Pharmaceutical composition for promoting angiogenesis and tissue regeneration and application thereof

Abstract

The invention discloses a pharmaceutical composition for promoting angiogenesis and tissue regeneration and application thereof. The pharmaceutical composition of the present invention comprises an acidic secretory protein rich in cysteine, a disaccharide chain proteoglycan, and an XV-type collagen alpha1 chain. The biological material system loaded with the three types of proteins can promote angiogenesis, and can improve the tissue regeneration efficiency of bone, cartilage and skin defect areas.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a pharmaceutical composition for promoting angiogenesis and tissue regeneration and its application. Background Art

[0002] Tissue regeneration is a biological process regulated by evolutionarily conserved mechanisms, and its repair efficacy benefits from the regulation among various signaling pathways in the microenvironment. Insufficient activation intensity of regeneration-related signals will lead to tissue repair dysfunction; while over-activation of signals will also cause pathological hyperplasia. Therefore, reconstructing the complex regeneration-related signal network is a decisive factor for achieving functional regeneration and also a major technical challenge to existing tissue engineering technologies.

[0003] It is worth noting that the unique biological feature of continuous angiogenesis in the tumor microenvironment is precisely the key regeneration signal that is generally lacking in the damaged healing microenvironment. Based on this, the "over-activated" microenvironment signal system in tumor formation will make up for the problem of insufficient functionality of key signals in the regeneration and repair process. By reconstructing microenvironment homeostasis, a breakthrough improvement in tissue regeneration efficiency can be achieved. Currently, existing technologies have attempted to achieve tissue regeneration by transplanting rapidly proliferating tumor cells or using tumor homogenate extracts to construct an immunosuppressive microenvironment to support the growth of ectopic organs. However, such solutions face serious clinical translation obstacles due to potential tumorigenic risks and uncontrollability of immune regulation. Summary of the Invention

[0004] To solve at least some of the above technical problems in the prior art, the present invention provides a pharmaceutical composition for promoting angiogenesis and tissue regeneration and its application. Specifically, the present invention includes the following contents.

[0005] In a first aspect of the present invention, there is provided a pharmaceutical composition for promoting angiogenesis and / or tissue regeneration, which comprises secreted protein acidic and rich in cysteine, biglycan, and type XV collagen alpha 1 chain.

[0006] In certain embodiments, for the pharmaceutical composition according to the present invention, wherein the secreted protein acidic and rich in cysteine, biglycan, and / or type XV collagen alpha 1 chain are respectively derived from the acellular extracellular matrix of tumor tissue.

[0007] In certain embodiments, for the pharmaceutical composition according to the present invention, wherein the secreted protein acidic and rich in cysteine in the pharmaceutical composition is 0.5 - 5 molar parts, biglycan is 0.5 - 5 molar parts, and type XV collagen alpha 1 chain is 1 molar part.

[0008] In certain embodiments, for the pharmaceutical composition according to the present invention, wherein it further comprises a pharmaceutically acceptable carrier.

[0009] In certain embodiments, for the pharmaceutical composition according to the present invention, the pharmaceutically acceptable carrier includes at least one of a scaffold material, a diluent, a filler, an absorbent, a wetting agent, a binder, a disintegrant, a lubricant, a preservative, and an antioxidant.

[0010] A second aspect of the present invention provides a method for preparing the pharmaceutical composition according to the present invention, which includes the step of separating secreted protein acidic and rich in cysteine, biglycan, and type XV collagen alpha 1 chain from the acellular extracellular matrix of tumor tissue.

[0011] In certain embodiments, for the preparation method according to the present invention, it further includes the step of preparing the acellular extracellular matrix of tumor tissue.

[0012] A third aspect of the present invention provides a method for promoting tissue regeneration in vitro, which includes the step of contacting the pharmaceutical composition according to the present invention with cells in vitro.

[0013] In certain embodiments, for the method for promoting tissue regeneration in vitro according to the present invention, the promoting tissue regeneration includes at least one of the following situations: (1) Promoting endothelial cell vascularization; (2) Promoting angiogenesis in the tissue defect area; (3) Promoting bone regeneration in the bone defect area; (4) Promoting tissue healing.

