A pharmaceutical composition for promoting angiogenesis and tissue regeneration and use thereof

Abstract

The application discloses a pharmaceutical composition for promoting angiogenesis and tissue regeneration and application thereof. The pharmaceutical composition comprises a cysteine-rich acidic secretory protein, a biglycan and a collagen XV alpha 1 chain. The application can promote angiogenesis and improve the tissue regeneration efficiency of bone, cartilage and skin defect areas through a biomaterial system loaded with the three types of proteins.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to a pharmaceutical composition that promotes angiogenesis and tissue regeneration and its application. Background Technology

[0002] Tissue regeneration is a biological process regulated by evolutionarily conserved mechanisms, and its repair efficiency benefits from the regulation of various signaling pathways within the microenvironment. Insufficient activation of regeneration-related signals leads to tissue repair dysfunction, while overactivation can trigger pathological hyperplasia. Therefore, reconstructing complex regeneration-related signaling networks is a decisive factor in achieving functional regeneration and a major technical challenge to existing tissue engineering techniques.

[0003] It is noteworthy that the unique biological characteristic of continuous angiogenesis in the tumor microenvironment is precisely a key regenerative signal that is generally lacking in the damaged healing microenvironment. Based on this, the "overactivated" microenvironmental signaling system formed by tumor formation can compensate for the functional deficiencies of key signals during the regeneration and repair process. By reconstructing microenvironmental homeostasis, a breakthrough improvement in tissue regeneration efficiency can be achieved. Currently, existing technologies have attempted to achieve tissue regeneration through the transplantation of rapidly proliferating tumor cells or by using tumor homogenate extracts to construct an immunosuppressive microenvironment to support the growth of ectopic organs. However, such approaches face serious clinical translational obstacles due to potential tumorigenic risks and the uncontrollability of immune regulation. Summary of the Invention

[0004] To address at least some of the 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.

[0005] In a first aspect, the present invention provides a pharmaceutical composition for promoting angiogenesis and / or tissue regeneration, comprising a cysteine-rich acidic secretory protein, a disaccharide proteoglycan, and an XV-type collagen α1 chain.

[0006] In some embodiments, according to the pharmaceutical composition of the present invention, the cysteine-rich acidic secretory protein, disaccharide proteoglycan, and / or XV-type collagen α1 chain are each derived from the extracellular matrix of tumor tissue.

[0007] In some embodiments, the pharmaceutical composition according to the present invention comprises 0.5-5 moles of cysteine-rich acidic secretory protein, 0.5-5 moles of disaccharide proteoglycan, and 1 mole of XV type collagen α1 chain.

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

[0009] In some embodiments, the pharmaceutical composition according to the present invention includes, wherein the pharmaceutically acceptable carrier comprises at least one selected from scaffold material, diluent, filler, absorbent, wetting agent, binder, disintegrant, lubricant, preservative and antioxidant.

[0010] A second aspect of the present invention provides a method for preparing the pharmaceutical composition of the present invention, comprising the steps of separating cysteine-rich acidic secretory protein, disaccharide proteoglycan, and XV-type collagen α1 chain from tumor tissue through decellularization of the extracellular matrix.

[0011] In some embodiments, the preparation method according to the present invention further includes a step of preparing a decellularized extracellular matrix of tumor tissue.

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

[0013] In some embodiments, the method for promoting tissue regeneration in vitro according to the present invention includes at least one of the following:

[0014] (1) Promotes endothelial cell vascularization;

[0015] (2) Promotes angiogenesis in areas of tissue loss;

[0016] (3) Promotes bone regeneration in areas of bone defects;

[0017] (4) Promotes tissue healing.

[0018] A fourth aspect of the invention provides the use of the pharmaceutical composition of the invention in the preparation of products that promote angiogenesis and / or tissue regeneration.

[0019] This invention achieves a technological breakthrough by identifying key functional components related to regeneration in the decellularized extracellular matrix of tumor tissue, thus clarifying functional targets. At the translational medicine level, this invention identifies key bioactive protein components with regenerative functions from the decellularized extracellular matrix of tumor tissue: cysteine-rich acidic secretory proteins, disaccharide proteoglycans, and XV-type collagen α1 chains. The biomaterial system loaded with these three types of proteins in this invention can promote angiogenesis and improve tissue regeneration efficiency in bone, cartilage, and skin defects. Furthermore, this invention ensures the functional reproducibility and biosafety of tumor-derived biomaterials at the molecular mechanism level. Attached Figure Description

[0020] Figure 1This demonstrates how different proteins promote budding of human umbilical vein endothelial cells.

