Recombinant III-type humanized collagen and application of composition containing same in eyes

Abstract

The invention relates to recombinant III-type humanized collagen and application of a composition containing the same to eyes. The invention provides an application of recombinant III type humanized collagen in preparation of a product for preventing and / or treating eye inflammation, a composition containing the recombinant III type humanized collagen, and an application of the composition in preparation of a product for preventing and / or treating eye inflammation and / or eye dryness. The recombinant III-type humanized collagen and the composition containing the recombinant III-type humanized collagen can obviously promote updating and repairing of corneal epithelial cells, and meanwhile, can inhibit release of inflammatory mediators, enhance eye tissue barriers, enhance tear film stability and relieve eye inflammation and eye dryness.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and particularly to the application of recombinant type III humanized collagen and compositions containing the same in the eye. Background Art

[0002] Contact lenses provide a convenient and aesthetic vision correction option for many patients with myopia, hyperopia or astigmatism, but they may cause some damage to the eyeball. For example, improper wearing of contact lenses may damage the corneal epithelium, causing problems such as corneal edema and neovascularization. At the same time, since contact lenses adhere to the cornea, substances such as tears and oils will deposit on the lens. Over time, this will lead to corneal hypoxia and corneal abrasion, resulting in a weakened barrier function of the corneal epithelium. Some bacteria and pathogens are more likely to invade the cornea, thus triggering pathological eye inflammations such as keratitis. At the same time, long-term wearing of contact lenses will also reduce the natural tear secretion of the eyes, causing dry and uncomfortable eyes. In severe cases, it can lead to eye infections, corneal injuries, opacities, etc. In addition, eye fatigue caused by long-term use of electronic products, reduced metabolic and immune functions due to irregular work and rest habits, pathogen infections caused by improper eye hygiene, as well as eye trauma and surgical complications, etc. will also trigger eye inflammatory reactions, and in severe cases, it can lead to vision loss, eye pain and other lesions.

[0003] Inflammation itself is the body's natural defense mechanism against harmful substances. Usually, the activation of immune cells and other non-immune cells is the most common cause of the inflammatory response. In the eye, corneal epithelial cells are involved in innate immunity and can directly respond to external stimuli, activating the expression of matrix metalloproteinases (MMPs), chemokines, and inflammatory cytokines. Metalloproteinases include MMP-9, etc., which itself has the function of maintaining and remodeling tissue structure, participating in the physiological shedding of corneal epithelial cells, healing injuries, and remodeling the extracellular matrix. Abnormal MMP-9 expression can reduce the tight junction degree of corneal epithelium and the basement membrane proteins connecting the cornea and conjunctiva. These changes can all damage the integrity of corneal epithelial cells. "Expression Changes and Significance of Inflammatory Cytokines and MMP-9 in Tear Fluids of Eyes with Fungal Keratitis and the Contralateral Uninfected Eyes" mentioned that MMP-9 is mainly distributed in the corneal stroma, participating in the repair and remodeling of epithelial cells and playing an important role in normal immune function; under pathological conditions, it is actively secreted by neutrophils and participates in corneal injury and the inflammatory process. This enzyme is overexpressed in corneal epithelial cells and causes severe tissue damage. "Study on the Expression of MMP-2 and MMP-9 and the Therapeutic Effect of Early Penetrating Keratoplasty after Severe Thermal Burn of Rabbit Cornea" mentioned that MMP-9 is mainly located in the stromal layer and epithelial cell layer of corneal tissue. In thermally burned corneal tissue, the expression of MMP-9 significantly increases. Inflammatory cytokines mainly include tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-10 (IL-10), etc. Stimulation by inflammatory factors or infection by pathogens will trigger an excessive inflammatory response. The excessive inflammatory response causes deep damage to cells, forming a cycle of inflammatory injury and leading to further exacerbation of the inflammatory response. In addition, there is literature indicating that the increase in inflammatory factors such as IL-6, TNF-α, and IL-1β will significantly cause inflammation in different parts of the ocular surface, including the eyelids, conjunctiva, and cornea.

[0004] Currently, chemical drugs (such as trehalose, sodium hyaluronate, chondroitin sulfate, hexylene glycol, salicylic acid, allantoin, etc.) and plant extracts are usually used to relieve ocular inflammation. However, these drugs have various side effects. For example, chondroitin sulfate is likely to cause gastrointestinal discomfort symptoms, hexylene glycol will produce a stimulating effect when in contact with the skin for a long time, and sodium hyaluronate may cause risks of excessive inflammatory reactions such as rashes and itching. Although some plant extracts have anti-inflammatory effects, the anti-inflammatory effects are relatively poor and difficult to maintain for a long time. Therefore, there is still a need in this field for a non-allergenic product with high safety and effective in improving ocular inflammation.

[0005] Citation Document 1 (ZL201811438582.6) discloses recombinant humanized collagen type III (rhCOL III) developed by Shanxi Jinbo Biopharmaceutical Co., Ltd., which has excellent biological properties such as biodegradability and cell proliferation promoting properties, and has been widely used in the field of medical beauty. Citation Document 2 (CN202110960230.2) and Citation Document 3 (CN202410340763.4) disclose the use of compositions containing the recombinant humanized collagen type III in ZL201811438582.6, such as eye drops, in the prevention and / or treatment of dry eye, but no study has explored its effect on ocular inflammation. Citation Document 4 (CN202311069893.0) discloses the recombinant humanized collagen type III developed by Shanxi Jinbo Biopharmaceutical Co., Ltd. and its use in preparing ophthalmic materials. Studies have shown that it can inhibit the amount of TNF-α secreted by macrophages and has a soothing and anti-inflammatory effect. Summary of the Invention

[0006] Problems to be Solved by the Invention

[0007] Contact lenses are common devices for correcting vision, but improper wearing of contact lenses can easily cause problems such as dry eyes, eye dryness, and ocular inflammation. Although prior art has studied drugs for treating ocular diseases or having an anti-inflammatory effect, such studies still cannot be said to be sufficient. For example, although the above-mentioned Citation Documents 2, 3, etc. disclose the role of recombinant humanized collagen type III in the treatment of dry eye, no study has been conducted on other symptoms of the eye. Although the above-mentioned Citation Document 4 discloses the inhibitory effect of recombinant humanized collagen type III on TNF-α, no study has been conducted on the symptoms of the eye. Therefore, there is still a need to develop safe and effective products with good therapeutic effects on ocular inflammation and other ocular diseases.

[0008] In the process of deeply studying the function of the recombinant humanized collagen type III disclosed in ZL201811438582.6, the present invention unexpectedly found that it can inhibit the release of inflammatory mediators, enhance the ocular tissue barrier, enhance the stability of the tear film, and relieve ocular dryness and ocular inflammation. Based on this, the object of the present invention is to provide the use of the recombinant humanized collagen type III in the prevention and / or treatment of ocular inflammation, especially in the prevention and / or treatment of ocular surface inflammation with tissue damage symptoms such as blepharitis, keratitis, conjunctivitis, etc., such as eyelid defect, conjunctival scarring, corneal ulcer, etc., and to provide a composition containing the recombinant humanized collagen type III and its use in the prevention and / or treatment of ocular inflammation and / or ocular dryness.

