUVECs migration by KV-7A. Fig. 9 A shows the migration of HUVECs in each group 12 hours after cell seeding, and the purple ones are cells stained with crystal violet; Fig. 9 B is a statistical diagram of the number of cells in each field of view of each group, with at least 3 fields of view in each group (*: P < 0.05, **: P < 0.01, ***: P < 0.001).

[0086] Fig.10 The results show that different concentrations of KV-7A inhibit the ability of HUVECs to form tubes. Fig.10 A shows the migration of HUVECs in each group after the cells were seeded in Matrigel and treated with different concentrations of KV-7A for 6 hours; Fig.10 B and Fig.10 C is a statistical diagram of the length of the nodes and tube networks connected by cells in each field of view in each group, with at least 3 fields of view in each group (ns: no statistically significant difference, **: P < 0.01, ***: P < 0.001).

[0087] Fig.11 The effect of KV-7A on the expression of VEGFA protein in HUVECs was shown. Fig.11 A shows the expression of VEGFA protein in HUVECs of each group at different treatment times; Fig.11 B is a statistical graph of the grayscale values ​​of the target protein bands in each group, with at least 3 samples in each group (*: P < 0.05, **: P < 0.01, ***: P < 0.001).

[0088] Fig.12 The effect of KV-7A on the expression of VEGF gene in HUVECs was shown. Fig.12 A shows the expression of VEGFA gene in cells of each group after 24 h of treatment; Fig.12 B shows the expression of VEGFC gene in cells of each group after 24 hours of treatment (**: P < 0.01, ***: P < 0.001, ****: P < 0.0001).

[0089] Fig.13 The figure shows the effect of KV-7A on the expression of VE-Cadherin in HUVECs, where VE-cadherin staining (red) is located on the cell membrane where cells contact each other and is a marker protein for endothelial cell junctions, and DAPI staining (blue) is located in the cell nucleus and is a marker protein for the cell nucleus.

[0090] Fig.14 The effect of KV-7A on the angiogenesis of chick embryo chorioallantoic membrane was shown. Fig.14 A is the angiogenesis in the drug-added area of ​​the chick embryo chorioallantoic membrane of each group, and the yellow dotted circle is the drug-added range; Fig.14B is a statistical chart showing the percentage of blood vessel area within the dosing range in each group, with at least 6 samples in each group (**: P < 0.01, ***: P < 0.001).

[0091] Fig.15 The suture-induced angiogenesis in rat cornea in Example 15 is shown.

[0092] Fig.16 The results show the inhibitory effect of different concentrations of KV-7A on suture-induced corneal neovascularization in rats in Example 16, wherein: Fig.16 A is a slit lamp photograph of suture-induced corneal neovascularization in rats treated with different concentrations of KV-7A, and FL is corneal sodium fluorescein staining; Fig.16 B is a statistical chart of the ratio of corneal neovascularization area to corneal area in each group; Fig.16 C is a statistical graph of the ratio of corneal neovascularization length to lateral corneal length in each group; Fig.16 D is the statistical chart of the inflammation index of each group; *: P < 0.05, ***: P < 0.001.

[0093] Fig.17 The effects of KV-7A and Conbercept on the angiogenesis of the chick embryo chorioallantoic membrane were shown. Fig.17 A is the angiogenesis in the drug-added area of ​​the chick embryo chorioallantoic membrane of each group, and the yellow dotted circle is the drug-added range; Fig.17 B is a statistical chart showing the percentage of blood vessel area within the dosing range in each group, with at least 6 samples in each group (ns: no statistical difference, **: P < 0.01, ***: P < 0.001).

[0094] Fig.18 The results show that KV-7A (15mM) was compared with Conbercept in treating suture-induced corneal neovascularization in rats at different time points. Fig.18 A is the slit lamp photos of corneal neovascularization of rats in each group at 3, 7, and 10 days; Fig.18 B is a statistical chart of the ratio of corneal neovascularization area to corneal area in each group; Fig.18 C is a statistical graph of the ratio of corneal neovascularization length to lateral corneal length in each group; Fig.18 D is the statistical chart of the inflammation index of each group; *: P < 0.05, **: P < 0.01, ***: P < 0.001.

[0095] Fig.19 The whole-mount staining of the rat cornea in different treatment groups is shown. CD146 is one of the markers of neovascularization and is widely expressed in vascular endothelial cells. The blood vessels show green fluorescence, and the white arrows point to corneal neovascularization.

[0096] Fig. 20 HE staining of corneal paraffin sections of rats in different treatment groups in Example 19 is shown.

[0097] Fig.21 The CD146 immunofluorescence staining of rat corneal paraffin sections of different treatment groups in Example 20 is shown.

[0098] Fig. 22 The results showed the effects of KV-7A on genes related to corneal angiogenesis in rats, among which, Fig. 22 A is a statistical graph of Vegfa gene expression in each group; Fig. 22 B is a statistical graph of Vegfb gene expression in each group; Fig. 22 C is a statistical graph of Vegfr2 gene expression in each group; Fig. 22 D is a statistical graph of Vegfr3 gene expression in each group; Fig. 22 E is the statistical graph of Il1b gene expression in each group; Fig. 22 F is the statistical graph of Mmp9 gene expression in each group; Fig. 22 G is a statistical chart of the expression of Mmp13 gene in each group; *: P < 0.05, **: P < 0.01, ***: P < 0.001.

[0099] Fig.23 The results of different concentrations of KV-7A inhibiting the proliferation of HPF (human lung fibroblasts) in Example 23 are shown; wherein, the significant difference of each group is compared with the model group (ie, the second column of bar graphs), ***: P < 0.001.

[0100] Fig.24 The Transwell experiment in Example 24 is shown to explore the inhibition of HPF migration by KV-7A at different concentrations; wherein, Fig.24 Figure A shows the migration of HPFs to the other side of the membrane in each group after 24 hours; Figure B shows the statistical diagram of the number of migrated cells in each group (*: P < 0.05, **: P < 0.01, ***: P < 0.001).

[0101] Fig.25 The results of αSMA immunofluorescence staining of HPFs in different treatment groups in Example 25 are shown.

[0102] Fig.26 The results of KV-7A treatment of mice OIR (oxygen-induced retinopathy) in Example 26 are shown; wherein, Fig.26 A is the condition of retinal blood vessels at 17 days, red fluorescence represents blood vessels, the white dotted line represents the central retinal non-vascular perfusion area, and the yellow dotted line represents the retinal neovascularization clusters; Fig.26 B is a statistical chart showing the ratio of the central non-perfused area of ​​the retina to the total retinal area in each group; Fig.26C is the statistical graph of the ratio of the area of ​​retinal neovascularization clusters to the total retinal area in each group (**: P < 0.01, ***: P < 0.001).

[0103] Sequence information

[0104] The information of the partial sequences involved in the present invention is provided in Table 1 below.

[0105] Table 1: Description of sequences

[0106] DETAILED DESCRIPTION

[0107] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

[0108] Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references. For example, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention can be found in Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., eds., (1987)); METHODS IN ENZYMOLOGY series (Academic Press): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)); and ANIMAL CELL CULTURE (Animal Cell Culture). CELL CULTURE) (RI Freshney, ed. (1987)).

[0109] In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

[0110] Example 1. Comparison of KV-7 and KV-7A in inhibiting the growth of blood vessels in the chick embryo chorioallantoic membrane

[0111] This example uses a chicken embryo chorioallantoic membrane experiment to compare the effects of KV-7 and KV-7A on inhibiting angiogenesis in an animal model. The specific experimental steps are as follows:

[0112] (1) Purchase fertilized eggs from the local market and place them with the air chamber facing upwards;

[0113] (2) After 24 hours, prepare a solution of Chlorhexidine (1:25) to clean the stains on the outside of the fertilized eggs, then rinse with warm water and dry;

[0114] (3) Place the dried fertilized eggs in an incubator, set the temperature to 38.4°C, the humidity to 60%, and turn the eggs every 240 seconds, which is counted as day 1;

[0115] (4) After 9 days of incubation, sterile 1x phosphate buffered saline (1xPBS) was prepared as the NC group, sterile 1xPBS solution containing 20 ng / ml VEGF was prepared as the VEGF group, sterile 1xPBS solution containing 20 ng / ml VEGF and 1000 μM KV-7 was prepared as the KV-7 group, and sterile 1xPBS solution containing 20 ng / ml VEGF and 1000 μM KV-7A was prepared as the KV-7A group. Appropriate amounts of filter paper pieces with a diameter of 3.5 mm were taken and soaked in the above solutions for use.

[0116] (5) Use ophthalmic scissors and micro toothless forceps to open the air chamber of the fertilized egg and fully expose the chorioallantoic membrane. Randomly take filter paper pieces containing different groups of solutions and place them on the chorioallantoic membrane.

[0117] (6) Use autoclaved medical gauze tape to seal the air chamber opening and place the fertilized egg with the air chamber opening facing upward in the incubator. The incubation conditions are the same as before.

[0118] (7) After 72 hours, remove the fertilized egg, use ophthalmic scissors to cut the tape, and observe whether the embryo is alive. The yolk of a living embryo should be yellow with a tinge of orange-red, and the egg white should be clear. Under slight stimulation or in a normal state, the embryo's movements and blood vessels and heartbeats can be seen. The yolk of a dead embryo is yellow, and the egg white is turbid. The embryo will not move under stimulation and may be accompanied by a foul odor.

[0119] (8) Discard dead embryos and fix the chorioallantoic membrane of surviving chicken embryos with 1-2 ml of 4% paraformaldehyde. After 20 minutes, discard the fixative and rinse twice with 1xPBS.

[0120] (9) Use corneal scissors and micro-toothless forceps to remove the chorioallantoic membrane, and use a stereo microscope to take pictures and compare the blood vessels in the filter paper;

[0121] (10) The images were processed using image J software, and the relative vascular area within the filter paper area (indicated by the yellow dotted circle) was calculated.

[0122] The results showed that the average vascular area ratio of the drug-treated area in the NC group was 51.652±8.605, and the average vascular area ratio of the VEGF group was 69.575±10.512. Compared with the NC group, the blood vessels were thicker and more tortuous, and the blood vessels increased significantly, with a statistically significant difference (P=0.0054). The average vascular area ratios of the VEGF+KV-7 (1000μM) and VEGF+KV-7A (1000μM) groups were 33.235±7.341 and 21.072±6.783, respectively. Compared with the VEGF group, the blood vessels in the drug-treated area of ​​the VEGF+KV-7 (1000μM) and VEGF+KV-7A (1000μM) groups were significantly reduced, with a statistically significant difference (P values ​​were all <0.001), and the mean value of the VEGF+KV-7A (1000μM) group was lower than that of the VEGF+KV-7 (1000μM) group. The results showed that both KV-7 and KV-7A could significantly inhibit the angiogenesis of chick embryo chorioallantoic membrane. At the same concentration, KV-7A showed a stronger inhibitory effect.