[0021] Figure 2 This study demonstrates how cysteine-rich acidic secretory proteins, disaccharide proteoglycans, and XV-type collagen α1 chains promote angiogenesis and bone regeneration in rat skull defect areas.

[0022] Figure 3 This study demonstrates how cysteine-rich acidic secretory proteins, disaccharide proteoglycans, and XV-type collagen α1 chains promote the recovery of cartilage defects in the patella of rats.

[0023] Figure 4 This study demonstrates how cysteine-rich acidic secretory proteins, disaccharide proteoglycans, and XV-type collagen α1 chains promote angiogenesis and skin healing in areas of skin defects in mice. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Pharmaceutical Composition

[0028] In one aspect, the present invention provides a pharmaceutical composition for promoting angiogenesis and / or tissue regeneration, comprising a cysteine-rich acidic secretory protein, a disaccharide proteoglycan, and an XV-type collagen α1 chain.

[0029] In this invention, "promotion" refers to a degree of promotion or improvement of at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% compared to a control group under equivalent conditions and measured by any standard technique. Beneficial or desired clinical outcomes include, but are not limited to, the following, whether detectable or undetectable, outcomes such as promotion of angiogenesis and / or tissue regeneration. Promotion of angiogenesis and tissue regeneration includes at least one of the following: (1) promotion of endothelial cell vascularization; (2) promotion of angiogenesis in areas of tissue defects; (3) promotion of bone regeneration in areas of bone defects; and (4) promotion of tissue healing.

[0030] In this invention, "angiogenesis" refers to the growth or formation of new blood vessels, including the growth of relatively small-diameter new blood vessels composed of endothelial cells. Angiogenesis is an integral part of many important biological processes, including wound healing, damaged tissue repair, vascular reconstruction, and remodeling. Angiogenesis can be determined in various ways, including but not limited to quantifying budding, vessel diameter, vessel length, number of branching points, and vessel density to assess angiogenesis.

[0031] In this 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, bones, muscles, and nerves. "Injury" refers to the damage to tissue structures such as skin, bone, cartilage, and organs caused by various traumatic factors, or the resulting local and systemic reactions. Injury includes physical injury, chemical injury, and biological injury. In this invention, the pharmaceutical composition is specifically used for bone injury (or bone defect), cartilage injury (or cartilage defect), or skin injury (or skin defect).

[0032] In a preferred embodiment, the cysteine-rich acidic secretory protein (Sparc), disaccharide proteoglycan (Bgn), and / or XV-type collagen α1 chain (Col15a1) are each derived from the decellularized extracellular matrix. The source of the decellularized extracellular matrix is ​​not particularly limited and can be derived from normal tissue, tumor tissue, etc. In a preferred embodiment, the decellularized extracellular matrix is ​​derived from tumor tissue.

[0033] In this invention, the tumor includes highly malignant and rapidly growing solid tumors, examples of which include, but are not limited to: breast cancer, melanoma, osteosarcoma, neuroblastoma, pancreatic cancer, lung cancer, rhabdomyosarcoma, Ewing sarcoma, bladder cancer, colon cancer, liver cancer, ovarian cancer, cervical cancer, nasopharyngeal carcinoma, laryngeal cancer, gastric cancer, kidney cancer, head and neck tumors, esophageal cancer, thyroid cancer, or brain cancer. In a preferred embodiment, the tumor includes epithelial malignant tumors, such as liver cancer, colon cancer, and lung cancer. In a preferred embodiment, the decellularized extracellular matrix is ​​derived from liver cancer tissue.

[0034] The present invention has found that cysteine-rich acidic secretory proteins, disaccharide proteoglycans, and XV-type collagen α1 chains have a synergistic effect in promoting angiogenesis and / or tissue regeneration. In a preferred embodiment, the molar ratio of the cysteine-rich acidic secretory protein, disaccharide proteoglycan, and XV-type collagen α1 chain is (0.5-5):(0.5-5):1, preferably (0.8-5):(1-5):1, and even more preferably (1-5):(1-5):1, for example 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.

[0035] In a preferred embodiment, the pharmaceutical composition of the present invention comprises a cysteine-rich acidic secretory protein, a disaccharide proteoglycan, and an XV-type collagen α1 chain, as well as a pharmaceutically acceptable carrier.