[0014] A fourth aspect of the present invention provides the use of the pharmaceutical composition according to the present invention in the preparation of a product for promoting angiogenesis and / or tissue regeneration.

[0015] The present invention has achieved a technical breakthrough by identifying key functional components related to regeneration in the acellular extracellular matrix of tumor tissue and clarifying the functional targets. At the level of translational medicine, the present invention has identified key bioactive protein components with pro-regenerative functions from the acellular extracellular matrix of tumor tissue: secreted protein acidic and rich in cysteine, biglycan, and type XV collagen alpha 1 chain. The biomaterial system loaded with the three types of proteins of the present invention can promote angiogenesis and improve the tissue regeneration efficiency in the bone, cartilage, and skin defect areas. In addition, the present invention ensures the functional reproducibility and biosafety of tumor-derived biomaterials at the molecular mechanism level. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Shows the sprouting of human umbilical vein endothelial cell spheres promoted by different proteins.

[0017] Figure 2It shows the promotion of angiogenesis and bone regeneration in the skull defect area of rats by secreted protein acidic and rich in cysteine, biglycan and type XV collagen alpha 1 chain.

[0018] Figure 3 It shows the promotion of the recovery of osteochondral defects in the patella of rats by secreted protein acidic and rich in cysteine, biglycan and type XV collagen alpha 1 chain.

[0019] Figure 4 It shows the promotion of angiogenesis and skin healing in the skin defect area of mice by secreted protein acidic and rich in cysteine, biglycan and type XV collagen alpha 1 chain. Detailed implementation mode

[0020] Now, various exemplary implementation modes of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0021] It should be understood that the terms described in the present invention are only for describing specific implementation modes and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Each intermediate value within any stated value or stated range and each smaller range between any other stated value or intermediate value within the stated range are also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0022] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0023] Drug composition In one aspect of the present invention, there is provided a pharmaceutical composition for promoting angiogenesis and / or tissue regeneration, which comprises secreted protein acidic and rich in cysteine, biglycan and type XV collagen alpha 1 chain.

[0024] In the present invention, "promote" means that, compared with an untreated reference group under the same conditions, as measured by any standard technique, the degree of such promotion or improvement is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100%. Beneficial or desired clinical outcomes include, but are not limited to, the following outcomes whether detectable or undetectable, such as promoting angiogenesis and / or tissue regeneration. The promoting angiogenesis and tissue regeneration include at least one of the following situations: (1) promoting endothelial cell vascularization; (2) promoting angiogenesis in the tissue defect area; (3) promoting bone regeneration in the bone defect area; (4) promoting tissue healing.

[0025] In the present invention, "angiogenesis" refers to the growth of new blood vessels or neovascularization, including the growth of new blood vessels with relatively small diameters composed of endothelial cells. Angiogenesis is an indispensable part of many important biological processes, and the biological processes include wound healing, damaged tissue repair, vascular reconstruction and remodeling, etc. Angiogenesis can be determined in a variety of ways, including but not limited to quantifying budding, blood vessel diameter, blood vessel length, number of branching points, and blood vessel density to evaluate angiogenesis.

[0026] In the present invention, "tissue regeneration" refers to the process of repair and regeneration of different types of tissues after injury. Examples of tissues include, but are not limited to, blood vessels, skin, bone, muscle, nerves, etc. "Injury" refers to the destruction of the organizational structures of the skin, bone, cartilage, organs, etc. or the local and systemic reactions brought about by various traumatic factors acting on the body. Injury includes physical injury, chemical injury, biological injury, etc. In the present invention, the pharmaceutical composition is particularly used for bone injury (or bone defect), cartilage injury (or cartilage defect) or skin injury (or skin defect).

[0027] In a preferred embodiment, the secreted protein acidic and rich in cysteine (Sparc), biglycan (Bgn) and / or type XV collagen alpha 1 chain (Col15a1) are each derived from the acellular extracellular matrix. Among them, the source of the acellular extracellular matrix is not particularly limited and can be derived from normal tissues, tumor tissues, etc. In a preferred embodiment, the acellular extracellular matrix is derived from tumor tissues.