[0123] Example 2. Comparison of KV-7 and KV-7A in inhibiting suture-induced corneal neovascularization

[0124] This example uses an animal model of suture-induced corneal neovascularization in rats to compare the effects of KV-7 and KV-7A at the same concentration of 15 mM in inhibiting corneal angiogenesis. The specific experimental steps are as follows:

[0125] (1) SPF-grade male Wistar rats weighing 150 g to 180 g were purchased from Shanghai Slake Experimental Animal Co., Ltd. After arrival, the rats were placed in the animal observation room of Xiamen University Medical School in a common barrier environment with a temperature of 24°C and a humidity of 40% to 70%;

[0126] (2) Before the experiment, the subjects were fasted and deprived of water for 4 hours, and all instruments and towels were sterilized by high pressure;

[0127] (3) Each rat was intraperitoneally injected with sodium pentobarbital injection according to its body weight (0.2 ml / 100 g), and one drop of compound tropicamide eye drops was instilled into each eye to dilate the pupil. The whiskers on both sides of the rat were trimmed with ophthalmic scissors;

[0128] (4) After anesthesia is in place, observe that the rat's pupils are dilated and the iris is at the corneal limbus, and then administer proparacaine hydrochloride eye drops to surface anesthetize the cornea;

[0129] (5) The rat was transferred to the operating microscope, and a perforated towel was placed around the eye. The surgeon used one hand to open the eyelid to fully expose the cornea. A 3 mm trephine drill was used to gently press the center of the cornea, and a ring-shaped pressure mark was visible.

[0130] (6) Use Lingqiao's 10-0 corneal spatula needle and suture to sew one stitch at each of the 3, 6, 9, and 12 o'clock positions of the circular indentation. The distance between the entry and exit of the needle is consistent, about 0.5 mm. The other eye is the same as before;

[0131] (7) After suturing, levofloxacin eye drops were applied to prevent infection, and the rats were placed in a lateral position on a rewarming blanket and returned to a normal barrier environment for feeding after awakening from anesthesia;

[0132] (8) On the second day after surgery, slit lamp imaging system was used to take frontal and lateral photographs of the rats' eyes. Rats with failed surgeries were eliminated based on the conditions of vascular growth and corneal inflammation. Rats with successful surgeries were randomly divided into PBS group, KV-7 group, and KV-7A group, and were given 10 μl of sterile 1xPBS solution, 1xPBS solution containing 15 mM KV-7, and 1xPBS solution containing 15 mM KV-7A, respectively, with eye drops four times a day.

[0133] (9) On the 7th day after surgery, anteroposterior and lateral photographs of the rat eyes were taken using a slit lamp imaging system, and the area and length of corneal neovascularization were compared among the groups to evaluate the anti-angiogenic effect.

[0134] (10) Evaluation of neovascularization and inflammation: Image J software was used to analyze the length and area of ​​neovascularization.

[0135] a. Measure the length of new blood vessels growing into the cornea (L) from the lateral image. Select the thickest and longest blood vessels growing toward the center of the cornea for measurement. The blood vessel length ratio is I = L / R*100%, where R is the highest length of the lateral cornea perpendicular to the corneal limbus.

[0136] b. The area of ​​new blood vessels growing into the cornea (S) was measured from the anteroposterior image using the following formula: S = 3.1416*[(r2-(r-l1)2)+…+(r2-(r-l4)2)] / 4. The formula for the ratio of the new blood vessel area was: i = S / (3.1416*r2)*100%, where r is the radius of the rat cornea, and l is the length of the new blood vessels growing from the corneal edge into the cornea at each suture.

[0137] The results show that Figure 2): The average vessel areas of the NC group, PBS group, KV-7 group, and KV-7A group were 0.000±0.000, 74.349±1.826, 68.718±0.635, and 53.778±3.615, respectively; the average vessel lengths were 0.000±0.000, 0.497±0.062, 0.441±0.027, and 0.327±0.095, respectively. Compared with the PBS group, the corneal neovascularization area of ​​the KV-7 and KV-7A groups was significantly reduced (P values ​​were <0.01) and the vessel length was shortened (P values ​​were <0.01, P values ​​were <0.001, respectively), and the differences were statistically significant. KV-7A was superior to KV-7 in inhibiting the vessel area and vessel length of suture-induced corneal neovascularization (P=0.010; P=0.0001). The results showed that KV-7A was superior to KV-7 in inhibiting corneal neovascularization.

[0138] Example 3. HE staining of corneal paraffin sections of CNV treated with KV-7 and KV-7A

[0139] In this example, paraffin sections of rat eyes in each group after corneal suture were stained with HE to compare the therapeutic effects of KV-7 and KV-7A on CNV from a microscopic perspective. The specific experimental steps are as follows:

[0140] Paraffin embedding: (1) Remove the rat eyeball and surrounding eyelid tissue using a blade and ophthalmic scissors, and fix them in 4% paraformaldehyde.

[0141] (2) After 24 hours, the tissue was repeatedly rinsed with 1xPBS solution, and the excess water on the surface of the eyeball was absorbed with filter paper. The eyeball was placed in a plastic paraffin embedding box for gradient dehydration: 70% ethanol overnight (≥8h) at room temperature, 80% ethanol, 95% ethanol, 100% ethanol I, 100% ethanol II, 100% ethanol III, xylene I, xylene II, and xylene III for 1h each; paraffin I, paraffin II, and paraffin III at 60°C for 1h each.

[0142] (4) Preheat the paraffin embedding machine and paraffin embedding bottom mold in advance;

[0143] (5) Take out the eyeball tissue to be embedded, place it in the paraffin embedding bottom mold at an angle such that the line connecting the two eyelids is perpendicular to the horizontal plane, add an appropriate amount of paraffin wax so that the eyeball tissue is completely immersed, and place it on a refrigeration table to solidify the paraffin wax;

[0144] (6) After the paraffin is completely solidified, separate the paraffin block from the embedding bottom mold and place it at room temperature.

[0145] Paraffin sectioning: (1) Before paraffin sectioning, pre-cool the paraffin block in an ice-water mixture and pre-heat the slide spreader and slide baking machine;

[0146] (2) Adjust the slice thickness of the paraffin slicer to 30 μm, fix the blade and paraffin block for rough cutting, and adjust the slice thickness to 6 μm for further slicing after cutting to a suitable plane;

[0147] (3) Use a blade to cut the long tissue slices into 3-4 slices / sheet, place them on a glass slide dripped with 30% ethanol, place the tissue slices on the liquid surface of a slide spreader, wait for the tissue slices to fully unfold, then use a clean glass slide to pick up the slices, and place them on a slide dryer to dry;

[0148] (4) After the paraffin sections are completely dry, place them in a dry section box and store them at room temperature for later use.

[0149] Hematoxylin-eosin (HE) staining: (1) Place the eyeball slices on a paraffin section dewaxing rack and place them in a 60°C oven for 1-2 hours before performing the following paraffin dewaxing steps: at room temperature, xylene 1 and xylene 2 for 15 minutes each, 100% ethanol 1 and 100% ethanol 2 for 15 minutes each, 95% ethanol, 80% ethanol, and 70% ethanol for 5 minutes each, and DDW three times for five minutes each.

[0150] (2) Place the slide on the staining rack and moisten the tissue with 1x PBS solution;

[0151] (3) Place the slide and staining rack into a pre-filtered hematoxylin staining tank and stain for 5 minutes;

[0152] (4) Rinse off excess dye with tap water and rinse with running water for 5 min, then differentiate in 1% hydrochloric acid alcohol solution for 1-2 s;

[0153] (5) Wash the slides in tap water for 5 minutes;

[0154] (6) Wipe off the excess water droplets on the slide and stain with eosin solution for 2 minutes;

[0155] (7) Rinse off excess dye on the staining rack with tap water, and perform paraffin section dehydration and transparentization steps: 70% ethanol, 80% ethanol, 95% ethanol, 100% ethanol, xylene 1, and xylene 2 at room temperature for 2 min each;

[0156] (8) Air-dry the slides in a fume hood and seal them with neutral resin (H-5000);

[0157] (9) Observe under an ordinary microscope and take photos.

[0158] The results show that Figure 3): The NC group was a normal corneal epithelium with neat and smooth arrangement, and the corneal stromal cells in the stromal layer were neat and dense, without inflammatory cell infiltration and blood vessel growth. The PBS group was the cornea after suture, with a significant increase in the overall corneal thickness, a rough corneal epithelium, enlarged and neatly arranged basal cells, a large number of new blood vessels and inflammatory cell infiltration in the stromal layer, the depth of which reached more than two-thirds of the stromal layer, and red blood cells in the blood vessels. The KV7 group had new blood vessels and inflammatory cell infiltration in the stromal layer, the depth of which reached about one-half of the stromal layer. The KV-7A group had thickened corneas, a slightly rough corneal epithelium, slightly larger and neatly arranged epithelial basal cells, and new blood vessels and inflammatory cell infiltration in the stromal layer, the depth of which reached between one-third and one-half of the stromal layer. The results showed that the depth of vascular infiltration and the degree of inflammatory infiltration in the KV-7A group were lighter than those in the KV-7 group.

[0159] Example 4. CD146 immunofluorescence staining of corneal paraffin sections of CNV treated with KV-7 and KV-7A

[0160] In this example, paraffin sections of rat eyeballs in each group after corneal suture were subjected to CD146 immunofluorescence staining to compare the therapeutic effects of KV-7 and KV-7A on CNV in terms of vascular infiltration depth. The specific experimental steps are as follows:

[0161] (1) The steps of paraffin embedding, sectioning and dewaxing of paraffin sections are the same as those in Example 3;

[0162] (2) Prepare 1x antigen retrieval solution (1mM Tris / EDTA solution, pH=9.0), immerse the dewaxed sections in the antigen retrieval solution, boil them in a microwave oven at high temperature for 10 min, immediately cool them in DDW after boiling, and then wash them three times with 1x PBS solution, each time for 5 min;

[0163] (3) Wipe off excess water droplets on the slide, circle the tissue with an immunohistochemical pen, incubate in 3% hydrogen peroxide solution for 20 min, and then wash three times with 1xPBS, 5 min each time;

[0164] (4) Dissolve 1 g of BSA powder in 50 ml of 1xPBS solution to prepare blocking solution. Take an appropriate amount of blocking solution to cover the tissue and block at room temperature for 1 h.

[0165] (5) Dissolve 0.5 g of BSA powder in 50 mL of 1x PBS solution to prepare an antibody diluent. Dilute the antibody diluent at 1:300 according to the instructions of CD146 (ab75769, Abcam) and set aside.