[0009] Solutions for Solving the Problems

[0010] [1]. Use of recombinant type III humanized collagen in the preparation of a product for preventing and / or treating ocular inflammation; wherein the amino acid sequence of the recombinant type III humanized collagen comprises n repeats of the sequence shown in SEQ ID NO.1, n is an integer greater than or equal to 1, and when n is an integer greater than or equal to 2, the repeat sequences are directly linked;

[0011] Preferably, the amino acid sequence of the recombinant type III humanized collagen comprises any one of the following (a) to (d):

[0012] (a) The sequence shown in SEQ ID NO.3;

[0013] (b) A sequence having 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with the sequence shown in SEQ ID NO.3, and retaining the cell adhesion effect of the sequence shown in SEQ ID NO.3;

[0014] (c) A sequence in which one or more amino acid residues are added, substituted or deleted in the sequence shown in SEQ ID NO.3, and retaining the cell adhesion effect of the sequence shown in SEQ ID NO.3;

[0015] (d) An amino acid sequence encoded by a nucleotide sequence that hybridizes under stringent conditions with a polynucleotide sequence encoding the sequence shown in SEQ ID NO.3, and the amino acid sequence retaining the cell adhesion effect of the sequence shown in SEQ ID NO.3, the stringent conditions being moderate stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

[0016] [2]. The use according to [1], wherein the ocular inflammation includes any one or more of blepharitis, keratitis, conjunctivitis, scleritis, hordeolum, dacryocystitis, optic neuritis and uveitis;

[0017] Preferably, the ocular inflammation is inflammation caused by wearing contact lenses.

[0018] [3]. The use according to [1] or [2], wherein the ocular inflammation is ocular surface inflammation, and the ocular surface inflammation includes any one or more of blepharitis, keratitis and conjunctivitis;

[0019] Preferably, the ocular surface inflammation is blepharitis, keratitis or conjunctivitis;

[0020] More preferably, the blepharitis has symptoms including eyelid defect, the keratitis has symptoms including corneal ulcer, and the conjunctivitis has symptoms including conjunctival scarring.

[0021] [4]. Use according to any one of [1] to [3], wherein the prevention and / or treatment of ocular inflammation includes any one or more of the following manifestations: reducing the content of ocular inflammatory mediators, promoting the proliferation, migration, and / or adhesion of any one or more of eyelid epithelial cells, corneal epithelial cells, and conjunctival epithelial cells, maintaining the normal structural morphology of any one or more of the eyelid, cornea, and / or conjunctiva, and enhancing the stability of the tear film.

[0022] [5]. Use according to [4], wherein the inflammatory mediators include any one or more of MMP-1, MMP-9, TNF-α, and IL-1β;

[0023] Preferably, the inflammatory mediators include MMP-1, MMP-9, TNF-α, and IL-1β;

[0024] More preferably, the inflammatory mediators are a combination of MMP-1, MMP-9, TNF-α, and IL-1β.

[0025] [6]. Use according to [4] or [5], wherein enhancing the stability of the tear film includes any one or more of increasing the mucin content in the tear fluid and reducing the osmotic pressure of the tear fluid.

[0026] [7]. A composition comprising the following components by mass percentage: recombinant type III humanized collagen 0.001% - 0.1%, osmotic pressure regulator 0.5% - 1.5%, buffer salt 0.01% - 0.5%, sodium hyaluronate 0.02% - 1%, and the balance being water;

[0027] Preferably, the composition is an eye drops.

[0028] [8]. Use of the composition according to [7] in the preparation of a product for preventing and / or treating ocular inflammation and / or ocular dryness.

[0029] [9]. Use according to [8], wherein the ocular inflammation includes any one or more of blepharitis, keratitis, conjunctivitis, scleritis, hordeolum, dacryocystitis, optic neuritis, and uveitis;

[0030] Preferably, the ocular inflammation and / or ocular dryness is caused by wearing contact lenses.

[0031]

[10] . Use according to [8] or [9], wherein the ocular inflammation is ocular surface inflammation, and the ocular surface inflammation includes any one or more of blepharitis, keratitis, and conjunctivitis;

[0032] Preferably, the ocular surface inflammation is blepharitis, keratitis or conjunctivitis;

[0033] More preferably, the blepharitis has symptoms including eyelid defect, the keratitis has symptoms including corneal ulcer, and the conjunctivitis has symptoms including conjunctival scarring.

[0034]

[11] . The use according to any one of [8] to

[10] , wherein the prevention and / or treatment of ocular inflammation and / or ocular dryness includes any one or more of the following manifestations: reducing the content of ocular inflammatory mediators, promoting the proliferation, migration and / or adhesion of any one or more of eyelid epithelial cells, corneal epithelial cells and conjunctival epithelial cells, maintaining the normal structural morphology of any one or more of the eyelid, cornea and / or conjunctiva, and enhancing the stability of the tear film.

[0035]

[12] . The use according to

[11] , wherein the inflammatory mediators include any one or more of MMP-1, MMP-9, TNF-α, IL-1β;

[0036] Preferably, the inflammatory mediators include MMP-1, MMP-9, TNF-α and IL-1β;

[0037] More preferably, the inflammatory mediators are a combination of MMP-1, MMP-9, TNF-α and IL-1β.

[0038]

[13] . The use according to

[11] or

[12] , characterized in that the enhancing the stability of the tear film includes any one or more of increasing the content of mucin in the tear fluid and reducing the osmotic pressure of the tear fluid.

[0039] Effects of the Invention

[0040] The present invention provides the use of recombinant type III humanized collagen in the preparation of a product for preventing and / or treating ocular inflammation. Research data shows that recombinant type III humanized collagen can significantly promote the renewal and repair of corneal epithelial cells, while inhibiting the release of inflammatory mediators, enhancing the ocular tissue barrier, enhancing the stability of the tear film, and alleviating ocular inflammation, thus providing a new idea for preventing and / or treating ocular diseases such as ocular inflammation caused by improper contact lens wear.

[0041] Meanwhile, the present invention also provides a composition comprising recombinant type III humanized collagen, and the use of the composition in the preparation of a product for preventing and / or treating ocular inflammation and / or dry eyes. Research data shows that the composition provided by the present invention has functions such as moisturizing, hydrating, repairing, and anti-inflammation, can relieve or treat ocular inflammation and / or dry eyes, and repair corneal injuries. At the same time, the composition provided by the present invention can supplement the components of the tear film, relieve the hyperosmotic condition of the tear fluid, enhance the stability of the tear film, thereby can relieve symptoms such as dry eyes and eye fatigue caused by reasons such as wearing contact lenses, and it can also play a lubricating role when wearing contact lenses. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 : Results of the cytotoxicity experiment in Example 1.