[0028] In the present invention, the tumor includes solid tumors with high malignancy and fast growth, and its examples include but are not limited to: breast cancer, melanoma, osteosarcoma, neuroblastoma, pancreatic cancer, lung cancer, rhabdomyosarcoma, Ewing's sarcoma, bladder cancer, colon cancer, liver cancer, ovarian cancer, cervical cancer, nasopharyngeal cancer, laryngeal cancer, gastric cancer, renal cancer, head and neck tumors, esophageal cancer, thyroid cancer or brain cancer. In a preferred embodiment, the tumor includes epithelial malignancies, such as liver cancer, colon cancer, lung cancer, etc. In a preferred embodiment, the acellular extracellular matrix is derived from liver cancer tissue.

[0029] It has been found through research in the present invention that secreted protein acidic and rich in cysteine, biglycan, and collagen type XV alpha 1 chain have a synergistic effect in promoting angiogenesis and / or tissue regeneration. In a preferred embodiment, the molar ratio of the secreted protein acidic and rich in cysteine, biglycan, and collagen type XV alpha 1 chain is (0.5 - 5):(0.5 - 5):1, preferably (0.8 - 5):(1 - 5):1, more preferably (1 - 5):(1 - 5):1, such as 1:1:1, 1.2:1:1, 1.4:1:1, 1.6:1:1, 1.8:1:1, 2:1:1, 2.2:1:1, 2.4:1:1, 2.6:1:1, 2.8:1:1, 3:1:1, 3.2:1:1, 3.4:1:1, 3.6:1:1, 3.8:1:1, 4:1:1, 4.2:1:1, 4.4:1:1, 4.6:1:1, 4.8:1:1, 5:1:1, 1:2:1, 1.2:2:1, 1.4:2:1, 1.6:2:1, 1.8:2:1, 2:2:1, 2.2:2:1, 2.4:2:1, 2.6:2:1, 2.8:2:1, 3:2:1, 3.2:2:1, 3.4:2:1, 3.6:2:1, 3.8:2:1, 4:2:1, 4.2:2:1, 4.4:2:1, 4.6:2:1, 4.8:2:1, 5:2:1, 1:3:1, 1.2:3:1, 1.4:3:1, 1.6:3:1, 1.8:3:1, 2:3:1, 2.2:3:1, 2.4:3:1, 2.6:3:1, 2.8:3:1, 3:3:1, 3.2:3:1, 3.4:3:1, 3.6:3:1, 3.8:3:1, 4:3:1, 4.2:3:1, 4.4:3:1, 4.6:3:1, 4.8:3:1, 5:3:1, 1:4:1, 1.2:4:1, 1.4:4:1, 1.6:4:1, 1.8:4:1, 2:4:1, 2.2:4:1, 2.4:4:1, 2.6:4:1, 2.8:4:1, 3:4:1, 3.2:4:1, 3.4:4:1, 3.6:4:1, 3.8:4:1, 4:4:1, 4.2:4:1, 4.4:4:1, 4.6:4:1, 4.8:4:1, 5:4:1, 1:5:1, 1.2:5:1, 1.4:5:1, 1.6:5:1, 1.8:5:1, 2:5:1, 2.2:5:1, 2.4:5:1, 2.6:5:1, 2.8:5:1, 3:5:1, 3.2:5:1, 3.4:5:1, 3.6:5:1, 3.8:5:1, 4:5:1, 4.2:5:1, 4.4:5:1, 4.6:5:1, 4.8:5:1, 5:5:1, or any ratio within the above range.

[0030] In a preferred embodiment, the pharmaceutical composition of the present invention comprises secreted protein acidic and rich in cysteine, biglycan, and collagen type XV alpha 1 chain, as well as a pharmaceutically acceptable carrier.