[0166] (6) Aspirate the blocking solution and wash three times with 1xPBS, 2 min each time;

[0167] (7) Add 20-30 μl of primary antibody dilution to each tissue on the slide and incubate at 4°C overnight;

[0168] (8) The next day, remove the primary antibody and wash three times with 1xPBS, 5 min each time;

[0169] (9) Protect from light. According to the instructions of the goat anti-rabbit secondary antibody conjugated with 488 fluorescent group (A32731, Thermo Fisher Scientific), dilute it with antibody diluent at 1:300, add 20-30 μl of secondary antibody diluent to each tissue, and incubate at room temperature in the dark for 1 h.

[0170] (10) Remove the secondary antibody and wash three times with 1xPBS, 5 min each time;

[0171] (11) After absorbing the water droplets on the slide, use DAPI (H-1200) to seal the slide and colorless nail polish to seal the edges. Store at 4°C away from light and observe and photograph under a fluorescence microscope.

[0172] The results show that Figure 4 ): The NC group was a normal cornea that was smooth and neat, with no blood vessels growing in. The PBS group was a cornea after sutures, with increased thickness, and tubular green fluorescence, i.e., new blood vessels, could be seen in the corneal stroma, with a large number. The KV-7 group had a significantly thickened cornea, with tubular green fluorescence visible in the corneal stroma, and the number was slightly less than that in the VEGF group. The KV-7A group had a thickened cornea, with a small amount of tubular green fluorescence visible in the corneal stroma, which was significantly less than that in the VEGF group. The results show that KV-7A is better than KV-7 in reducing the depth of corneal neovascularization.

[0173] Example 5. Inhibitory effect of different concentrations of KV-7A on the proliferation of HUVECs

[0174] This example uses CCK8 experiment to study the inhibitory effect of different concentrations of KV-7A on the proliferation of HUVECs (human umbilical vein endothelial cells). The specific experimental steps are as follows:

[0175] (1) The cell density of HUVECs was observed to be 70%-80% under a microscope. The cells were digested with 0.25% trypsin and centrifuged. The cells were resuspended to 50,000 cells / ml in complete ECM culture medium.

[0176] (2) 200 μl of cells were seeded into each well of a 96-well plate, gently shaken to evenly distribute the cells, and then placed in an incubator at 37°C and 5% CO2;

[0177] (3) After the cells adhered to the wall, the culture medium in the 96-well plate was discarded and replaced with basal medium containing 2% FBS, and placed in an incubator for 2 h;

[0178] (4) The basal medium containing 5% FBS was used as the control group, and the basal medium containing 5% FBS containing 20 ng / ml VEGF was used as the model group (where 20 ng / ml VEGF was the optimal induction concentration for HUVECs). The basal medium containing 5% FBS containing 20 ng / ml VEGF was used to prepare culture medium containing different concentrations of KV-7A (10 μM, 50 μM, 100 μM, 500 μM, 1000 μM, 1500 μM, 2000 μM, 5000 μM);

[0179] (5) Discard the culture medium in the original 96-well plate and add the culture medium of each group mentioned above, 200 μl per well, 6 replicates per group, and place in the incubator;

[0180] (6) According to the instructions of the CCK8 kit (C0038, Bio-Tech Biotech Co., Ltd.), the CCK8 solution was prepared with basal culture medium at a ratio of 1:10;

[0181] (7) After 24 h, the culture medium in the 96-well plate was discarded, 100 μl of CCK8 solution was added to each well, and the cells were incubated in an incubator at 37°C in the dark for 2-4 h.

[0182] (8) The OD value at a wavelength of 450 nm was measured using an enzyme-labeled instrument and statistically analyzed using GraphPad Prism 8.0.

[0183] The results show that Figure 5 ): Control group (corresponding to Figure 5 The average proliferation rate of the first column) was 1.000±0.162, and the model group (corresponding to Figure 5 The second column) The average relative proliferation rate was 1.837±0.298, which was significantly higher than that of the control group, and the difference was statistically significant (P<0.0001). The groups containing different concentrations of KV-7A (10μM, 50μM, 100μM, 500μM, 1000μM, 1500μM, 2000μM, 5000μM) (corresponding to Figure 5The average relative proliferation rates of the third to tenth columns were 1.694±0.312, 1.640±0.445, 1.570±0.410, 1.433±0.536, 1.215±0.369, 1.120±0.420, 0.863±0.244, and 0.563±0.140, respectively, which were lower than those of the model group. There were no significant differences between the 10μM and 50μM groups and the model group, with P values ​​of 0.1550 and 0.0868, respectively; there were significant differences between the 100μM and 500μM groups and the model group, with P values ​​of 0.0178 and 0.0042, respectively; there were extremely significant differences between the 1000μM, 1500μM, 2000μM, and 5000μM groups and the model group, with P values ​​all less than 0.0001. The results showed that KV-7A could significantly inhibit the proliferation of HUVECs in a dose-dependent manner.

[0184] Example 6. Effects of different concentrations of KV-7A on HUVECs cell viability

[0185] In order to study whether different concentrations of KV-7A have an effect on the cell viability of HUVECs, we treated the cells with drugs for 24 hours and then detected the cell viability by CCK8 assay. The specific experimental steps were the same as those in Example 5, and the drug addition conditions were as follows: a basal medium containing 5% FBS was used as a control group, and a culture medium containing 5% FBS was used to prepare culture solutions with different concentrations of KV-7A (10 μM, 50 μM, 100 μM, 500 μM, 1000 μM, 1500 μM, 2000 μM, 5000 μM).

[0186] The results show that Figure 6 ): Control group (corresponding to Figure 6 The average cell viability of the first column was 0.995 ± 0.043, and the 100 μM, 500 μM, 1000 μM, 1500 μM, 2000 μM, 2500 μM and 5000 μM groups (corresponding to Figure 6The average relative cell viability of the second to eighth columns was 1.035±0.075, 1.028±0.052, 1.014±0.090, 0.978±0.084, 0.950±0.089, 0.902±0.057 and 0.820±0.105, and the cell viability of different concentrations of KV-7A groups was above 80%. Among them, 100μM, 500μM, 1000μM, 1500μM and 2000μM had no significant decrease compared with the control group, and the difference was not statistically significant (P was 0.0698, 0.0674, 0.4551, 0.4668 and 0.0672, respectively), and the cell viability of 2500μM and 5000μM was reduced compared with the control group, and the difference was statistically significant (P<0.0001, P<0.0001). The results showed that KV-7A had a certain effect on the cell viability of HUVECs, but had no obvious toxic effect.

[0187] Example 7. Effects of different concentrations of KV-7A on HCEs (human corneal epithelial cells) cell viability

[0188] In this example, in order to study whether different concentrations of KV-7A have an effect on the cell viability of HCEs, we treated the cells with drugs for 24 hours and then tested the cell viability by CCK8 assay. The specific experimental steps are as follows:

[0189] (1) The cell density of HCEs was observed to be 70%-80% under a microscope. The cells were digested with 0.25% trypsin and centrifuged. The cells were resuspended to 50,000 cells / ml in complete DEME / F12 culture medium.

[0190] (2) 200 μl of cells were seeded into each well of a 96-well plate, gently shaken to evenly distribute the cells, and then placed in an incubator at 37°C and 5% CO2;

[0191] (3) After the cells adhered to the wall, the culture medium in the 96-well plate was discarded and replaced with basal medium containing 2% FBS, and placed in an incubator for 2 h;

[0192] (4) Using basal medium containing 5% FBS as the control group, culture medium containing 5% FBS was used to prepare culture solutions with different concentrations of KV-7A (10 μM, 50 μM, 100 μM, 500 μM, 1000 μM, 1500 μM, 2000 μM, 5000 μM);

[0193] (5) Discard the culture medium in the original 96-well plate and add the culture medium of each group mentioned above, 200 μl per well, 6 replicates per group, and place in the incubator;

[0194] (6) According to the instructions of the CCK8 kit (C0038, Bio-Tech Biotech Co., Ltd.), the CCK8 solution was prepared with basal culture medium at a ratio of 1:10;

[0195] (7) After 24 h, the culture medium in the 96-well plate was discarded, 100 μl of CCK8 solution was added to each well, and the cells were incubated in an incubator at 37°C in the dark for 2-4 h.

[0196] (8) The OD value at a wavelength of 450 nm was measured using an enzyme-labeled instrument and statistically analyzed using GraphPad Prism 8.0.

[0197] The results show that Figure 7 ): Control group (corresponding to Figure 7 The average cell viability of the first column was 1.000 ± 0.043, and the 100 μM, 500 μM, 1000 μM, 1500 μM, 2000 μM, 2500 μM and 5000 μM groups (corresponding to Figure 7 The average relative cell viability of the second to eighth columns was 1.013±0.047, 0.974±0.048, 0.977±0.044, 0.952±0.031, 0.918±0.043, 0.911±0.039 and 0.841±0.053, and the cell viability of each group was above 80%. Compared with the control group, the relative cell viability of the cells in the 100μM, 500μM, and 1000μM groups did not decrease significantly, and the difference was not statistically significant (P was 0.4938, 0.2010, and 0.2424, respectively). The cell viability of the 1500μM, 2000μM, 2500μM, and 5000μM groups was slightly reduced compared with the control group, and the difference was statistically significant (P<0.0001). The results show that KV-7A has a certain effect on the cell viability of HCEs, but has no obvious toxic effect.

[0198] Example 8. Scratch assay to detect the inhibitory effect of KV-7A on HUVECs migration

[0199] In this example, KV-7A concentrations (500 μM and 1000 μM) that had no significant effect on the cell viability of HUVECs and HCEs were used to study the inhibitory effect of KV-7A on HUVECs migration through a scratch test. The specific experimental steps are as follows:

[0200] (1) The cell density of HUVECs was observed to be 70%-80% under a microscope. The cells were digested with 0.25% trypsin and centrifuged. The cells were resuspended to 100,000 cells / ml in complete ECM culture medium.

[0201] (2) Use a marker to draw a straight line on the bottom of the 6-well plate as a marking line for taking photos;

[0202] (3) Take 2 ml of the cell resuspension and add it to a 6-well plate. Shake well to make the cells evenly distributed. Incubate in an incubator for 24 h at 37°C and 5% CO2.

[0203] (4) The culture medium and non-adherent cells in the 6-well plate were discarded and the cells were treated with ECM culture medium containing 2% FBS for 2 h;

[0204] (5) The basal medium containing 5% FBS was used as the NC group, the basal medium containing 5% FBS containing 20 ng / ml VEGF was used as the VEGF group, and the basal medium containing 5% FBS containing 20 ng / ml VEGF was used to prepare culture medium containing different concentrations of KV-7A (500 μM, 1000 μM) as the VEGF+KV-7A (500 μM) and VEGF+KV-7A (1000 μM) groups, respectively;

[0205] (6) Use a 200 μl pipette tip to make a scratch perpendicular to the bottom mark line of the 6-well plate, rinse the floating cells twice with sterile 1x PBS, and then add 2 ml of the culture medium of the above different groups, with at least 3 replicates for each group;

[0206] (7) Take photos using an ordinary optical microscope at 0 h, 6 h, and 12 h. The photographed area selected at each time point should be consistent with the marking line on the back. Use image J software to measure the average scratch width of different groups at different time points, and perform statistical analysis using GraphPad Prism 8.0.