[0043] Figure 2 : Results of the cell adhesion experiment in Example 1.

[0044] Figure 3 : Results of the cell scratch experiment in Example 1.

[0045] Figure 4 : Results of the tear crystallization of each group of animals in Example 2.

[0046] Figure 5 : Gross eye photos of each group of animals in Example 4.

[0047] Figure 6 : Results of the staining of corneal and conjunctival pathological sections of each group of animals in Example 4. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0048] The following describes the implementation embodiments of the present invention, but the present invention is not limited thereto. Various changes can be made within the scope claimed by the present invention, and the implementation embodiments and examples obtained by appropriately combining the technical means disclosed in different implementation embodiments and examples are also included in the technical scope of the present invention.

[0049] I. TERM DEFINITIONS

[0050] In the present invention, the "some specific / preferred implementation embodiments", "some other specific / preferred implementation embodiments", "implementation embodiments", etc. mentioned refer to the specific elements (for example, features, structures, properties, and / or characteristics) related to the implementation embodiment, which are included in at least one of the implementation embodiments described herein, and may or may not exist in other implementation embodiments. In addition, it should be understood that the elements can be combined in any suitable manner in various implementation embodiments.

[0051] In the present invention, the meaning expressed by using "can" includes the meanings of both performing a certain treatment and not performing a certain treatment.

[0052] In the present invention, the terms "comprising", "having", "including", or "containing" may refer to inclusive or open-ended, and do not exclude additional, unrecited components or method steps. At the same time, "comprising", "having", "including", or "containing" may also mean closed-ended, excluding additional, unrecited components or method steps.

[0053] In the present invention, the term "optional" or "optionally" means that the subsequent described event or situation may or may not occur, or the subsequent described step or component may or may not be included in the embodiments described herein, and this description includes the situation where the event occurs and the situation where the event does not occur, and also includes the situation where the embodiments include and do not include the step or component.

[0054] In the present invention, the terms "individual", "patient", or "subject" include mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0055] In the present invention, the term "prevent" means that before suffering from a disease (such as ocular inflammation), by exposing a subject to the recombinant type III humanized collagen or composition described in the present invention, the probability of suffering from the disease and / or the symptoms after suffering from the disease are reduced compared to not being exposed, and it does not necessarily mean complete inhibition of the disease. Suffering from a disease means that the body shows symptoms of the disease.

[0056] In the present invention, the term "treat" means that after suffering from a disease (such as ocular inflammation), exposing (such as administering) a subject to the recombinant type III humanized collagen or composition described in the present invention, so that the symptoms of the disease are alleviated compared to not being exposed, and it does not necessarily mean complete inhibition of the symptoms of the disease.

[0057] In the present invention, the terms "administer", "give", and "treat" when applied to animals, humans, experimental subjects, cells, tissues, or organs, etc., refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, experimental subject, cell, tissue, or organ.

[0058] In the present invention, the term "effective amount" includes an amount sufficient to improve or prevent the symptoms or conditions of a medical disease. The effective amount for a specific subject may vary depending on factors such as the condition to be treated, the overall health of the subject, the method of administration, the dosage, and the severity of side effects. The effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

[0059] In the present invention, the term "identity" refers to the percentage of identical amino acids between two or more polypeptides. The sequence identity between two or more polypeptides can be determined by the following method: aligning the amino acid sequences of the polypeptides and scoring the number of positions containing identical amino acid residues in the aligned polypeptides, and comparing it with the number of positions containing different amino acid residues in the aligned polypeptides. The sequence identity can be calculated by dividing the number of positions containing identical amino acid residues by the total number of amino acid residues in the polypeptide.

[0060] In the present invention, amino acid addition may refer to adding 1, 2, or more than 3 amino acids at any position at the C-terminus, N-terminus, or between the C-terminus and N-terminus of an amino acid sequence, as long as the modified sequence completely or partially retains the activity of the original amino acid sequence.

[0061] In the present invention, amino acid substitution may refer to replacing an amino acid at a certain position in an amino acid sequence with another amino acid, as long as the modified sequence completely or partially retains the activity of the original amino acid sequence. Amino acid substitution can be a conservative amino acid substitution, which means that compared with the original amino acid sequence, several amino acids are replaced by amino acids with similar or close properties to form a peptide (conservative variant peptide). Exemplarily, these conservative variant peptides can be generated according to the following amino acid substitutions: substitution of Ala by Val, Leu, or Ile; substitution of Arg by Lys, Gln, Asn, or His; substitution of Asn by Gln, His, Lys, or Arg; substitution of Asp by Glu or Asn; substitution of Cys by Ser or Ala; substitution of Gln by Asn or Glu; substitution of Glu by Asp or Gln; substitution of Gly by Ala; substitution of His by Asn, Lys, Gln, or Arg; substitution of Ile by Leu, Met, Ala, Val, Phe, or norleucine; substitution of Leu by Ile, Met, Ala, Val, Phe, or norleucine; substitution of Lys by Asn, Gln, or Arg; substitution of Met by Ile, Leu, or Phe; substitution of Phe by Leu, Val, Ile, Ala, or Tyr; substitution of Pro by Ala; substitution of Ser by Thr; substitution of Thr by Ser or Val; substitution of Trp by Phe or Tyr; substitution of Tyr by Trp, Phe, Thr, or Ser; and substitution of Val by Phe, Ala, Met, Ile, Leu, or norleucine. Amino acid substitution can also be a non-conservative amino acid substitution.

[0062] In the present invention, amino acid deletion may refer to deleting 1, 2, or more than 3 amino acids from an amino acid sequence, as long as the modified sequence completely or partially retains the activity of the original amino acid sequence.

[0063] In the present invention, "hybridization" means the ability of a polynucleotide or oligonucleotide to bind to a substantially complementary sequence under stringent conditions, without non-specific binding to non-complementary targets under these conditions. The terms "medium stringency conditions", "medium-high stringency conditions", "high stringency conditions" or "very high stringency conditions" as used in the present invention describe the conditions for nucleic acid hybridization and washing. For example, specific hybridization conditions are as follows: (1) Low stringency hybridization conditions are in 6× sodium chloride / sodium citrate (SSC), at about 45°C, and then at at least 50°C, washed twice in 0.2× SSC, 0.1% SDS (for low stringency conditions, the washing temperature can be raised to 55°C); (2) Medium stringency hybridization conditions are in 6× SSC, at about 45°C, and then at 60°C, washed once or more in 0.2× SSC, 0.1% SDS; (3) High stringency hybridization conditions are in 6× SSC, at about 45°C, and then at 65°C, washed once or more and preferably in 0.2× SSC, 0.1% SDS; (4) Very high stringency hybridization conditions are 0.5 M sodium phosphate, 7% SDS, at 65°C, and then at 65°C, washed once or more in 0.2× SSC, 1% SDS.