[0031] In the present invention, the carrier is "acceptable", which means that it is compatible with the other components of the formulation (such as secreted protein acidic and rich in cysteine, biglycan, or collagen type XV alpha 1 chain) and does not harm the patient. The pharmaceutically acceptable carrier includes at least one of a scaffold material, a diluent, a filler, an absorbent, a wetting agent, a binder, a disintegrant, a lubricant, a preservative, and an antioxidant. Among them, the scaffold material includes but is not limited to natural polymers (such as but not limited to collagen, gelatin, chitosan, etc.), synthetic polymers (such as but not limited to PVA, PLGA, PCL, PEG, etc.), hydrogels (such as but not limited to sodium alginate, hyaluronic acid, polyacrylamide), inorganic materials (such as but not limited to hydroxyapatite, bioactive glass, etc.), nano-scaffold materials (such as but not limited to electrospun nanofibers, graphene composites, etc.). Examples of diluents include but are not limited to normal saline, aqueous buffer solutions, solvents, dispersion media, etc.; fillers include but are not limited to starch, lactose, mannitol, microcrystalline cellulose, etc.; absorbents include but are not limited to calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; wetting agents include but are not limited to water, ethanol, etc.; binders include but are not limited to hypromellose, povidone, microcrystalline cellulose, etc.; disintegrants include but are not limited to croscarmellose sodium, crospovidone, surfactants, low-substituted hydroxypropyl cellulose, etc.; lubricants include but are not limited to magnesium stearate, talc, polyethylene glycol, sodium lauryl sulfate, colloidal silicon dioxide, talc, etc.; preservatives include but are not limited to parabens, chlorobutanol, phenol, sorbic acid, etc.; antioxidants include but are not limited to ascorbic acid, methionine, etc.

[0032] In the present invention, the angiogenesis and tissue regeneration are achieved by administering a therapeutically effective amount of the pharmaceutical composition to a subject. The subjects include but are not limited to mammals, and examples of mammals include but are not limited to humans, mice, rabbits, cats, dogs, cows, sheep, pigs, etc.

[0033] In the present invention, there is no particular limitation on the mode of administration of the drug, and representative modes of administration include but are not limited to: parenteral (intravenous, intramuscular or subcutaneous) and topical administration. Correspondingly, the pharmaceutical composition of the present invention can be made into various clinically acceptable dosage forms, including injection dosage forms, topical dosage forms or external use dosage forms, etc. In some embodiments, the pharmaceutical composition of the present invention can be a composite film, patch, gel, microneedle, porous sponge, fiber scaffold, 3D printed structure, injection, etc.

[0034] The therapeutically effective amount described in the present invention refers to the pharmaceutically recognized effective dosage, that is, the amount of the pharmaceutical composition of the present invention is sufficient to significantly improve the condition without causing serious side effects. The daily dosage of the pharmaceutical composition of the present invention is usually 0.01 - 500 mg / Kg, or 0.01 - 400 mg / Kg, or 0.01 - 300 mg / Kg, or 0.01 - 200 mg / Kg, or 0.01 - 150 mg / Kg, or 0.01 - 100 mg / Kg, or 0.01 - 50 mg / Kg, or 0.01 - 40 mg / Kg, or 0.01 - 30 mg / Kg, and most preferably 0.01 - 20 mg / Kg. Exemplary effective dosages are as follows: 0.01 mg / Kg, 0.05 mg / Kg, 0.1 mg / Kg, 0.2 mg / Kg, 0.3 mg / Kg, 0.4 mg / Kg, 0.5 mg / Kg, 0.75 mg / Kg, 0.95 mg / Kg, 1 mg / Kg, 1.25 mg / Kg, 1.5 mg / Kg, 1.75 mg / Kg, 2 mg / Kg, 2.5 mg / Kg, 2.75 mg / Kg, 3 mg / Kg, 3.25 mg / Kg, 3.5 mg / Kg, 3.75 mg / Kg, 4 mg / Kg, 4.25 mg / Kg, 4.5 mg / Kg, 4.75 mg / Kg, 5 mg / Kg, 5.25 mg / Kg, 5.5 mg / Kg, 5.75 mg / Kg, 6 mg / Kg, 6.25 mg / Kg, 6.5 mg / Kg, 6.75 mg / Kg, 7 mg / Kg, 7.25 mg / Kg, 7.5 mg / Kg, 7.75 mg / Kg, 8 mg / Kg, 8.25 mg / Kg, 8.5 mg / Kg, 8.75 mg / Kg, 9 mg / Kg, 9.25 mg / Kg, 9.5 mg / Kg, 9.75 mg / Kg, 10 mg / Kg, 11 mg / Kg, 12 mg / Kg, 13 mg / Kg, 14 mg / Kg, 15 mg / Kg, 16 mg / Kg, 17 mg / Kg, 18 mg / Kg, 19 mg / Kg, 20 mg / Kg, or any dosage within the above range. It can be administered as a single dose once a day, administered in multiple doses per day, or used intermittently.