[0207] The results show that Figure 8 ): Figure 8 A is the result of the scratch test, which shows that the width of the scratch in each group decreases with time. At 6 h, the average migration rate of the NC group was 0.262±0.018, and the average migration rates of the VEGF, VEGF+KV-7A (500μM) and VEGF+KV-7A (1000μM) groups were 0.383±0.012, 0.332±0.015, and 0.297±0.028; at 12 h, the average migration rate of the NC group was 0.447±0.032, and the average migration rates of the VEGF, VEGF+KV-7A (500μM) and VEGF+KV-7A (1000μM) groups were 0.583±0.045, 0.499±0.016, and 0.476±0.035. Compared with the NC group, the migration rate of the VEGF group increased significantly at 6h and 12h, and the difference was statistically significant (P values ​​were all <0.001). Compared with the VEGF group, the migration rate of the VEGF+KV-7A (500μM) and VEGF+KV-7A (1000μM) groups decreased significantly at 6h and 12h, and the difference was statistically significant (P values ​​were all <0.001). The results showed that KV-7A could significantly inhibit the migration of HUVECs.

[0208] Example 9. Transwell assay to detect the ability of KV-7A to inhibit VEGF165-induced HUVECs migration

[0209] This example uses a Transwell experiment to explore the ability of KV-7A to inhibit the migration of HUVECs. The specific experimental steps are as follows:

[0210] (1) The cell density of HUVECs was 70%-80% under microscopy and treated with serum-free basal medium for 24 h;

[0211] (2) Digest the cells with 0.25% trypsin and centrifuge, then resuspend the cells to 100,000 cells / ml in serum-free basal medium;

[0212] (3) The basal medium containing 5% FBS was used as the NC group, the basal medium containing 5% FBS containing 20 ng / ml VEGF was used as the VEGF group, and the basal medium containing 5% FBS containing 20 ng / ml VEGF was used to prepare culture medium containing different concentrations of KV-7A (500 μM, 1000 μM) as the VEGF+KV-7A (500 μM) and VEGF+KV-7A (1000 μM) groups, respectively;

[0213] (4) Add 600 μl of the above culture medium to the lower chamber of the Transwell and place the upper chamber in the well;

[0214] (5) Add 100 μl of cell resuspension into the upper chamber of the Transwell and culture in an incubator for 24 h at 37°C and 5% CO2;

[0215] (6) dissolving crystal violet in methanol and preparing a 0.5% crystal violet staining solution as a storage solution, which is then diluted to a 0.05% crystal violet staining solution before use;

[0216] (7) Remove the upper chamber of the Transwell, remove the culture medium, and wash three times with 1xPBS, 5 min each time;

[0217] (8) Soak the Transwell membrane in 4% paraformaldehyde for 30 min, discard it, and then wash it three times with 1xPBS, each time for 5 min;

[0218] (9) Aspirate the liquid from the Transwell membrane, stain with 0.05% crystal violet stain for 10 min, wash the staining solution in the upper chamber with 1x PBS, and gently wipe off the cells on the inner surface of the upper chamber membrane with a moistened cotton swab;

[0219] (10) Observe and take photos under a conventional microscope to record the cell number, and perform statistical analysis using GraphPad Prism 8.0.

[0220] The results show that Fig. 9 ): Fig. 9A is the microscopic photo of the Transwell membrane of each group. After 24 hours of treatment with different groups, the average number of cells in the NC group was 1.000±1.414, and the average number of migrated cells in the VEGF, VEGF+KV-7A (500μM) and VEGF+KV-7A (1000μM) groups was 220.667±34.586, 135.333±8.807 and 31.000±7.483. Compared with the NC group, the number of migrated cells in the VEGF group increased significantly, with significant differences (P=0.009). The number of cells in VEGF+KV-7A (500μM) and VEGF+KV-7A (1000μM) groups decreased significantly compared with the VEGF group, and the difference was statistically significant (P=0.0278 and 0.0020, respectively). The results show that KV-7A can significantly inhibit the migration of HUVECs, and it is dose-dependent within a certain concentration range.

[0221] Example 10. Matrigel tube formation assay to detect the inhibitory effect of KV-7A on HUVECs tube formation ability

[0222] This example uses Matrigel gel tube formation experiment to explore the inhibition of KV-7A on HUVECs tube formation. The specific experimental steps are as follows:

[0223] (1) HUVECs were seeded on a 100 mm dish and cultured in a basal medium containing 5% FBS as the NC group, a basal medium containing 20 ng / ml VEGF in 5% FBS as the VEGF group, and a basal medium containing 20 ng / ml VEGF in 5% FBS prepared with culture medium containing different concentrations of KV-7A (500 μM, 1000 μM) as the VEGF+KV-7A (500 μM) and VEGF+KV-7A (1000 μM) groups, respectively. The cells were cultured in an incubator at 37°C and 5% CO2 for 24 h.

[0224] (2) Pre-cool a 48-well plate and a 200 μl pipette tip, take the Matrigel out of the -20°C refrigerator, place it on a crushed ice box, and place it in a 4°C refrigerator to melt;

[0225] (3) Take 100 μl of Matrigel per well and add it to a pre-cooled 48-well plate. Place it in an ice box. After the plate is leveled, place it in a 37°C incubator to solidify for 30 min.

[0226] (4) After 24 h of different treatments, the cells were digested with 0.25% trypsin and centrifuged, and the cells were resuspended to 500,000 cells / ml in serum-free ECM medium;

[0227] (5) Add the cell suspension to the solidified Matrigel, 100 μl per well, and culture for 6-9 h;

[0228] (6) Observe and take pictures under a normal microscope, use image J software to analyze the nodes and blood vessel lengths of each group of cells, and use GraphPad Prism 8.0 for statistical analysis. The results show that ( Fig.10 ): The cells in the NC group, VEGF group, VEGF+KV-7A (500μM) group and VEGF+KV-7A (1000μM) group all showed the phenomenon of cell connection into a network and tube formation. The average number of cell connection nodes in the NC group was 67.667±1.247, and the average number of cell connection nodes in the VEGF, VEGF+KV-7A (500μM) and VEGF+KV-7A (1000μM) groups was 105.000±7.348, 69.667±6.600 and 67.000±3.742. Compared with the NC group, the number of cell connection nodes in the VEGF group increased significantly, and there was a significant difference (P=0.0021). The number of cell connection nodes in the VEGF+KV-7A (500 μM) group and the VEGF+KV-7A (1000 μM) group was significantly reduced compared with the VEGF group (P=0.0072, P=0.0029).

[0229] The average length of the NC group was 16636.263±1171.460, and the average number of cell connection nodes in the VEGF, VEGF+KV-7A (500μM) and VEGF+KV-7A (1000μM) groups was 32878.447±2350.366, 27562.117±2116.874 and 24891.387±565.608. Compared with the NC group, the length of the VEGF group was significantly increased, and there was a significant difference (P=0.009). Compared with the VEGF group, the length of the VEGF+KV-7A (500μM) group did not decrease significantly (P=0.1540), and the length of the VEGF+KV-7A (1000μM) group was significantly reduced, and the difference was statistically significant (P=0.0095). The results showed that KV-7A could significantly inhibit the effect of VEGF on HUVECs tube formation in a dose-dependent manner within a certain concentration range.

[0230] Example 11. Effect of KV-7A on the expression of VEGFA protein in HUVECs

[0231] Based on the experimental results in the previous article, we used normal culture medium as the control group, VEGF (20ng / ml) as the model group, and VEGF (20ng / ml) + KV-7A (1000μM) as the drug group. After treating HUVECs cells for 12h, 24h and 48h, the total cell protein was extracted and Western Blot experiments were performed. The specific experimental steps are as follows:

[0232] (1) Total protein extraction: After digestion and centrifugation of the cells, they were seeded on a 6-well plate and cultured in a 37°C, 5% CO2 incubator. When the cells grew to 80%-90% under a microscope, the culture medium was removed, and the cells were washed twice with warm sterile 1xPBS and discarded. 100 μl of protein lysis buffer was added to each well, and the cells were scraped off with a cell scraper. All the liquid in the well was transferred to a 1.5 ml centrifuge tube and stored at -80°C; centrifuged at 4°C for 10 min at 14000 rpm / min. After centrifugation, the supernatant was taken and placed in a new centrifuge tube;

[0233] (2) Protein quantification using the BCA method:

[0234] a. Prepare the standard sample with standard solution according to the gradient: 2000, 1500, 1000, 500, 250, 125, 62.5, 31.25, 0 μg / ml;

[0235] b. Take liquid A and liquid B in the kit and mix them evenly at a volume ratio of A:B=50:1;

[0236] c. Take 10 μl of the gradient standard and each sample and add them to a 96-well plate, then add AB mixture, 200 μl per well, and incubate at 37°C in the dark for 30 minutes;

[0237] d. Measure the OD value at a wavelength of 570 nm using an enzyme reader, and make a standard curve based on the gradient standard;

[0238] e. Substitute the OD value of the sample into the standard curve equation to calculate the protein concentration of the sample;

[0239] f. Dilute the sample to a consistent concentration with protein lysis buffer according to the protein concentration, add 4x protein denaturation loading, boil at 100℃ for 10min, and store at -80℃;

[0240] (3) SDS-PAGE gel preparation and electrophoresis:

[0241] a. Clean the glass plate with detergent, scrape off the residual glue, check whether there are impurities in the cut surface, rinse with DDW, and put it in the oven to dry;

[0242] b. Insert a set of glass plates into the plastic plate clamp, make sure the bottom is flush, clamp it tightly, and clamp it vertically into the plastic rack;

[0243] c. Prepare the separation gel in a 50ml centrifuge tube according to the instructions of the rapid gel preparation kit. Each plate needs 10ml. Shake up and down to mix evenly, and then pour the separation gel into the gap between the glass plates;

[0244] d. Pour an appropriate amount of isopropanol on top of the separation gel to press the gel and make the separation gel coagulate faster;

[0245] e. After the separation gel solidifies, you can see a clear fold line between the isopropanol and the separation gel. Pour out the isopropanol on top, rinse with DDW, and invert for a few minutes to dry.

[0246] f. Prepare concentrated gel according to the instructions, 4 ml per plate, mix well, pour concentrated gel on separation gel, vertically insert sample well comb, and wait for gel to coagulate at room temperature for about 40 minutes;

[0247] g. Take out the sample and place it on ice to melt. After shaking, centrifuge briefly. Take the electrophoresis gel from the gel preparation rack and put it into the electrophoresis tank. Fill the electrophoresis tank with electrophoresis liquid. Gently pull out the comb vertically upwards and prepare for sample loading.