[0064] In the present invention, the unit of "parts by mass" can be any weight unit such as g, mg, kg or t, etc.

[0065] Unless otherwise defined, other technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention pertains.

[0066] II. Recombinant type III humanized collagen and its preparation method

[0067] The recombinant type III humanized collagen described in the present invention is recorded in the patent with the application number 201811438582.6 and the invention name "Polypeptide, its production method and use".

[0068] In some embodiments, the amino acid sequence of the recombinant type III humanized collagen comprises n repeats of the sequence shown in SEQ ID NO.1, where n is an integer greater than or equal to 1, and n is preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 24 or 32. When n is an integer greater than or equal to 2, the respective repeat sequences are directly linked.

[0069] In some embodiments, the amino acid sequence of the recombinant type III humanized collagen comprises the sequence shown in SEQ ID NO.2 or does not comprise the sequence shown in SEQ ID NO.2; preferably, the amino acid sequence of the recombinant type III humanized collagen does not comprise the sequence shown in SEQ ID NO.2.

[0070] In some specific embodiments, the amino acid sequence of the recombinant type III humanized collagen comprises any one of the following (a) to (d):

[0071] (a) The sequence shown in SEQ ID NO.3;

[0072] (b) A sequence having 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with the sequence shown in SEQ ID NO.3, and retaining the cell adhesion effect of the sequence shown in SEQ ID NO.3;

[0073] (c) A sequence with one or more amino acid residues added, substituted or deleted in the sequence shown in SEQ ID NO.3, and retaining the cell adhesion effect of the sequence shown in SEQ ID NO.3;

[0074] (d) An amino acid sequence encoded by a nucleotide sequence that hybridizes with the polynucleotide sequence encoding the sequence shown in SEQ ID NO.3 under stringent conditions, and the amino acid sequence retains the cell adhesion effect of the sequence shown in SEQ ID NO.3, and the stringent conditions are moderate stringent conditions, medium-high stringent conditions, high stringent conditions or very high stringent conditions.

[0075] In some specific embodiments, the amino acid sequence of the recombinant type III humanized collagen comprises the sequence shown in SEQ ID NO.3.

[0076] In some specific embodiments, the amino acid sequence of the recombinant type III humanized collagen is the sequence shown in SEQ ID NO.3.

[0077] In some embodiments, the amino acid sequence of the recombinant type III humanized collagen further comprises an amino acid sequence that can be excised by TEV protease, and the amino acid sequence that can be excised by TEV protease is the sequence shown in SEQ ID NO.4.

[0078] In some specific embodiments, the amino acid sequence that can be excised by TEV protease is located at the N-terminus of the amino acid sequence of the recombinant type III humanized collagen.

[0079] In some specific embodiments, the amino acid sequence of the recombinant type III humanized collagen comprises the sequence shown in SEQ ID NO.5.

[0080] In some specific embodiments, the amino acid sequence of the recombinant type III humanized collagen is the sequence shown in SEQ ID NO.5.

[0081] The recombinant type III humanized collagen described in the present invention can be prepared by conventional methods in the art. For example, it can be prepared by the following steps: (1) construction of an Escherichia coli genetic engineering bacterium; (2) fermentation culture of the Escherichia coli genetic engineering bacterium; (3) induced expression of the recombinant type III humanized collagen; and (4) purification and optional enzymatic digestion of the recombinant type III humanized collagen.

[0082] III. Composition and Its Preparation Method

[0083] The present invention provides a composition comprising the following components: recombinant type III humanized collagen, an osmotic pressure regulator, a buffer salt, sodium hyaluronate, and water; wherein, the recombinant type III humanized collagen is as described in the above Part II.

[0084] In some embodiments, the composition comprises the following components in mass percentage: 0.001% - 0.1% of recombinant type III humanized collagen, 0.5% - 1.5% of the osmotic pressure regulator, 0.01% - 0.5% of the buffer salt, 0.02% - 1% of sodium hyaluronate, and the balance of water.

[0085] In order to achieve a good preventive and / or therapeutic effect on ocular inflammation and / or dry eyes, in some embodiments, in terms of mass percentage, the composition comprises 0.005% - 0.05% of recombinant type III humanized collagen, preferably 0.01%.

[0086] In some embodiments, the osmotic pressure regulator comprises at least one of sodium nitrate, potassium nitrate, sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, glycerol, propylene glycol, mannitol, sorbitol, glucose.

[0087] In some embodiments, the osmotic pressure regulator comprises sodium chloride.

[0088] In some specific embodiments, the osmotic pressure regulator is sodium chloride.

[0089] In some embodiments, the buffer salt comprises at least one of phosphates, carbonates, bicarbonates, borates, citrates, tartrates.

[0090] In some embodiments, the buffer salt comprises sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate anhydrous.

[0091] In some specific embodiments, the buffer salt is sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate anhydrous.

[0092] Considering the usage scenarios of the composition, for example, the composition can be used as an eye drops in the human physiological environment, the content of the pH regulator in the composition needs to ensure that the pH of the finished composition is 6-8, preferably 6.5-7.5. In some specific embodiments, calculated by mass percentage, the composition contains 0.02%-0.1% of buffer salts, preferably 0.03%. In some more specific embodiments, calculated based on the total mass of the composition, the composition contains 0.005%-0.02% of sodium dihydrogen phosphate monohydrate and 0.01%-0.05% of disodium hydrogen phosphate anhydrous, preferably contains 0.01% of sodium dihydrogen phosphate monohydrate and 0.02% of disodium hydrogen phosphate anhydrous.

[0093] In some more specific embodiments, calculated by mass percentage, the composition contains 0.05%-0.5% of sodium hyaluronate, preferably 0.1%.

[0094] In some embodiments, the composition is an eye drops.

[0095] The present invention also provides a preparation method of the composition, which includes the following steps: dissolving the formulated amount of osmotic pressure regulator, buffer salts, and sodium hyaluronate in the formulated amount of water, mixing until homogeneous, sterilizing, adjusting the pH value to 6-8, adding the recombinant type III humanized collagen filtered through a membrane in proportion, and mixing evenly to obtain the product.

[0096] In some embodiments, the preparation process of the composition is carried out in a sterilized environment; preferably, the preparation environment is sterilized by ultraviolet irradiation, and the utensils required for preparation are sterilized by treatment at 121°C for 15 minutes.

[0097] In some embodiments, the sterilization conditions of the homogeneous mixture of osmotic pressure regulator, buffer salts, sodium hyaluronate and water are 121°C for 15 minutes.

[0098] IV. Medical Use

[0099] In some embodiments, the present invention provides the use of the recombinant type III humanized collagen in the preparation of a product for preventing and / or treating ocular inflammation; wherein, the recombinant type III humanized collagen is as described in the above Part II.