[0036] In this invention, the carrier is "acceptable," meaning it is compatible with other components of the formulation (e.g., cysteine-rich acidic secretory proteins, disaccharide proteoglycans, or XV-type collagen α1 chains) and does not harm the patient. The pharmaceutically acceptable carrier includes at least one of the following: scaffold materials, diluents, fillers, absorbents, wetting agents, binders, disintegrants, lubricants, preservatives, and antioxidants. Scaffold materials include, but are not limited to, natural polymers (e.g., but not limited to, collagen, gelatin, chitosan, etc.), synthetic polymers (e.g., but not limited to, PVA, PLGA, PCL, PEG, etc.), hydrogels (e.g., but not limited to, sodium alginate, hyaluronic acid, polyacrylamide), inorganic materials (e.g., but not limited to, hydroxyapatite, bioactive glass, etc.), and nanoscaffold materials (e.g., but not limited to, electrospun nanofibers, graphene composites, etc.). Examples of diluents include, but are not limited to, physiological saline, aqueous buffer solutions, solvents, and dispersion media; fillers include, but are not limited to, starch, lactose, mannitol, and microcrystalline cellulose; absorbents include, but are not limited to, calcium sulfate, dicalcium phosphate, and calcium carbonate; wetting agents include, but are not limited to, water and ethanol; binders include, but are not limited to, hydroxypropyl methylcellulose, povidone, and microcrystalline cellulose; disintegrants include, but are not limited to, croscarmellose sodium, croscarmellose, surfactants, and low-substituted hydroxypropyl cellulose; lubricants include, but are not limited to, magnesium stearate, talc, polyethylene glycol, sodium dodecyl sulfate, micronized silica gel, and talc; preservatives include, but are not limited to, parabens, chlorobutanol, phenol, and sorbic acid; and antioxidants include, but are not limited to, ascorbic acid and methionine.

[0037] In this invention, angiogenesis and tissue regeneration are achieved by administering a therapeutically effective amount of the pharmaceutical composition to a subject. Subjects include, but are not limited to, mammals, including but not limited to, humans, mice, rabbits, cats, dogs, cattle, sheep, and pigs.

[0038] In this invention, the method of administration of the drug is not particularly limited, and representative methods include, but are not limited to, parenteral (intravenous, intramuscular, or subcutaneous) and local administration. Accordingly, the pharmaceutical compositions of this invention can be formulated into various clinically acceptable dosage forms, including injectable dosage forms, local administration dosage forms, or topical dosage forms. In some embodiments, the pharmaceutical compositions of this invention can be composite films, patches, gels, microneedles, porous sponges, fiber scaffolds, 3D printed structures, injectable formulations, etc.

[0039] The therapeutically effective dose described in this invention refers to a pharmaceutically recognized effective dosage, meaning that the amount of the pharmaceutical composition of this invention is sufficient to significantly improve the condition without causing serious side effects. The daily dosage of the pharmaceutical composition of this invention is typically 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, with 0.01-20 mg / kg being the most preferred. 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. The recommended dosage is 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 daily dose, divided into multiple daily doses, or at intervals.

[0040] Those skilled in the art will understand that the pharmaceutical compositions of the present invention can also be used in combination with other drugs to prepare combined drugs for promoting angiogenesis and / or tissue regeneration. These other drugs are not specifically limited and can be any suitable drug for angiogenesis and / or tissue regeneration.

[0041] Preparation method

[0042] The preparation method of the cysteine-rich acidic secretory protein, disaccharide proteoglycan, and XV-type collagen α1 chain derived from the extracellular matrix of the present invention is not particularly limited, and can be prepared using methods known in the art. Those skilled in the art can also directly purchase known commercial products, and this is not particularly limited.