[0248] h. When loading the sample, the tip of the pipette should be close to the glass plate and slowly added without generating bubbles;

[0249] i. Close the electrophoresis tank cover tightly, adjust the voltage to 80V, and after the protein runs into the separation gel, adjust the voltage to 120V. Determine whether to terminate the electrophoresis based on the marker and loading of the protein electrophoresis;

[0250] (4) Wet transfer:

[0251] a. Prepare the transfer solution, pre-cool it in the refrigerator, pour it into the plate after the electrophoresis is finished, put the transfer clip, two cotton pads and two filter papers into the plate, and activate the PVDF membrane with methanol for 1 minute;

[0252] b. Separate the two glass plates, remove the gel with a cutter, and clamp the transfer clamp in the order of transfer clamp (black) - cotton pad - filter paper - gel - PVDF membrane - filter paper - cotton pad - transfer clamp (white). During this process, be sure to use a roller to drive away bubbles and impurities;

[0253] c. Place the transfer clip with the black side facing the black side of the transfer tank into the transfer tank and clamp it tightly. Fill it with pre-cooled transfer solution. Since a lot of heat will be generated during the transfer, put it in an ice box or ice bag to cool it down. After closing the lid, put the entire transfer tank into a foam box with crushed ice.

[0254] d. Adjust the voltage and time of membrane transfer according to the molecular weight of the protein;

[0255] (5) Immune response:

[0256] a. Take out the membrane from the transfer folder, place it with the protein side facing up, and immerse it in 5% BSA blocking solution. Incubate it on a shaker at room temperature for 2 hours or at 4°C overnight.

[0257] b. According to the instructions of VEGFA (ab46154, Abcam), 5 ml of primary antibody dilution was prepared with antibody diluent at a dilution ratio of 1:10000 in a 50 ml centrifuge tube. The blocking solution in the incubation box was discarded, and the membrane was placed in a 50 ml centrifuge tube with the protein side facing inwards. Incubate overnight on a rotating shaker at 4°C.

[0258] c. The next day, remove the membrane and place it in an incubation box. Wash it three times with TBST solution on a shaker, 10 minutes each time.

[0259] d. According to the instructions of goat anti-rabbit (32460, Thermo Fisher Scientific), prepare the secondary antibody diluent at 1:5000 with antibody diluent, discard the TBST solution in the incubation box, pour the secondary antibody diluent to immerse the PVDF membrane, and incubate slowly at room temperature on a shaker for 1 hour;

[0260] e. Discard the secondary antibody dilution in the incubation box, add appropriate amount of TBST solution and wash three times on a shaker, each time for 10 minutes;

[0261] f. Take the ECL chemiluminescence kit, prepare the developer in a 1:1 ratio according to the instructions, and mix well;

[0262] g. Place a plastic wrap in the tray of the gel imager, then lay the washed PVDF membrane flat on the designated position, use a pipette to absorb a certain amount of developer to cover the membrane, set the exposure time and the number of exposure pictures, take chemiluminescent photos and white light photos, and use image J software for processing and grayscale analysis, and use GraphPad Prism 8.0 for statistical analysis. The results show ( Fig.11 ): After 12h, 24h and 48h of cell treatment, the expression of VEGFA in the model group was significantly increased compared with the normal group (P=0.0094, P=0.0014, P=0.0044), and the difference was statistically significant. After 12h, 24h and 48h, the expression of VEGFA in the drug-added group was significantly decreased compared with the model group (P=0.0021, P=0.0143, P=0.0101), and the difference was statistically significant. The results show that KV-7A can downregulate the protein expression of VEGFA in HUVECs.

[0263] Example 12. Effect of KV-7A on VEGF gene expression in HUVECs

[0264] In this example, ordinary ECM culture medium was used as the control group, VEGF (20 ng / ml) was used as the model group, and VEGF (20 ng / ml) + KV-7A (1000 μM) was used as the drug addition group. After treating HUVECs cells for 24 hours, mRNA was extracted and real-time fluorescence PCR was performed after reverse transcription. The specific experimental steps are as follows:

[0265] (1) After digestion and centrifugation, the cells were seeded on a 24-well plate and cultured in a 37°C, 5% CO2 incubator. When the cells grew to 70%-80% under a microscope, the culture medium was removed, and the cells were washed twice with warm sterile 1xPBS and discarded. 1 ml of Trizol was added to each well, and the cells were repeatedly blown up and down with a pipette. All the liquid in the well was transferred to a 1.5 ml enzyme-free centrifuge tube and stored at -80°C.

[0266] (2) Wear a mask and gloves before the experiment. The centrifuge tubes and reagents used are not treated with enzymes. The operation is performed on a crushed ice box to prevent RNA degradation.

[0267] (3) Melt the sample on ice and pre-cool the cryogenic grinder;

[0268] (4) Add enzyme-free grinding beads to each tube of sample and place it in a cryogenic grinder and grind three times, 60 seconds each time;

[0269] (5) Add 500 μl of chloroform to each tube, shake quickly to mix thoroughly, and centrifuge at 4°C for 5 min at 12,000 rpm / min;

[0270] (6) After centrifugation, take the upper layer of liquid and place it in a new enzyme-free centrifuge tube. Add 700 μl of isopropanol to each tube, gently mix by inverting, and place at -80°C.

[0271] (7) After 20 min, remove the sample and centrifuge at 4°C for 10 min, 12,000 rpm / min;

[0272] (8) Prepare 70% ethanol solution using DEPC water and anhydrous ethanol for later use;

[0273] (9) After centrifugation, discard the liquid in the tube, add 700 μl of 70% ethanol to each tube, and centrifuge again at 4°C for 3 min, 12000 rpm / min. Repeat this step twice;

[0274] (10) After discarding the liquid in the tube, centrifuge at 12000 rpm / min at 4°C for 2 min;

[0275] (11) Use a pipette to remove the liquid in the tube. After air drying, add an appropriate amount of enzyme-free water to each tube and gently tap to dissolve the RNA. Add 0.5 μl of RNA inhibitor to each tube. After simple centrifugation, measure the RNA concentration and adjust the concentration to 100 ng / μl. It can be stored in a -80°C refrigerator for a long time.

[0276] (12) According to the rapid RNA reverse transcription reaction system, add the following reagents into a 0.2 ml enzyme-free centrifuge tube: 5x PrimeScript RT Master Mix—4 μl, mRNA—10 μl, DEPC water—6 μl;

[0277] (13) After simple centrifugation, place the tube in a PCR instrument and perform reverse transcription under the following reaction conditions: 37°C for 15 min, 85°C for 5 s, 4°C for Pause;

[0278] (14) After the reaction is completed, the cDNA can be stored in a -20°C refrigerator.

[0279] (15) Add the following reagents into the PCR reaction tube according to the real-time fluorescence PCR system: 2x Taq Pro UniversalSYBR qPCR Master Mix—5 μl, Froward Primer—0.2 μl, Reverse Primer—0.2 μl, cDNA—1 μl, DEPC water—3.6 μl;

[0280] (16) After simple centrifugation, the reaction tube was placed in a PCR instrument and real-time fluorescence quantitative PCR was performed according to the following real-time fluorescence PCR reaction conditions: a. Pre-denaturation: 95°C-3 min once; b. Cycling reaction: 95°C-10 s, 60°C-30 s 40 times; c. Melting curve: 95°C-15 s, 60°C-60 s, 95°C-15 s once;

[0281] (17) Result analysis: The data were exported from the machine, and the expression level difference between each target gene and the internal reference gene was calculated using the ΔΔCT method. GraphPad Prism 8.0 was used for statistical analysis. The results show ( Fig.12 ): Compared with the normal group, the expression of VEGFA in the model group was significantly increased (P=0.0097, P<0.0001), and the difference was statistically significant. Compared with the model group, the expression of VEGFA in the drug-added group was significantly decreased (P=0.0026, P=0.001), and the difference was statistically significant. The results showed that KV-7A could downregulate the gene expression of VEGFA and VEGFC in HUVECs.

[0282] Example 13. VE-Cadherin staining of HUVECs

[0283] In this example, VE-Cadherin immunofluorescence staining was used to observe the effect of KV-7A on the junction between HUVECs and endothelial cells. The specific experimental steps are as follows:

[0284] (1) Cells were seeded on a 30 mm confocal culture dish, and after the cell density reached 50%-60%, the following culture media were added: ordinary culture medium containing 5% FBS was used as the control group, ordinary culture medium containing 5% FBS containing 20 ng / ml VEGF was used as the model group, and ordinary culture medium containing 5% FBS containing 20 ng / ml VEGF and 1000 μM KV-7A was used as the drug-added group;

[0285] (2) Place the cells in a cell culture incubator at 37°C and 5% CO2 for 24 h, rinse twice with 1xPBS, and fix with 4% paraformaldehyde for 30 min;

[0286] (3) Rinse three times with 1xPBS, 5 min each time, and add appropriate amount of 0.2% Triton X-100 to permeabilize the membrane for 15 min;

[0287] (4) Dissolve 1 g of BSA powder in 50 ml of 1xPBS solution to prepare blocking solution. Rinse the cells three times with 1xPBS, 5 min each time, add appropriate amount of blocking solution, and block at room temperature for 1 h.

[0288] (5) Dissolve 0.5 g of BSA powder in 50 mL of 1x PBS solution to prepare an antibody diluent. Dilute the antibody diluent at 1:100 according to the instructions of VE-Cadherin (ab33168, Abcam) and set aside.

[0289] (6) Aspirate the blocking solution and wash three times with 1xPBS, 5 min each time;

[0290] (7) Add 100 μl of primary antibody dilution and incubate overnight at 4°C on a shaker;

[0291] (8) The next day, remove the primary antibody and wash three times with 1xPBS, 5 min each time;

[0292] (9) Protect from light. Follow the instructions of the goat anti-rabbit secondary antibody conjugated with 594 fluorescent group (A32740, Thermo Fisher Scientific), dilute it with antibody diluent at 1:300, add 100 μl of secondary antibody diluent to each well, and incubate at room temperature in the dark for 1 h.

[0293] (10) Remove the secondary antibody and wash three times with 1xPBS, 5 min each time;

[0294] (11) After absorbing the liquid in the culture dish, use DAPI (H-1200) to cover the slides and store them at 4°C away from light. Observe and photograph under a fluorescence microscope.

[0295] The results show that Fig.13 ): The VE-cadherin staining (red) of HUVECs in the control group was clear and continuous. Compared with the normal group, the VE-cadherin staining (red) of the model group was discontinuous, and the fluorescence intensity was significantly weakened. Compared with the model group, the VE-cadherin staining (red) of the drug-added group was clearer, with enhanced intensity and continuous. The results show that KV-7A can inhibit the migration of HUVECs by enhancing the connection between vascular endothelial cells.