[0100] In some embodiments, the present invention provides the use of the composition in the preparation of a product for preventing and / or treating ocular inflammation and / or ocular dryness; wherein, the composition is as described in the above Part III.

[0101] In some embodiments, the recombinant type III humanized collagen of the present invention is used for preventing and / or treating ocular inflammation; wherein, the recombinant type III humanized collagen is as described in the above Part II.

[0102] In some embodiments, the composition of the present invention is used for preventing and / or treating ocular inflammation and / or dry eye; wherein, the composition is as described in the above Part III.

[0103] In some embodiments, the present invention provides a method for preventing and / or treating ocular inflammation, the method comprising administering an effective amount of the recombinant type III humanized collagen to an individual in need thereof; wherein, the recombinant type III humanized collagen is as described in the above Part II, and the composition is as described in the above Part III.

[0104] In some embodiments, the present invention provides a method for preventing and / or treating ocular inflammation and / or dry eye, the method comprising administering an effective amount of the composition to an individual in need thereof; wherein, the composition is as described in the above Part III.

[0105] In some embodiments, the ocular inflammation includes any one or more of blepharitis, keratitis, conjunctivitis, scleritis, hordeolum, dacryocystitis, optic neuritis, and uveitis.

[0106] In some embodiments, the ocular inflammation and / or dry eye is inflammation caused by wearing contact lenses.

[0107] In some embodiments, the ocular inflammation is ocular surface inflammation, and the ocular surface inflammation includes any one or more of blepharitis, keratitis, and conjunctivitis.

[0108] In some embodiments, the ocular inflammation is keratitis, blepharitis, or conjunctivitis.

[0109] In some embodiments, the blepharitis has symptoms including eyelid defect, the keratitis has symptoms including corneal ulcer, and the conjunctivitis has symptoms including conjunctival scarring.

[0110] In some embodiments, the prevention and / or treatment of ocular inflammation includes any one or more of the following manifestations: reducing the content of ocular inflammatory mediators, promoting the proliferation, migration, and / or adhesion of any one or more of eyelid epithelial cells, corneal epithelial cells, and conjunctival epithelial cells, maintaining the normal structural morphology of any one or more of the eyelid, cornea, and / or conjunctiva, and enhancing the stability of the tear film.

[0111] In some embodiments, the prevention and / or treatment of ocular inflammation simultaneously includes the following manifestations: reducing the content of ocular inflammatory mediators, promoting the proliferation, migration, and / or adhesion of any one or more of eyelid epithelial cells, corneal epithelial cells, and conjunctival epithelial cells, maintaining the normal structural morphology of any one or more of the eyelid, cornea, and / or conjunctiva, and enhancing the stability of the tear film.

[0112] In some embodiments, the prevention and / or treatment of ocular inflammation simultaneously includes the following manifestations: reducing the content of ocular inflammatory mediators, promoting the proliferation, migration, and / or adhesion of any one or more of eyelid epithelial cells, corneal epithelial cells, and conjunctival epithelial cells, maintaining the normal structural morphology of any one or more of the eyelid, cornea, and / or conjunctiva.

[0113] In some embodiments, the prevention and / or treatment of dry eye includes the following manifestation: enhancing the stability of the tear film.

[0114] In some embodiments, the product reduces the content of ocular inflammatory mediators by inhibiting the generation and / or release of inflammatory mediators.

[0115] In some embodiments, the inflammatory mediators include any one or more of MMP-1, MMP-9, TNF-α, and IL-1β. In some specific embodiments, the inflammatory mediators simultaneously include MMP-1, MMP-9, TNF-α, and IL-1β. In some specific embodiments, the inflammatory mediators are a combination of MMP-1, MMP-9, TNF-α, and IL-1β.

[0116] Studies on corneal / conjunctival epithelial cells cultured in vitro have found that the inflammatory cytokine TNF-α can promote the production of MMPs, and MMPs can in turn activate potential inflammatory cytokines such as IL-1β, forming a cycle of corneal / conjunctival epithelial inflammation. And studies have shown that the inflammatory cytokine IL-1β is involved in the pathogenesis of various ocular inflammations. In addition, studies have shown that in the pathological process of keratitis / conjunctivitis, especially in infectious keratitis / conjunctivitis caused by bacterial biofilms, the inflammatory mediator MMP-9 plays an important role. However, the present invention discovers that the recombinant type III humanized collagen and the composition have inhibitory effects on MMP-1, MMP-9, TNF-α, and IL-1β, especially an unexpected inhibitory effect on MMP-9.

[0117] In some embodiments, enhancing the stability of the tear film includes any one or more of increasing the content of mucin in the tear fluid and reducing the osmotic pressure of the tear fluid. In some embodiments, enhancing the stability of the tear film simultaneously includes increasing the content of mucin in the tear fluid and reducing the osmotic pressure of the tear fluid.

[0118] In some embodiments, the product comprises a pharmaceutical composition, a medical device, or a combination of a drug and a device.

[0119] V. Amino Acid Sequences Related to the Present Invention

[0120] SEQ ID NO.1: GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0121] SEQ ID NO.2: GPPGPCCGGG.

[0122] SEQ ID NO.3: GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0123] SEQ ID NO.4: ENLYFQ.

[0124] SEQ ID NO.5: ENLYFQGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP。

[0125] Example

[0126] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those not specified in the examples, they are carried out under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the materials or instruments used in the examples are all conventional products that can be obtained through commercial purchase.

[0127] The amino acid sequence of the recombinant type III humanized collagen (rhCol III) used in the examples is shown in SEQ ID NO.3.

[0128] Example 1: rhCol III Promotes the Renewal and Repair of Corneal Epithelial Cells

[0129] (1) Cytotoxicity experiment:

[0130] The cultured human corneal epithelial cells (HCE-T) were digested with 0.25% trypsin solution to prepare a cell suspension of 5000 cells / mL, inoculated into a 96-well plate (100 μL / well), and after culturing for 24 h, the supernatant was discarded. Recombinant type III humanized collagen at different concentrations (0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1 mg / ml) was prepared. At the same time, a positive control well (1% DMSO solution) and a negative control well (DMEM medium) were set up, with 6 replicates for each concentration, surrounded by D-PBS, and cultured at 37 °C and 5% CO 2 After culturing for 24 h, after discarding the supernatant, CCK8 and DMEM medium were added and cultured for 2 h. The absorbance value (OD) at 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the cell survival rate (%) was calculated as follows: (OD value of the sample well - OD value of the blank well) / (OD value of the negative control well - OD value of the blank well) × 100.