[0043] For the preparation of decellularized extracellular matrix, an exemplary preparation method includes the following steps: (1) obtaining tumor tissue; (2) chopping the tumor tissue, immersing it in double-distilled water, and shaking it at 0-10°C and 50-300 rpm for 5-20 hours to obtain a tissue sample; (3) sequentially placing the tissue sample in the following buffer solutions for continuous stirring: first, reacting overnight in hypotonic Tris buffer (1-50 mM Tris, 1-10 mM EDTA) at 50-300 rpm; second, washing with ddH2O for 0.5-5 hours at 50-300 rpm; then, reacting for 20-30 hours in hypertonic Tris buffer (20-80 mM Tris, 0.5-2 M NaCl, 1-20 mM EDTA) at 30-40°C and 50-300 rpm; then, reacting at 100-300 rpm. Wash with ddH2O for 0.5-5 hours at rpm; then treat with 0.5-5% (v / v) TritonX-100 for 12-24 hours at 30-40°C and 50-300 rpm; wash with ddH2O for 0.5-5 hours at 50-300 rpm; treat with DNase I for 3-6 hours at 30-40°C and 50-300 rpm; finally, wash with ddH2O overnight at 50-300 rpm; (4) Supercritical carbon dioxide extraction was used, with the following operating conditions: pressure 200-400 bar, temperature 30-40°C, ethanol as an auxiliary agent (concentration 1-10%), total extraction flow rate 20-40 g / L, extraction time 1-5 hours. After extraction, the obtained decellularized extracellular matrix was ground into powder for later use.

[0044] Those skilled in the art are familiar with how to prepare cysteine-rich acidic secretory proteins, disaccharide proteoglycans, and XV-type collagen α1 chains, for example, by physical, chemical (acidic solutions, such as acetic acid, citric acid; neutral salt solutions, etc.) or enzymatic (e.g., pepsin, collagenase) methods. Exemplary methods include: (1) optionally washing the decellularized extracellular matrix before extraction: rinsing with PBS or deionized water to remove residual decellularization reagents, followed by obtaining the above-mentioned proteins by a suitable method, such as acid extraction, enzymatic hydrolysis, etc.; (2) separating and purifying, for example by salting out, dialysis, or further purification by combining ion exchange chromatography or gel filtration chromatography. The temperature, pH, enzyme concentration, and other conditions in the above process are not particularly limited, and those skilled in the art can adjust them as needed. In addition, the qualitative and quantitative analysis of the above-mentioned proteins is known in the art, for example, by SDS-PAGE (electrophoresis), hydroxyproline content determination, or Western blot.

[0045] Methods to promote tissue regeneration in vitro

[0046] One aspect of the present invention provides a method for promoting tissue regeneration in vitro, comprising 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 therapeutic purposes, such as disease mechanism research, drug screening and evaluation, organoid model construction, organ-on-a-chip construction, and artificial blood vessel development.

[0047] In a preferred embodiment, tissue regeneration includes at least one of the following: (1) promoting endothelial cell vascularization; (2) promoting angiogenesis in tissue defect areas; (3) promoting bone regeneration in bone defect areas; and (4) promoting tissue healing.

[0048] application

[0049] One aspect of the present invention provides the use of the pharmaceutical composition described herein in the preparation of products that promote angiogenesis and / or tissue regeneration. Examples of such products include, but are not limited to, pharmaceuticals, health supplements, medical materials, and medical devices.

[0050] Example 1

[0051] The following examples illustrate the role of different proteins in promoting angiogenesis.

[0052] 1. Experimental Methods

[0053] Proteomics was used to analyze differentially expressed proteins between hepatocellular carcinoma (HCC) tissues and liver tissues (screening criteria: |log2FC|≥2, adj.p<0.05). A differential protein-protein interaction network was constructed based on the STRING database, and topological analysis was performed. Key nodes in the network were identified using a topological centrality algorithm. After multi-dimensional screening combining differential expression fold-over and biological validation, cysteine-rich acidic secretory proteins, disaccharide proteoglycans, and XV-type collagen α1 chains were identified as potential key protein combinations for promoting regeneration.

[0054] Experimental groups: (1) Gelatin group (control group): 2.5% gelatin (GelMA); (2) Cysteine-rich acidic secretory protein group: 2.5% gelatin with 2.71 nM cysteine-rich acidic secretory protein; (3) Disaccharide proteoglycan group: 2.5% gelatin with 2.71 nM disaccharide proteoglycan; (4) XV-type collagen α1 chain group: 2.5% gelatin with 2.71 nM XV-type collagen α1 chain; (5) Tri-protein group: 2.5% gelatin (GelMA) with 1.16 nM cysteine-rich acidic secretory protein, 1.19 nM disaccharide proteoglycan, and 0.36 nM XV-type collagen α1 chain. Each group was mixed with human umbilical vein endothelial cell spheres, light-cured for 1 min, and then added to endothelial cell culture medium. The mixtures were incubated for 12 h.