[0035] Those skilled in the art will understand that the pharmaceutical composition of the present invention can also be used in combination with other drugs to prepare drugs for jointly promoting angiogenesis and / or tissue regeneration. Among them, the other drugs are not specifically limited and can be any suitable drugs for angiogenesis and / or tissue regeneration.

[0036] Preparation method The preparation method of cysteine-rich acidic secretory protein, biglycan and type XV collagen α1 chain derived from acellular extracellular matrix of the present invention is not particularly limited, and can be prepared by methods known in the art. Those skilled in the art can also directly purchase from known commercial products, and this is not particularly limited.

[0037] For the preparation of acellular extracellular matrix, exemplary preparation methods include the following steps: (1) Obtain tumor tissue; (2) Chop the tumor tissue, immerse it in double-distilled water, and oscillate at 0-10°C and 50-300 rpm for 5-20 hours to obtain a tissue sample; (3) Place the tissue sample in the following buffers in sequence for continuous stirring treatment: First, react overnight in a hypotonic Tris buffer (1-50 mM Tris, 1-10 mM EDTA) at 50-300 rpm; Second, wash with ddH2O at 50-300 rpm for 0.5-5 hours; Then, react in a hypertonic Tris buffer (20-80 mM Tris, 0.5-2 M NaCl, 1-20 mM EDTA) at 30-40°C and 50-300 rpm for 20-30 hours; Then, wash with ddH2O at 100-300 rpm for 0.5-5 hours; Subsequently, treat with 0.5-5% (v / v) TritonX-100 at 30-40°C and 50-300 rpm for 12-24 hours; Wash with ddH2O at 50-300 rpm for 0.5-5 hours; Treat with DNase I at 30-40°C and 50-300 rpm for 3-6 hours, and finally, wash with ddH2O at 50-300 rpm overnight; (4) Use supercritical carbon dioxide extraction, and its operating conditions are: pressure 200-400 bar, temperature 30-40°C, ethanol as an adjuvant (concentration 1-10%), total extraction flow rate 20-40 g / L, extraction time 1-5 hours. After extraction, grind the obtained acellular extracellular matrix into powder for standby.

[0038] Those skilled in the art are familiar with how to prepare cysteine-rich secretory protein, biglycan, and type XV collagen alpha 1 chain. For example, physical, chemical (acid solutions such as acetic acid, citric acid; neutral salt solutions, etc.), or enzymatic hydrolysis (such as pepsin, collagenase) methods can be used. Exemplary methods include: (1) Optionally cleaning the acellular extracellular matrix before extraction: rinsing with PBS or deionized water to remove residual acellular reagents, and then obtaining each of the above proteins by appropriate methods such as acid extraction, enzymatic hydrolysis, etc.; (2) Isolation and purification, for example, further purifying by salting out, dialysis, or further combining ion exchange chromatography or gel filtration chromatography. The conditions such as temperature, pH, enzyme concentration, etc. in the above process are not particularly limited, and those skilled in the art can adjust them according to needs. In addition, the qualitative and quantitative analysis of each of the above proteins is known in the art. For example, it can be determined by SDS-PAGE (electrophoresis), hydroxyproline content determination, or immunoblot (Western blot).