[0131] The results of the cytotoxicity experiment are as Figure 1 shown. According to the detection results by the CCK-8 method, the larger the OD value, the higher the activity. Compared with the positive control group and the negative control group, as the sample concentration increased, the activity of human corneal epithelial cells in the sample wells supplemented with recombinant type III humanized collagen gradually increased. Recombinant type III humanized collagen can reduce corneal epithelial cell damage and promote corneal epithelial cell repair.

[0132] (2) Cell adhesion experiment:

[0133] Recombinant type III humanized collagen was diluted to 1.00 mg / ml, 0.5 mg / ml, 0.1 mg / ml, 0.05 mg / ml, 0.01 mg / ml with D-PBS. A positive control (1 mg / ml bovine type I collagen) and a negative control (D-PBS) were added and placed in an ELISA well, 100 μL per well, with 5 replicates for each group, and incubated overnight at 4 °C. The supernatant was discarded, 100 μL of 1% BSA (inactivated at 56 °C for 30 min) was added, and incubated at 37 °C for 1 h. The supernatant was discarded and washed 3 times with D-PBS. 1 × 10 6 well-cultured HCE-T resuspended in D-PBS was added to each well and incubated at 37 °C for 2 h. Washed 2 times with D-PBS. The absorbance at OD492 nm was detected with an LDH detection kit (Roche, 04744926001). According to the value of the blank control, the cell adhesion rate can be calculated. The calculation formula is as follows: Cell adhesion rate = (test well - blank well) / (positive well - blank well) × 100%.

[0134] The results of the cell adhesion experiment are as Figure 2As shown, compared with the positive control and negative control, recombinant type III humanized collagen at different concentrations all has better adhesion activity. Compared with the positive control, when the concentration of recombinant type III humanized collagen is 1.00 mg / ml, 0.5 mg / ml, 0.1 mg / ml, and 0.01 mg / ml, there are significant differences in cell adhesion activity (P < 0.001, P < 0.001, P < 0.01, P < 0.05). There is no significant difference in cell adhesion activity in the 0.05 mg / ml recombinant type III humanized collagen group (P > 0.05). Recombinant type III humanized collagen significantly improves cell adhesion activity. In subsequent experiments, the concentration of recombinant type III humanized collagen was determined to be 0.5 mg / ml (0.05%). Cell adhesion is the basis for maintaining tissue structure. Recombinant type III humanized collagen provides a high-quality external environment for cells in a short time, helps cells adhere to the wall, and has excellent cell adhesion activity.

[0135] (3) Cell scratch assay:

[0136] Experimental grouping: The experiment was divided into a PBS group, a commercial group (commercial type III collagen group, purchased from Jilin Guoda Bioengineering Co., Ltd.), and a 0.5 mg / ml recombinant type III humanized collagen group. 0.4% serum medium was used as the negative control (NC), and 10 ng / ml epidermal growth factor (EGF) was used as the positive control (PC).

[0137] Use a marker pen to draw a line on the back of the 12-well plate for convenient subsequent positioning and photographing. Add 1 mL of HCE-T cell suspension containing 1×10 6 / mL to each well of the 12-well plate and incubate in an incubator for 24 h. Discard the supernatant, add 1 mL of 0.4% serum medium for starvation treatment for 12 h. After the cells are confluent, use a 200 μL yellow pipette tip to vertically mark a scratch along the marker line. Discard the supernatant, wash 3 times with PBS, add 1 ml of each group of samples respectively, and observe the cell healing situation at 0, 12, and 24 h. Use Image J software to estimate the scratch healing area.

[0138] The results of the cell scratch assay are as Figure 3 shown. Through an inverted microscope, it was observed that from 0 h to 24 h, the cell migration rate in the recombinant type III humanized collagen group was better than that in the other four groups, and the entire field of view was basically covered at 24 h. By calculating the average migration rate of cells at 12 h and 24 h using software, it can be proved that the migration area in the recombinant type III humanized collagen group is the largest, thus proving that recombinant type III humanized collagen can enhance the migration ability of corneal epithelial cells and has the effect of promoting corneal injury repair.

[0139] Example 2: rhCol III eye drops enhance the stability of the tear film

[0140] Preparation of rhCol III group: Before the experiment, a sterile environment was prepared. The ultra-clean workbench was sterilized by ultraviolet light. The instruments required for the experiment were sterilized in an autoclave at 121 °C for 15 minutes. In the ultra-clean workbench, 0.1 part of sodium hyaluronate, 0.85 part of sodium chloride, 0.02 part of disodium hydrogen phosphate anhydrous, and 0.01 part of sodium dihydrogen phosphate monohydrate were weighed according to the ratio and dissolved in the formulated amount of water (the amount of water should ensure that the total volume of the eye drops is 100 parts). The solution was placed in a sterilized container and stirred thoroughly until it was uniformly mixed and reached a homogeneous state. Then it was sterilized at 121 °C for 15 minutes to obtain the matrix. After the matrix cooled down, its pH was measured and should be between 6 and 8. 0.05% recombinant type III humanized collagen filtered through a membrane was added. The commercially available type III collagen group was prepared in the same way.

[0141] Several healthy Japanese white rabbits with large ears were selected and randomly divided into an experimental group (recombinant type III humanized collagen group), a commercial group (commercially available type III collagen group, purchased from Jilin Guoda Bio-Engineering Co., Ltd.), a model group (intervened with buffer salt solution), and a normal group (without any intervention).

[0142] All Japanese white rabbits with large ears were adaptively fed in the animal house for 1 week, during which they had free access to water. The rabbits were fixed, and 2 mL·kg-1 chloral hydrate was injected into the marginal ear vein for general anesthesia. Then, 2 drops of proparacaine hydrochloride eye drops were instilled into the local eye area. After the anesthesia took effect, the top of the third eyelid was lifted with forceps, and part of the tissue was cut off. After the operation, 2 drops of tobramycin eye drops were instilled 3 times a day for 4 days. After the wound healed, 3 rabbits were randomly selected as the normal group without any treatment (hereinafter referred to as the normal group). The other groups were instilled with 0.2% benzalkonium chloride, 2 drops each time, 3 times a day, for 7 consecutive days. 7 days after modeling, it was observed that the eye surface secretions of the rabbits gradually increased, the eyelids were swollen and red, the conjunctiva was severely congested, and the cornea was turbid, ensuring the successful establishment of the inflammation model. The experimental group, the commercial group, and the model group were treated with eye drops, 2 drops each time, 3 times a day, for 7 days and 14 days continuously.

[0143] Detection of tear film stability: A disposable micro blood collection pipette was used to suck the tears of the rabbits from the conjunctival sac of the lower eyelid onto a glass slide, spread evenly, and air-dried. The tear film stability was judged according to the morphology of the tear crystals (classified from regular, complete, and dense fern-like crystals to irregular, amorphous, or even no crystals in turn as grades I-IV. Grades I and II are normal tear crystals, and grades III and IV are abnormal crystals). Mucin in normal tears can form a branched fern-like image.