[0296] Example 14. Chicken embryo chorioallantoic membrane assay to detect the anti-angiogenic effect of KV-7A

[0297] In order to detect the effect of KV-7A on inhibiting angiogenesis in animal models, we first conducted a chick embryo chorioallantoic membrane experiment. The specific experimental steps were as in Example 1, and the groups were divided into NC group (sterile PBS), VEGF group (20ng / ml VEGF) and different concentrations of KV-7A group (20ng / ml VEGF + 500μM or 1000μM or 1500μM KV-7A), and the dosing range was as follows: Fig.14 As shown by the yellow dotted line in A, the drug was added and incubated for 48 hours before taking photos and observing.

[0298] The results are as follows ( Fig.14 ): Angiogenesis was observed in the NC group. The blood vessels in the VEGF group were larger and more tortuous than those in the NC group, and the number of blood vessels was significantly increased (P=0.0074). Compared with the VEGF group, the number of blood vessels in the drug-treated area in the VEGF+KV-7A (500μM), VEGF+KV-7A (1000μM) and VEGF+KV-7A (1500μM) groups was significantly reduced, and the difference was statistically significant (P values ​​were all <0.001). The results showed that 500-1500μM KV-7A could significantly inhibit the effect of VEGF on promoting angiogenesis in the chick embryo chorioallantoic membrane.

[0299] Example 15. Construction of suture-induced corneal neovascularization model in rats

[0300] To further study the effect of KV-7A on corneal neovascularization, we constructed a suture-induced rat corneal neovascularization model, observed for 14 days, and performed slit lamp photography on days 1, 3, 5, 7, 10, and 14 to find the best and most appropriate observation time. The specific experimental steps are as in Example 2.

[0301] The observation results are as follows ( Fig.15): The cornea of ​​the NC group without sutures was transparent and without edema. No blood vessels were seen in the cornea under the green filter. When observed from the side, there was no obvious filling and tortuosity of the conjunctival blood vessels. One day after the suture, the knot was in place, the cornea was slightly edematous, very few new blood vessels were seen at the limbus under the green filter, and the conjunctival blood vessels were filled and tortuosity when observed from the side. Three days after the suture, the knot was in place, the cornea was edematous, a few new blood vessels were seen growing into the cornea at the limbus, and the conjunctival blood vessels were filled and tortuosity. Five days after the suture, the knot was in place, the cornea was obviously edematous, and new blood vessels were seen growing into the cornea, about half the distance from the limbus to the suture knot, and the conjunctival blood vessels were filled and tortuosity. Seven days after the suture, the knot was in place, the cornea was edematous, and new blood vessels grew close to the suture knot. A little epithelium and secretions were seen attached to the knot, and the conjunctival blood vessels were filled and tortuosity. Ten days after the suture, the knot was in place, the cornea was edematous, the density of new blood vessels decreased, the growth length reached and slightly exceeded the suture knot, secretions were visible in the conjunctival sac, and the conjunctival blood vessels were filled and tortuous. Fourteen days after the suture, the knot was in place, the cornea was slightly edematous, and the new blood vessels were mainly around the knot. The density of new blood vessels not located at the knot was low and slender, and the conjunctival blood vessels were filled and tortuous.

[0302] Example 16. Inhibitory effect of different concentrations of KV-7A on suture-induced corneal neovascularization in rats

[0303] According to the suture-induced rat corneal neovascularization model in Example 15, 7 days is the time point when corneal neovascularization grows most vigorously, so we chose this time point to observe and evaluate the inhibitory effect of different concentrations of KV-7A on suture-induced rat corneal neovascularization. The specific experimental steps are as in Example 2.

[0304] The results show that Fig.16 ): Compared with the negative control PBS group, the area of ​​corneal neovascularization in the 10mM, 15mM, 25mM and 35mM KV-7A groups was reduced (P=0.0068, P<0.001, P<0.001, P=0.0038), the length of blood vessels was shortened (all P values ​​were <0.001), and the inflammatory index was reduced (P=0.0039, P<0.001, P<0.001, P=0.0082), and the differences were statistically significant. This shows that 10mM, 15mM, 25mM and 35mM KV-7A all have the effect of inhibiting corneal neovascularization, among which 15mM KV-7A has the best effect of inhibiting corneal neovascularization. Corneal fluorescein sodium staining of each group showed that the cornea was stained at the suture site, the epithelium was lost, and the cornea was not stained at the unsuture site, and the epithelium was intact. This indicates that 10mM, 15mM, 25mM and 35mM KV-7A have no obvious toxic effect on the corneal epithelium, and can inhibit the area and length of corneal neovascularization, as well as inhibit inflammation.

[0305] Example 17. Comparison of KV-7A and Conbercept in inhibiting neovascularization of chick embryo chorioallantoic membrane

[0306] In this example, in order to compare the effects of KV-7A and Conbercep on the inhibition of neovascularization of chick embryo chorioallantoic membrane, we set up NC group (sterile PBS), VEGF group (20 ng / ml VEGF), Conbercep group (20 ng / ml VEGF + 0.1 mg / ml Conbercep) and KV-7A group (20 ng / ml VEGF + 1000 μM KV-7A). Fig.17 A is indicated by a yellow circle, and the mixture is incubated for 48 hours under appropriate conditions. The specific experimental steps are as in Example 1.

[0307] The results are as follows ( Fig.17 ): The average vascular area ratio of the NC group and the VEGF group was 52.183±9.084, and the average vascular area ratio of the VEGF group was 69.441±11.821. Compared with the NC group, the blood vessels were thick and tortuous, and the number of blood vessels was significantly increased, and the difference was statistically significant (P=0.0150). The average vascular area ratio of the Conbercept group and the KV-7A group was 21.072±6.783 and 22.139±6.836. Compared with the VEGF group, the blood vessels in the KV-7A group and the Conbercept group were significantly reduced in the drug-treated area, and the difference was statistically significant (P values ​​were all <0.001), and there was no significant difference between the KV-7A and Conbercept groups. The results show that both KV-7A and Conbercept can significantly inhibit the formation of blood vessels in the chick embryo chorioallantoic membrane, and the degree of inhibition of the two is similar.

[0308] Example 18. Comparison of KV-7A (15 mM) and Conbercept in the treatment of suture-induced corneal neovascularization in rats

[0309] We selected 15mM KV-7A and 1% Conbercept for comparison, and used the PBS group as a negative control, and observed and evaluated the effects of treating suture-induced corneal neovascularization in rats on days 3, 7, and 10. The specific experimental steps are as in Example 2.

[0310] The results show that Fig.18 ): On day 3, the average vascular areas of the PBS group, KV-7A group, and Conbercept group were 26.409±3.509, 20.621±2.121, and 27.368±3.201, respectively; the average vascular lengths were 13.503±3.463, 10.854±2.961, and 14.136±2.869; the average inflammatory indexes were 0.687±0.155, 0 .500±0.118 and 0.583±0.160. At 7 days, the average vascular areas of the PBS group, KV-7A group and Conbercept group were 76.123±5.190, 57.976±8.088 and 75.690±4.385, respectively; the average vascular lengths were 54.034±9.801, 34.769±10.046 and 50.701±6.658

[0311] ; The average inflammatory index was 0.708±0.110, 0.521±0.130 and 0.639±0.062. At 10 days, the average vascular area of ​​the PBS group, KV-7A group and Conbercept group was 90.429±3.139, 67.429±11.881 and 87.800±5.832, respectively; the average vascular length was 69.834±6.866, 45.154±15.780 and 67.821±13.583; the average inflammatory index was 0.646±0.155, 0.333±0.167 and 0.500±0.096.

[0312] Compared with the PBS group, the area of ​​corneal neovascularization in the KV-7A group was significantly reduced (P=0.0022, P<0.001, P<0.001), the length of blood vessels was significantly shortened (all P values ​​were <0.001), and the inflammatory index was significantly reduced (P=0.0230, P=0.0114, P=0.0027) at 3, 5, and 7 days, and the differences were statistically significant. However, the area of ​​corneal neovascularization, blood vessel length, and inflammatory index in the Conbercept group were reduced compared with the PBS group, but there was no statistically significant difference (all P values ​​were >0.05). The results showed that KV-7A had a good therapeutic effect on suture-induced CNV in rats, while Conbercept had no obvious effect on suture-induced CNV in rats.

[0313] Example 19. Whole-mount staining of rat corneas in different treatment groups

[0314] Ten days after corneal suture, the corneas of rats in the NC group, PBS group, 15mM KV-7A group, and 1% Conbercept group were subjected to whole-mount staining of CD146. The specific experimental steps are as follows:

[0315] (1) After the rats were killed by cervical dislocation, the corneas were removed using micro-toothless forceps and corneal scissors and placed in pre-cooled 1xPBS;

[0316] (2) Fix the cornea with 4% paraformaldehyde solution for 20 min and wash it three times with 1xPBS, each time for 5 min;

[0317] (3) Place the cornea in 0.5% Triton X-100 solution to permeabilize the membrane and incubate in a 4°C refrigerator overnight;

[0318] (4) Remove excess liquid and wash three times with 1xPBS, 5 min each time;

[0319] (5) According to the CD146 (ab75769, Abcam) instructions, the primary antibody dilution was prepared with 1% BSA, each cornea was covered with an appropriate amount of the primary antibody dilution, and incubated in a refrigerator at 4°C overnight;

[0320] (6) Remove the primary antibody and wash three times with 1xPBS, 5 min each time;

[0321] (5) Protect from light. Follow the instructions of the goat anti-rabbit secondary antibody conjugated with 594 fluorescent group (A32740, Thermo Fisher Scientific) and dilute it with antibody diluent at 1:300. Cover each cornea with an appropriate amount of secondary antibody diluent and incubate in a refrigerator at 4°C overnight.

[0322] (6) Remove the secondary antibody and wash three times with 1xPBS, 5 min each time;

[0323] (7) Move the cornea under the operating microscope, cut the cornea into four petals with corneal scissors, flatten them on a glass slide, remove excess water droplets, and seal the slide with glycerol (H-1000);

[0324] (8) Observe and take photos under a fluorescence microscope.

[0325] The results show that Fig.19 ): The NC group was a normal cornea without sutures, with no obvious strip-shaped green fluorescence. In the PBS group, a large number of blood vessels (green) growing from the corneal edge to the central line knot were observed. In the KV-7A group, small blood vessels were observed at the corneal edge, which was less than that in the PBS group. In the Conbercept group, thin and long blood vessels were observed growing at the corneal edge, which was less than that in the PBS group. The results showed that KV-7A was better at inhibiting the growth of blood vessels in suture-induced rat CNV.