[0039] Method for promoting tissue regeneration in vitro In one aspect of the present invention, a method for promoting tissue regeneration in vitro is provided, which includes the step of contacting the pharmaceutical composition of the present invention with cells in vitro. The method of the present invention can be used for non-diagnostic and non-therapeutic purposes, such as disease mechanism research, drug screening and evaluation, organoid model construction, organ-on-a-chip construction, artificial blood vessel development, etc.

[0040] In a preferred embodiment, tissue regeneration includes at least one of the following situations: (1) promoting endothelial cell vascularization; (2) promoting angiogenesis in the tissue defect area; (3) promoting bone regeneration in the bone defect area; (4) promoting tissue healing.

[0041] Application In one aspect of the present invention, an application of the pharmaceutical composition of the present invention in the preparation of products for promoting angiogenesis and / or tissue regeneration is provided. Among them, examples of the products include, but are not limited to, drugs, health products, medical materials, medical devices, etc.

[0042] Example 1 The following exemplarily shows the angiogenesis-promoting situations of different proteins.

[0043] 1. Experimental method The significantly differentially expressed proteins in liver cancer tissues compared to liver tissues were analyzed using proteomic techniques (screening criteria: |log2FC|≥2, adj.p<0.05). Based on the STRING database, a differential protein interaction network was constructed and topological analysis was performed. Key nodes of the network were identified according to the topological centrality algorithm. After multi-dimensional screening combining the fold change of differential expression and biological verification, the acidic secretory protein rich in cysteine, biglycan, and collagen type XV alpha 1 chain were finally determined as the key protein combinations potentially promoting regeneration.

[0044] Experimental grouping: (1) The gelatin group (control group) was 2.5% gelatin (GelMA); (2) The acidic secretory protein rich in cysteine group was 2.5% gelatin plus 2.71 nM of the acidic secretory protein rich in cysteine; (3) The biglycan group was 2.5% gelatin plus 2.71 nM of biglycan; (4) The collagen type XV alpha 1 chain group was 2.5% gelatin plus 2.71 nM of collagen type XV alpha 1 chain; (5) The three-protein group was 2.5% gelatin (GelMA) plus 1.16 nM of the acidic secretory protein rich in cysteine, 1.19 nM of biglycan, and 0.36 nM of collagen type XV alpha 1 chain. Each of the above groups was mixed with human umbilical vein endothelial cell spheres, photocured for 1 min, then endothelial cell medium was added, and cultured in an incubator for 12 h.

[0045] 2. Experimental results The results were as Figure 1 shown. The quantitative results of the bud length (micrometers) were: gelatin group: 131.944±17.534, acidic secretory protein rich in cysteine group: 187.568±20.571, biglycan group: 126.689±12.952, collagen type XV alpha 1 chain group: 99.528±16.457, S / B / C group: 235.781±56.832 (P<0.05). The quantitative results of the number of buds were: gelatin group: 33.1±3.071, acidic secretory protein rich in cysteine group: 41±3.921, biglycan group: 28±2.362, collagen type XV alpha 1 chain group: 25±7.223, three-protein group: 49±4.055 (P<0.05).

[0046] The results indicate that the acidic secretory protein rich in cysteine, biglycan, and collagen type XV alpha 1 chain can synergistically promote the budding of endothelial cell spheres.

[0047] Example 2 The following exemplarily shows that the three-protein combination can promote angiogenesis and bone regeneration.

[0048] 1. Experimental methods At the rat skull defect site, collagen-hydroxyapatite (COL+HA) was applied to load 11.6 nM of secreted protein acidic and rich in cysteine, 11.9 nM of biglycan, and 3.6 nM of collagen type XV alpha 1 chain. The control group was collagen-hydroxyapatite. After 4 weeks of treatment, tissue sections of the bone defect area were analyzed.

[0049] 2. Experimental results The results were as Figure 2 shown. The fluorescence quantitative results of CD34 (tip cell marker) were: the triple-protein group was 5.763 times that of the control group. The fluorescence quantitative results of OCN (late osteogenesis marker) were: the triple-protein group was 2.023 times that of the control group.