[0144] The experimental results are as Figure 4As shown, on the 0th day of treatment, except for the normal group, only a small amount of irregular crystals could be observed in other groups, mainly grade III and grade IV. On the 7th day of treatment, the crystals in the recombinant humanized collagen type III eye drops group were more regular in shape and increased in number compared with the model group. On the 14th day of treatment, complete and thick fern-like crystals could be observed in the recombinant humanized collagen type III eye drops group. It was proved that after using the recombinant humanized collagen type III eye drops, the tear crystal morphology was closer to normal (grade I and II), indicating that it could supplement the tear film components, relieve the hyperosmotic situation of tears, and enhance the tear film stability.

[0145] Through Example 1 and Example 2, it was proved that recombinant humanized collagen type III had the ability to promote corneal epithelial renewal and repair. After injury occurred at the conjunctiva / cornea / eyelid position, an immune response was triggered, and a series of inflammatory factors such as interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and matrix metalloproteinases (MMPs) would be released. Example 3 detected the effect of recombinant humanized collagen type III on inflammatory factors through cell experiments to detect whether recombinant humanized collagen type III acted on these inflammatory factors, so as to be able to play an inhibitory and therapeutic role at the site of ocular inflammation.

[0146] Example 3: Cell experiment on rhCol III inhibiting inflammation

[0147] Enzyme-linked immunosorbent assay:

[0148] Experimental grouping: normal control group (DMEM / F12 medium), inflammation group (LPS 1 μM), experimental group (0.05% recombinant humanized collagen type III), commercial group (0.05% commercial collagen type III group, purchased from Jilin Guoda Biotechnology Co., Ltd.). Experimental steps: Use complete medium to seed HCE-T cells in the logarithmic growth phase at 1.5×10 5Inoculate 1 mL per well at a density of [number of cells] / mL into a 6-well plate. After the cells adhere, sequentially add 700 μL of the corresponding group solution and continue culturing for 24 h. Collect the cell supernatant. Take out the ELISA kit from the refrigerator and let it equilibrate at room temperature for a period of time, usually about 30 minutes, to allow the reagents in the kit to return to room temperature to ensure the stable performance of the reagents. Dilute the capture antibody to 5 μg / mL with coating buffer. Add the diluted capture antibody to the wells of the enzyme-linked immunosorbent assay (ELISA) plate, 100 - 200 μL per well. Then seal the ELISA plate with a sealing film and place it in a 4°C refrigerator overnight to allow the antibody to fully adsorb onto the well walls of the ELISA plate. After overnight coating, take out the ELISA plate and discard the liquid in the wells. Add 200 - 300 μL of washing PBS buffer to the wells. Let it stand for a moment and then discard the washing buffer. Repeat the washing step 3 - 5 times to ensure thorough washing. An automated washer can be used for washing to ensure the consistency of washing. Add blocking solution (PBS buffer containing 1% - 5% BSA) to the washed wells of the ELISA plate, 200 - 300 μL per well. Place the ELISA plate in an ELISA reader and select an appropriate wavelength (such as 450 nm) for absorbance detection. The ELISA reader will measure the absorbance value in each well. Detect the content levels of inflammatory factors TNF-α, IL-1β, MMP-1, and MMP-9.

[0149] The lower the content of these four inflammatory factors, the milder the inflammatory response. The experimental results are shown in Table 1. The content of the four inflammatory factors was detected. Compared with the commercial group, the recombinant type III humanized collagen experimental group had an inhibitory effect on all four inflammatory factors, and the inhibitory effect on MMP-9 was the most obvious, demonstrating that recombinant type III humanized collagen has the effect of inhibiting inflammation.

[0150] Table 1 Results of detecting the content of inflammatory factors by enzyme-linked immunosorbent assay

[0151]

[0152] Example 3 shows that recombinant type III humanized collagen acts on these inflammatory factors, and it has an obvious inhibitory effect on MMP-9, while the above four factors, especially MMP-9, are significantly elevated in the inflammatory symptoms of the eyelid / cornea / conjunctiva. Next, Example 4 further detects the effect of recombinant type III humanized collagen on an animal model of ocular inflammation by establishing an animal experimental model of eyelid / cornea / conjunctiva inflammation.

[0153] Example 4: Animal experiment on the inhibition of inflammation by rhCol III

[0154] (1) Gross appearance photos:

[0155] A number of healthy Japanese white rabbits were randomly divided into an experimental group (0.05% recombinant type III humanized collagen), a commercially available group (0.05% commercially available type III collagen group, purchased from Jilin Guoda Bioengineering Co., Ltd.), a model group (intervened with buffer salt solution), and a normal group (without any intervention).

[0156] All Japanese white rabbits were adaptively fed in the animal house for 1 week, during which they had free access to water. The Japanese white rabbits were fixed, and 2 mL·kg was injected into the marginal ear vein -1 Chloral hydrate was used for general anesthesia, and then 2 drops of proparacaine hydrochloride eye drops were dropped in the surgical area for local eye anesthesia. After the anesthesia took effect, the top of the third eyelid was lifted with forceps, and part of the tissue was cut off. After the operation, 2 drops of tobramycin eye drops were dropped 3 times a day for a total of 4 days. After the wound healed, 3 rabbits were randomly selected as the normal group without any treatment (hereinafter referred to as the normal group). The remaining groups were dropped with 0.2% benzalkonium chloride, 3 times a day, 2 drops each time, for 7 consecutive days. 7 days after modeling, it was observed that the ocular surface secretions of the rabbits gradually increased, the eyelids were red and swollen, the conjunctiva was severely congested, and the cornea was cloudy, ensuring the successful establishment of the inflammation model.

[0157] The experimental group, the commercially available group, and the model group were all in the form of solution, 2 drops each time, 3 times a day, and the changes in the rabbits' eyes were observed after continuous treatment for 7 days and 14 days.

[0158] The experimental results are as Figure 5 shown. On the 7th day of treatment, the secretions in the experimental group were reduced compared with the model group, and the symptoms of eye congestion and edema were alleviated. On the 14th day of treatment, there were no secretions in both the experimental group and the commercially available group; in terms of the degree of corneal opacity, eyelid and conjunctival edema, the recovery effect of the experimental group was better than that of the commercially available group. It was proved that recombinant type III humanized collagen had a good effect in the treatment of ocular inflammation.

[0159] (2) Observation of corneal / conjunctival pathological sections:

[0160] After the treatment, the rabbits were sacrificed, and the eyeballs were fixed in a fixative (such as 4% paraformaldehyde) for 48 h. After routine dehydration, clearing, wax infiltration, and embedding, sections with a thickness of 4-6 μm were made. Hematoxylin-eosin (HE) staining was performed, and the changes in the corneal / conjunctival tissue structure were observed under a light microscope.