[0326] Example 20. CD146 immunofluorescence staining of corneal paraffin sections of rats in different treatment groups

[0327] Seven days after corneal suture, corneal paraffin sections were prepared from the whole eyeballs of rats in the NC group, PBS group, 15 mM KV-7A group and 1% Conbercept group, and then immunofluorescence staining of CD146 was performed. The specific experimental steps are as in Example 4.

[0328] The results show that Fig. 20 ): The corneal epithelium of the NC group was smooth, and there was no green fluorescence in the corneal stroma, that is, there were no blood vessels. The PBS group was the cornea after sutures, with increased thickness, and tubular green fluorescence, that is, new blood vessels, could be seen in the corneal stroma. The number was large and the depth reached more than two-thirds of the corneal stroma. The cornea of ​​the KV-7A group was thickened, and a small amount of tubular green fluorescence could be seen in the corneal stroma, which was significantly less than that of the VEGF group. The cornea of ​​the Conbercept group was significantly thickened, and tubular green fluorescence could be seen in the corneal stroma, and the number was less than that of the VEGF group. The results show that KV-7A can effectively reduce the density and depth of blood vessel growth in suture-induced rat CNV.

[0329] Example 21. HE staining of corneal paraffin sections of rats in different treatment groups

[0330] Seven days after corneal suture, the whole eyeballs of rats in the NC group, PBS group, 15mM KV-7A group and 1% Conbercept group were taken to prepare corneal paraffin sections, which were then stained with HE and 10x microphotographs were taken. The specific experimental steps are as in Example 3.

[0331] The results show that Fig.21 ): The NC group was a normal corneal epithelium with neat and smooth arrangement, and the corneal stromal cells in the stromal layer were neat and dense, without inflammatory cell infiltration and blood vessel growth. The PBS group was the cornea after suture, with a significant increase in the overall corneal thickness, a rough corneal epithelium, enlarged and neatly arranged basal cells, a large number of new blood vessels and inflammatory cell infiltration in the stromal layer, the depth of which reached more than two-thirds of the stromal layer, and red blood cells in the blood vessels. The KV-7A group had thickened corneas, a slightly rough corneal epithelium, slightly larger and neatly arranged epithelial basal cells, and new blood vessels and inflammatory cell infiltration in the stromal layer, the depth of which reached between one-third and one-half of the stromal layer. The Conbercept group had significantly thickened corneas, smooth and neat epithelial layers, and new blood vessels and inflammatory cell infiltration in the stromal layer, the depth of which reached about one-half of the stromal layer. The results show that compared with Conbercept, KV-7A can not only better reduce the density and depth of vascular growth in suture-induced rat CNV, but also reduce inflammation and corneal edema.

[0332] Example 22. Effect of KV-7A on genes related to corneal angiogenesis in rats

[0333] Seven days after corneal suture, the corneas of rats in the NC group, PBS group, and 15 mM KV-7A group were respectively taken to extract mRNA and then perform real-time fluorescence quantitative PCR. The specific experimental steps are as in Example 12.

[0334] The results show that Fig. 22 ): The NC group had normal cornea. Compared with the PBS group, the expressions of Vegfa and Vegfb increased (P<0.001, P=0.0245), the expressions of Vegfr2 and Vegfr3 increased (P=0.0048, P<0.001), Il1b increased (P<0.001), and Mmp9 and Mmp13 increased (P<0.001, P<0.001). The differences were statistically significant. Compared with the PBS group, the expression of Vegfb in the KV-7A group was decreased (P=0.2575), with no significant statistical difference. The expression of Vegfa was decreased (P=0.0323), the expression of Vegfr2 and Vegfr3 was decreased (P=0.0236, P=0.0091), Il1b was decreased (P=0.0281), Mmp9 and Mmp13 were decreased (P=0.0030, P<0.001), and the differences were statistically significant. The results showed that KV-7A could significantly downregulate the gene expression of Vegfa and its receptors Vegfr2 and Vegfr3 in the rat cornea after suture, and downregulate the gene expression of Il1b, Mmp9 and Mmp13.

[0335] Example 23. CCK8 assay to detect the inhibitory effect of KV-7A on HPF (human lung fibroblast) proliferation

[0336] In this example, the inhibitory effect of different concentrations of KV-7A on HPF proliferation was detected by CCK8 experiment. The specific experimental steps are as follows:

[0337] (1) HPFs were seeded in 96-well plates at 1000 cells / well;

[0338] (2) After 24 h, the original culture medium was discarded and 100 μl of DMEM / F12 culture medium containing 2% FBS was added to each well to starve the cells for 2 h;

[0339] (3) The culture medium was discarded, and a culture medium containing different concentrations of KV-7A and 1 ng / ml bFGF was prepared with 5% FBS+DMEM / F12 culture medium, 200 μl was added to each well, and cultured in a 5% CO2+37°C incubator for 24 h;

[0340] (4) Discard the old culture medium and replace with 100 μl of DMEM / F12 culture medium containing 10% CCK8, incubate at 37°C in the dark for 1-4 h;

[0341] (5) The OD values ​​were measured at 450 nm using an enzyme reader and statistical analysis was performed using Graphpad Prism 8.0.

[0342] The results are as follows: Control group (corresponding to Fig.23 The average proliferation rate of the first column) was 1.000±0.032, and the model group (corresponding to Fig.23 The average relative proliferation rate was 1.249±0.054, which was significantly higher than that of the control group, and the difference was statistically significant (P<0.001). Fig.23 The average relative proliferation rates of the third to seventh columns were 1.078±0.033, 0.975±0.031, 0.944±0.019, 0.898±0.017, and 0.843±0.025, respectively, which were all lower than those of the model group, with statistically significant differences, and P values ​​were all less than 0.001. The results showed that KV-7A could significantly inhibit the proliferation of HPF.

[0343] Example 24. Transwell Experimental testing KV-7A right HPF Inhibition of migration

[0344] In this example, the inhibitory effect of different concentrations of KV-7A on HPF migration was detected by Transwell assay. The specific experimental steps are as follows:

[0345] (1) HPF were cultured in serum-free basal medium at 37°C and 5% CO2;

[0346] (2) Add 600 μl of culture medium containing different concentrations of KV-7A and 1 ng / ml bFGF prepared in advance into the lower chamber of the Transwell, and place the upper chamber into the well;

[0347] (3) After the HPF grew to 70%-80%, the cells were digested with 0.25% trypsin, centrifuged and resuspended in DMEM medium without FBS to 100,000 cells / ml;

[0348] (4) Take 100 μl of the resuspension and inoculate it in the upper chamber of the cell, place it in a 37°C, 5% CO2 incubator, remove the culture medium after 24 hours, wash it once with 1xPBS, and fix the Transwell membrane with 4% paraformaldehyde for 30 minutes;

[0349] (5) Wash three times with 1xPBS, 5 min each time, aspirate the liquid from the Transwell membrane, and stain with 0.05% crystal violet stain for 10 min;

[0350] (6) Wash the staining solution in the upper chamber with 1xPBS, and gently wipe the cells on the inner surface of the upper chamber membrane with a moist cotton swab. Observe and photograph the cells under a normal microscope to record the number of cells, and perform statistical analysis with Graphpad Prism 8.0.

[0351] The results show that Fig.24 ): Fig.24 A is a 10x ordinary optical micrograph of each group of Transwell membranes. The purple spindle-shaped structures are human fibroblasts HPF that migrated to the other side of the Transwell membrane. Fig.24B is a statistical chart of the number of migrating cells in each group. The number of cells in the NC group, bFGF group, bFGF+KV-7A1mM, bFGF+KV-7A1.5mM, bFGF+KV-7A 2mM and bFGF+KV-7A 2.5mM groups were 118.5±33.0, 250.5±17.3, 183.0±46.2, 148.0±50.2, 147.8±40.8 and 119.8±15.7, respectively. Compared with the NC group, the number of migrating cells in the bFGF group was significantly increased (P<0.001). Compared with the bFGF group, the number of migrating cells in the bFGF+KV-7A 1mM group was slightly reduced, but the difference was not statistically significant (P=0.1099), and the number of migrating cells in the bFGF+KV-7A1.5mM, bFGF+KV-7A2mM and bFGF+KV-7A2.5mM groups was significantly reduced, and the differences were statistically significant (P=0.0160, 0.0089 and 0.0006, respectively). The results showed that KV-7A has the effect of inhibiting HPF migration.

[0352] Example 25. α-SMA staining of HPF

[0353] In this example, α-SMA immunofluorescence staining was used to observe the effect of KV-7A on HPF actin synthesis and distribution. The specific experimental steps are as follows:

[0354] (1) Cells were seeded on a 30 mm confocal culture dish, and after the cell density reached 50%-60%, the following culture media were added: ordinary culture medium containing 5% FBS as the control group, ordinary culture medium containing 5% FBS with 1 ng / ml bFGF as the model group, and ordinary culture medium containing 5% FBS with 1 ng / ml bFGF and 2.5 mM KV-7A as the drug-added group;

[0355] (2) Place the cells in a cell culture incubator at 37°C and 5% CO2 for 24 h, rinse twice with 1xPBS, and fix with 4% paraformaldehyde for 30 min;

[0356] (3) Rinse three times with 1xPBS, 5 min each time, and add appropriate amount of 0.2% Triton X-100 to permeabilize the membrane for 15 min;

[0357] (4) Dissolve 1 g of BSA powder in 50 ml of 1xPBS solution to prepare blocking solution. Rinse the cells three times with 1xPBS, 5 min each time, add appropriate amount of blocking solution, and block at room temperature for 1 h.

[0358] (5) Dissolve 0.5 g of BSA powder in 50 mL of 1x PBS solution to prepare an antibody diluent. Dilute the antibody diluent at 1:200 according to the instructions of α-SMA (#19245, CST) and set aside.

[0359] (6) Aspirate the blocking solution and wash three times with 1xPBS, 5 min each time;

[0360] (7) Add 100 μl of primary antibody dilution and incubate overnight at 4°C on a shaker;

[0361] (8) The next day, remove the primary antibody and wash three times with 1xPBS, 5 min each time;

[0362] (9) Protect from light. Follow the instructions of the goat anti-rabbit secondary antibody conjugated with 488 fluorescent group (A32731, Thermo Fisher Scientific), dilute it with antibody diluent at 1:300, add 100 μl of secondary antibody diluent to each well, and incubate at room temperature in the dark for 1 h.

[0363] (10) Remove the secondary antibody and wash three times with 1xPBS, 5 min each time;

[0364] (11) After absorbing the liquid in the culture dish, use DAPI (H-1200) to cover the slides and store them at 4°C away from light. Observe and photograph under a fluorescence microscope.

[0365] The results show that Fig.25 ): α-SMA staining (green) is located in the HPF cytoplasm, and DAPI (blue) is located in the nucleus. The fluorescence intensity of α-SMA staining of HPF in the model group increased compared with the normal group, and the fluorescence intensity of the drug-added group decreased compared with the model group. The results show that KV-7A can inhibit the synthesis of HPF α-SMA.