[0050] The results indicated that the triple-protein combination could synergistically promote angiogenesis and bone regeneration in the skull defect area.

[0051] Example 3 The following exemplarily shows that the triple-protein combination can promote the regeneration of rat knee osteochondral.

[0052] 1. Experimental methods At the rat knee defect site, gelatin was applied to load 11.6 nM of secreted protein acidic and rich in cysteine, 11.9 nM of biglycan, and 3.6 nM of collagen type XV alpha 1 chain protein. The control group was gelatin. After 4 weeks of treatment, gross observations and tissue sections of angiogenesis were analyzed.

[0053] 2. Experimental results The results were as Figure 3 shown. The ICRS gross cartilage score results were: gelatin group: 5.0 ± 1.000, triple-protein group: 9.8 ± 0.8367 (P<0.05). The fluorescence intensity results of CD31 (vascular marker) were: gelatin group: 79.973 ± 12.376, triple-protein group: 144.323 ± 17.362 (P<0.05).

[0054] The results indicated that the triple-protein combination could synergistically promote the regeneration of rat knee osteochondral.

[0055] Example 4 The following exemplarily shows that the triple-protein combination can promote angiogenesis and skin healing in the skin defect area of mice.

[0056] 1. Experimental methods The acidic cysteine-rich secretory protein at 11.6 nM, biglycan at 11.9 nM, and type XV collagen alpha 1 chain at 3.6 nM were loaded on PVA and applied to the skin defect area of mice. The control group was PVA. After 10 days of treatment, the skin healing rate and histological sections of angiogenesis were analyzed.

[0057] 2. Experimental results The results were as Figure 4 shown. The results of blood vessel density ( / mm 2 ) were: PVA group: 69.415 ± 18.350, triple-protein group: 130.427 ± 5.358 (P < 0.05); the results of the skin wound healing rate (%) on the 10th day were: PVA group: 82.523 ± 1.462, triple-protein group: 94.544 ± 2.472 (P < 0.05).

[0058] The results indicate that the triple-protein combination can synergistically promote angiogenesis and skin healing in the skin defect area of mice.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. And these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pharmaceutical composition for promoting angiogenesis and / or tissue regeneration, characterized in that, It includes secreted protein acidic and rich in cysteine, biglycan, and collagen type XV alpha 1 chain.

2. The pharmaceutical composition according to claim 1, characterized in that, The secreted protein acidic and rich in cysteine, biglycan, and / or collagen type XV alpha 1 chain are respectively derived from acellular extracellular matrix.

3. The pharmaceutical composition according to claim 1, characterized in that, In the pharmaceutical composition, the secreted protein acidic and rich in cysteine is 0.5 - 5 mole parts, biglycan is 0.5 - 5 mole parts, and collagen type XV alpha 1 chain is 1 mole part.

4. The pharmaceutical composition according to claim 1, wherein It further includes a pharmaceutically acceptable carrier.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutically acceptable carrier includes at least one of scaffold material, diluent, filler, absorbent, wetting agent, binder, disintegrant, lubricant, preservative, and antioxidant.

6. A method for preparing the pharmaceutical composition according to any one of claims 1-5, characterized in that, It includes the step of separating the secreted protein acidic and rich in cysteine, biglycan, and collagen type XV alpha 1 chain from acellular extracellular matrix.

7. The preparation method according to claim 6, characterized in that, It further includes the step of preparing acellular extracellular matrix.

8. A method for promoting tissue regeneration in vitro, characterized in that, It includes the step of contacting the pharmaceutical composition according to any one of claims 1 - 5 with cells in vitro.

9. The method for promoting tissue regeneration in vitro according to claim 8, wherein The promoting of tissue regeneration includes at least one of the following situations: (1) Promoting endothelial cell vascularization; (2) Promoting angiogenesis in tissue defect areas; (3) Promoting bone regeneration in bone defect areas; (4) Promoting tissue healing.

10. Use of the pharmaceutical composition according to any one of claims 1 - 5 in the preparation of a product for promoting angiogenesis and / or tissue regeneration.

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