[0161] The experimental results are as Figure 6As shown, pathological section observation shows that for corneal tissue, the normal corneal epithelium has 2-3 layers of squamous epithelial cells, arranged neatly and evenly. On the 0th day of treatment, except for the normal group, the corneal epithelium of other groups was partially or completely exfoliated, the corneal endothelium was edematous, and the posterior limiting lamina was detached. On the 14th day of treatment, the corneal epithelium of the experimental group was more complete than that of the model group and the commercial group, the cells were arranged neatly, and the morphology was relatively normal. No edema or exfoliation was observed in the corneal endothelium. For conjunctival tissue, the normal conjunctival tissue was tightly connected, and the columnar epithelium had regular morphology and neat arrangement. On the 0th day of treatment, the tissue spaces of the experimental group, the model group, and the commercial group were widened, and the stroma was loose. On the 14th day of treatment, the cells of the experimental group were arranged more neatly than those of the model group and the commercial group. It is proved that the experimental group has a good repair effect on the damage of corneal / conjunctival cell tissue, and it is proved that recombinant humanized collagen type III has a therapeutic effect on corneal / conjunctival inflammation models.

[0162] From the perspective of cells, recombinant humanized collagen type III can significantly improve the proliferation and differentiation of corneal epithelial cells and stromal cells, accelerate the renewal and repair of epithelial cells, so as to cover the damaged area faster; and through the tear film experiment, recombinant humanized collagen type III can significantly improve the effect of eye dryness. Its moisturizing effect can further provide an external environment for the proliferation and repair of damaged cells. In addition, recombinant humanized collagen type III can reduce the release of inflammatory mediators, inhibit the infiltration and activation of inflammatory cells, thereby reducing the inflammatory reaction of the eyelid / cornea / conjunctiva, which is beneficial to the repair of eye tissues. The repaired eye tissues can better perform their physiological functions.

Claims

1. Use of recombinant humanized type III collagen in the preparation of a product for preventing and / or treating ocular inflammation; wherein: The amino acid sequence of the recombinant humanized type III collagen comprises n repetitions of the sequence shown in SEQ ID NO.1, where n is an integer greater than or equal to 1, wherein when n is an integer greater than or equal to 2, each repetitive sequence is directly connected; Preferably, the amino acid sequence of the recombinant humanized type III collagen comprises any one of the following (a) to (d): (a) the sequence shown in SEQ ID NO.3; (b) a sequence having 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with the sequence shown in SEQ ID NO. 3, and which retains the cell adhesion effect of the sequence shown in SEQ ID NO. 3; (c) a sequence in which one or more amino acid residues are added, substituted or deleted in the sequence as shown in SEQ ID NO.3, and which retains the cell adhesion effect of the sequence as shown in SEQ ID NO.3; (d) an amino acid sequence encoded by a nucleotide sequence, wherein the nucleotide sequence hybridizes with a polynucleotide sequence encoding the sequence as shown in SEQ ID NO.3 under stringent conditions, and the amino acid sequence retains the cell adhesion effect of the sequence as shown in SEQ ID NO.3, wherein the stringent conditions are medium stringent conditions, medium-high stringent conditions, high stringent conditions or very high stringent conditions.

2. The use according to claim 1, characterized in that The ocular inflammation includes any one or more of blepharitis, keratitis, conjunctivitis, scleritis, hordeolum, dacryocystitis, optic neuritis and uveitis; Preferably, the ocular inflammation is inflammation caused by wearing contact lenses.

3. The use according to claim 1 or 2, characterized in that The ocular inflammation is ocular surface inflammation, which includes any one or more of blepharitis, keratitis and conjunctivitis; Preferably, the ocular surface inflammation is blepharitis, keratitis or conjunctivitis; More preferably, the blepharitis has symptoms including eyelid coloboma, the keratitis has symptoms including corneal ulceration, and the conjunctivitis has symptoms including conjunctival scarring.

4. The use according to any one of claims 1 to 3, characterized in that The prevention and / or treatment of ocular inflammation includes any one or more of the following manifestations: reducing the content of ocular inflammatory mediators, promoting the proliferation, migration and / or adhesion of any one or more of eyelid epithelial cells, corneal epithelial cells and conjunctival epithelial cells, maintaining the normal structural morphology of any one or more tissues in the eyelid, cornea and / or conjunctiva, and enhancing the stability of the tear film.

5. The use according to claim 4, characterized in that The inflammatory mediators include any one or more of MMP-1, MMP-9, TNF-α, and IL-1β; Preferably, the inflammatory mediators include MMP-1, MMP-9, TNF-α and IL-1β; More preferably, the inflammatory mediator is a combination of MMP-1, MMP-9, TNF-α and IL-1β.

6. The use according to claim 4 or 5, characterized in that The enhancement of tear film stability includes any one or more of increasing the mucin content in tears and reducing the osmotic pressure of tears.

7. A composition comprising the following components in percentage by mass: 0.001%-0.1% of recombinant humanized type III collagen, 0.5%-1.5% of an osmotic pressure regulator, 0.01%-0.5% of a buffer salt, 0.02%-1% of sodium hyaluronate, and the balance of water; Preferably, the composition is an eye lotion.

8. Use of the composition according to claim 7 in the preparation of a product for preventing and / or treating ocular inflammation and / or ocular dryness.

9. The use according to claim 8, characterized in that The ocular inflammation includes any one or more of blepharitis, keratitis, conjunctivitis, scleritis, hordeolum, dacryocystitis, optic neuritis and uveitis; Preferably, the ocular inflammation and / or ocular dryness is caused by wearing contact lenses.

10. The use according to claim 8 or 9, characterized in that The ocular inflammation is ocular surface inflammation, which includes any one or more of blepharitis, keratitis and conjunctivitis; Preferably, the ocular surface inflammation is blepharitis, keratitis or conjunctivitis; More preferably, the blepharitis has symptoms including eyelid coloboma, the keratitis has symptoms including corneal ulceration, and the conjunctivitis has symptoms including conjunctival scarring.

11. The use according to any one of claims 8 to 10, characterized in that: The prevention and / or treatment of ocular inflammation and / or dry eyes includes any one or more of the following manifestations: reducing the content of ocular inflammatory mediators, promoting the proliferation, migration and / or adhesion of any one or more of eyelid epithelial cells, corneal epithelial cells and conjunctival epithelial cells, maintaining the normal structural morphology of any one or more tissues in the eyelid, cornea and / or conjunctiva, and enhancing the stability of the tear film.

12. The use according to claim 11, characterized in that The inflammatory mediators include any one or more of MMP-1, MMP-9, TNF-α, and IL-1β; Preferably, the inflammatory mediators include MMP-1, MMP-9, TNF-α and IL-1β; More preferably, the inflammatory mediator is a combination of MMP-1, MMP-9, TNF-α and IL-1β.

13. The use according to claim 11 or 12, characterized in that The enhancement of tear film stability includes any one or more of increasing the mucin content in tears and reducing the osmotic pressure of tears.

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