[0366] Example 26. Effect of KV-7A on OIR (oxygen-induced retinopathy) in mice ) Model of induced retinal neovascularization Influence

[0367] In this example, a mouse OIR model was established to induce fundus neovascularization, and sterile 1xPBS solution, 15Mm KV-7A and 10mg / ml Conbercept ophthalmic injection were injected into the vitreous cavity for treatment. The retina was taken for whole-mount staining of Lectin to evaluate the therapeutic effect of KV-7A on oxygen-induced retinal neovascularization in mice. The specific experimental steps are as follows:

[0368] (1) Select C57BL / 6 mice born in a normal barrier environment and record the date of birth. Place the mice on day 7 after birth (p7) together with their mothers in an oxygen chamber with 75% oxygen for 5 days;

[0369] (2) On p12, the mice were returned to a normal oxygen environment with an oxygen content of about 21%. At the same time, preparations were made for intravitreal injection therapy: after the mice were anesthetized with 1% Sumenxin 0.1ml / 100g, tropicamide hydrochloride eye drops were used to dilate the pupils, and a sterile insulin microneedle was used to puncture the lower corneal margin and inject the drug into the vitreous cavity with a microsyringe, with a drug volume of 0.1ul. After surgery, the mice were placed on a rewarming blanket for rewarming, and returned to a normal barrier environment for breastfeeding after waking up from anesthesia;

[0370] (3) Five days later, i.e., p17, the mice were weighed, and dysplastic mice weighing less than 6 g should not be included in the statistics because of their abnormal phenotypes.

[0371] (4) After cardiac perfusion with 4% paraformaldehyde at p17, the eyeballs were removed and fixed with 4% paraformaldehyde for 1 h;

[0372] (5) The mouse retina was completely removed and rinsed twice in 1xPBS solution, each time for 5 min. The retina was placed in 0.5% Triton X-100 at 4°C overnight for permeabilization;

[0373] (6) The next day, the retina was rinsed three times with 1xPBS solution, each time for 5 min;

[0374] (7) Protect from light. Prepare 5 ug / ml working solution with antibody diluent according to the instructions of Lectin (L32473, Thermo Fisher Scientific) coupled with 594 fluorescent group. Take an appropriate amount of working solution and incubate overnight at 4°C.

[0375] (8) On the third day, the retina was rinsed three times with 1x PBS solution, each time for 5 min. The retina was cut open under a surgical microscope, spread on a slide, and photographed under a fluorescence microscope.

[0376] The results show that Fig.26 ): Fig.26 A is a flat mount of the retina of OIR mice in each group, red represents blood vessels, the white dotted line represents the central retinal area without vascular perfusion, and the yellow dotted line represents the retinal neovascularization cluster; Fig.26 B and C are the statistical graphs of the ratio of the central non-vascular perfusion area and the area of ​​retinal neovascularization clusters to the total retinal area in each group of OIR mice; the average ratio of the central non-vascular perfusion area to the retinal area in the OIR group and the KV-7A group was 11.31±1.12 and 18.15±2.86, and the average ratio of the retinal neovascularization clusters to the retinal area was 13.40±2.29 and 4.19±0.65; compared with the OIR group, the central non-vascular perfusion area in the KV-7A group increased (P=0.0021) and the area of ​​the neovascularization clusters decreased (P<0.001), and the differences were statistically significant. The results show that KV-7A can significantly inhibit oxygen-induced retinal neovascularization.

[0377] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. A polypeptide, wherein the third amino acid residue in the amino acid sequence corresponding to SEQ ID NO: 1 is mutated to alanine.

2. A polypeptide composition comprising the polypeptide of claim 1 and one or more additional polypeptides.

3. The polypeptide composition of claim 2, wherein the additional polypeptide is selected from human plasminogen Kringle1 or a conserved fragment thereof, human plasminogen Kringle2 or a conserved fragment thereof, human plasminogen Kringle3 or a conserved fragment thereof, human plasminogen Kringle4 or a conserved fragment thereof, human plasminogen Kringle5 or a conserved fragment thereof, KV-7, or any combination thereof.

4. The polypeptide composition of claim 2, wherein the sequence of the additional polypeptide is selected from SEQ ID NO: 1, SEQ ID NO: 3, or any combination thereof.

5. A nucleic acid molecule comprising or consisting of a nucleotide sequence encoding the polypeptide of claim 1.

6. The nucleic acid molecule of claim 5, wherein the nucleotide sequence of the nucleic acid molecule is codon-optimized according to the host cell preference. A vector comprising the nucleic acid molecule according to claim 5 .

8. The vector of claim 7, having one or more characteristics selected from the group consisting of: (1) The vector is selected from plasmid, phagemid, artificial chromosome phage; and viral vector; (2) The vector is used to express the protein encoded by the nucleic acid molecule; (3) The vector is a vector for gene therapy.

9. The vector of claim 8, wherein the vector has one or more characteristics selected from the group consisting of: (1) The artificial chromosome is selected from a yeast artificial chromosome, a bacterial artificial chromosome or a P1-derived artificial chromosome; (2) The bacteriophage is λ phage or M13 phage; (3) The viral vector is a retrovirus vector, adenovirus vector, adeno-associated virus vector or herpes virus vector; (4) The vector is used for expression in cells in vitro or in vivo in a subject.

10. A host cell comprising the nucleic acid molecule according to claim 5 or 6 or the vector according to any one of claims 7 to 9. The host cell of claim 10 , wherein the host cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell.

12. The host cell of claim 10, wherein the host cell is selected from the group consisting of Escherichia coli cells, yeast cells, insect cells or plant cells.

13. The host cell of claim 10, which is a mammalian cell. The host cell of claim 10 , which is a mouse cell or a human cell.

15. A pharmaceutical composition comprising an effective amount of the polypeptide according to claim 1, or the polypeptide composition according to any one of claims 2 to 4, or the nucleic acid molecule according to claim 5 or 6, or the vector according to any one of claims 7 to 9, or the host cell according to any one of claims 10 to 14.

16. The pharmaceutical composition of claim 15, further comprising a pharmaceutically acceptable carrier and / or excipient.

17. The pharmaceutical composition according to claim 15, wherein The pharmaceutical composition exists in any of the following forms: ophthalmic preparation, ophthalmic injection, and sustained-release preparation.

18. The pharmaceutical composition according to claim 17, wherein The pharmaceutical composition is in any of the following forms: eye drops, eye ointment, gel, intraocular injection or fundus injection.

19. The pharmaceutical composition according to any one of claims 15 to 18, further comprising a pharmaceutically acceptable excipient.

20. The pharmaceutical composition of claim 19, wherein the pharmaceutically acceptable excipient is selected from a pH regulator, a solubilizing agent, an osmotic pressure regulator, a viscosity regulator, an antioxidant, an antibacterial preservative, a buffer, a suspending agent, a local anesthetic, a surfactant, a solubilizing agent, a wetting agent, an emulsifier, a filler, a gel matrix component, or any combination thereof.

21. The pharmaceutical composition of claim 20, having one or more characteristics selected from the following: (1) The pH adjuster is selected from hydrochloric acid, lactic acid, sulfuric acid, citric acid, sodium citrate, boric acid, sodium borate, triethanolamine, phosphates or mixtures thereof, or any combination thereof; (2) the osmotic pressure regulator is selected from sodium chloride, glucose, mannitol, glycerol or propylene glycol or a mixture thereof, or any combination thereof; (3) the antibacterial preservative is selected from benzalkonium chloride, benzalkonium bromide, methylparaben, ethylparaben, propylparaben or a mixture thereof, or any combination thereof; (4) The gel matrix component is selected from sodium hyaluronate, carbomer, poloxamer, chitosan, hydroxypropyl methylcellulose and mixtures thereof, or any combination thereof.

22. The pharmaceutical composition of any one of claims 15-18, further comprising an anti-inflammatory drug, an aminoglycoside antibiotic, polyvinyl pyrrolidone, or any combination thereof.

23. The pharmaceutical composition of claim 22, wherein The anti-inflammatory drug is selected from indomethacin, diclofenac sodium, ketorolac tromethamine, flurbiprofen sodium, pranoprofen, or any combination thereof.

24. The pharmaceutical composition of claim 22, wherein The aminoglycoside antibiotic is selected from tobramycin, gentamicin, neomycin, amikacin, netilmicin, kanamycin, or any combination thereof.

25. The pharmaceutical composition according to any one of claims 15 to 18, wherein The pharmaceutical composition has any one or more forms selected from the following: liquid, solid, semi-solid, aerosol.

26. Use of the polypeptide according to claim 1, the polypeptide composition according to any one of claims 2 to 4, the nucleic acid molecule according to claim 5 or 6, the vector according to any one of claims 7 to 9, or the host cell according to any one of claims 10 to 14 in preparing a pharmaceutical composition, wherein the pharmaceutical composition is used to alleviate or inhibit corneal neovascularization, or to treat symptoms caused by corneal neovascularization, or to treat diseases related to ocular neovascularization.

27. The use according to claim 26, wherein the disease is selected from decreased vision, blindness, corneal opacity, or any combination thereof.

28. The use according to claim 26, wherein the ocular neovascularization-related disease is selected from the group consisting of: corneal neovascularization-related disease, iris neovascularization-related disease, choroidal neovascularization-related disease, and retinal neovascularization-related disease.

29. The use according to claim 28, wherein the disease associated with corneal neovascularization is selected from pterygium, corneal foreign body and trauma, corneal chemical injury and infectious, immune, malnutrition and exposure keratitis.

30. The use of claim 28, wherein the disease associated with retinal neovascularization is selected from retinal vein occlusion, diabetic retinopathy, idiopathic choroidal neovascular disease, retinopathy of prematurity and neovascular age-related macular degeneration.

31. The use according to claim 26, wherein the pharmaceutical composition is used for: (1) Alleviate or inhibit the formation of corneal neovascularization; (2) Alleviate or inhibit the proliferation, migration and / or tube-forming ability of corneal neovascularization cells; (3) Reduce or inhibit the expression of VEGF.

32. The use according to claim 31, wherein the alleviation or inhibition of the formation of corneal neovascularization is alleviation or inhibition of the area and / or length of corneal neovascularization.

33. The use according to claim 31, wherein the VEGF is VEGFA or VEGFC.

34. A method for expressing or producing the polypeptide of claim 1, the method comprising culturing the host cell of any one of claims 10 to 14 under conditions allowing protein expression, and optionally, recovering or purifying the expressed polypeptide.

35. The method of claim 34, comprising the steps of: (1) constructing an expression vector comprising a nucleic acid sequence encoding the polypeptide of claim 1; (2) introducing the expression vector into a host cell and culturing the host cell under conditions that allow protein expression; (3) isolating and recovering the polypeptide from the cell culture of the host cell.

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