[0055] 2. Experimental Results

[0056] The results are as follows Figure 1 As shown, the quantitative results of budding length (micrometers) were: gelatin group: 131.944±17.534, cysteine-rich acidic secretory protein group: 187.568±20.571, disaccharide-chain proteoglycan group: 126.689±12.952, XV type collagen α1 chain group: 99.528±16.457, S / B / C group: 235.781±56.832 (P<0.05). The quantitative results of budding number were: gelatin group: 33.1±3.071, cysteine-rich acidic secretory protein group: 41±3.921, disaccharide-chain proteoglycan group: 28±2.362, XV type collagen α1 chain group: 25±7.223, triprotein group: 49±4.055 (P<0.05).

[0057] The results showed that cysteine-rich acidic secretory proteins, disaccharide proteoglycans, and XV-type collagen α1 chains synergistically promote budding of endothelial cell spheroids.

[0058] Example 2

[0059] The following examples illustrate how a combination of three proteins can promote angiogenesis and bone regeneration.

[0060] 1. Experimental Methods

[0061] In rats, collagen-hydroxyapatite (COL+HA) was used to load 11.6 nM of cysteine-rich acidic secretory protein, 11.9 nM of disaccharide proteoglycan, and 3.6 nM of XV type collagen α1 chain onto the skull defect site. The control group was collagen-hydroxyapatite. The treatment lasted for 4 weeks, and tissue sections of the bone defect area were analyzed.

[0062] 2. Experimental Results

[0063] The results are as follows Figure 2 As shown, the quantitative fluorescence results for CD34 (a marker of advanced cell formation) were 5.763 times that of the control group. The quantitative fluorescence results for OCN (a marker of late osteogenic formation) were 2.023 times that of the control group.

[0064] The results showed that the combination of the three proteins could synergistically promote angiogenesis and bone regeneration in the skull defect area.

[0065] Example 3

[0066] The following example demonstrates how a combination of three proteins can promote the regeneration of cartilage in the rat patella.

[0067] 1. Experimental Methods

[0068] In rats, gelatin was used to load 11.6 nM of cysteine-rich acidic secretory protein, 11.9 nM of disaccharide proteoglycan, and 3.6 nM of XV type collagen α1 chain protein at the patellar bone defect site. The control group was gelatin. The treatment lasted for 4 weeks, and the gross appearance and angiogenesis tissue sections were analyzed.

[0069] 2. Experimental Results

[0070] The results are as follows Figure 3 As shown, the ICRS gross cartilage score was: gelatin group: 5.0±1.000, three-protein group: 9.8±0.8367 (P<0.05). The CD31 (vascular marker) fluorescence intensity was: gelatin group: 79.973±12.376, three-protein group: 144.323±17.362 (P<0.05).

[0071] The results showed that the combination of the three proteins synergistically promoted the regeneration of cartilage in the rat patella.

[0072] Example 4

[0073] The following example demonstrates how a combination of three proteins can promote angiogenesis and skin healing in areas of skin defects in mice.

[0074] 1. Experimental Methods

[0075] In mice, PVA loaded with 11.6 nM of cysteine-rich acidic secretory protein, 11.9 nM of disaccharide proteoglycan, and 3.6 nM of XV type collagen α1 chain was applied to the skin defect area. The control group was PVA. After 10 days of treatment, the skin healing rate and angiogenesis were analyzed in tissue sections.

[0076] 2. Experimental Results

[0077] The results are as follows Figure 4 As shown, the blood vessel density results ( / mm) 2 The results of the skin wound healing rate (%) on day 10 were as follows: PVA group: 69.415±18.350, Tri-protein group: 130.427±5.358 (P<0.05); PVA group: 82.523±1.462, Tri-protein group: 94.544±2.472 (P<0.05).

[0078] The results showed that the combination of the three proteins synergistically promoted angiogenesis and skin healing in areas of skin defects in mice.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to 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, characterized by, 0.5-5 molar parts of the cysteine-rich acidic secretory protein, 0.5-5 molar parts of the biglycan and 1 molar part of the collagen type XV alpha 1 chain.

2. The pharmaceutical composition of claim 1, wherein, Each of the cysteine-rich acidic secretory protein, the biglycan and / or the collagen type XV alpha 1 chain is derived from a decellularized extracellular matrix, respectively.

3. The pharmaceutical composition of claim 1, wherein, Further comprising a pharmaceutically acceptable carrier.

4. The pharmaceutical composition of claim 3, wherein, The pharmaceutically acceptable carrier comprises 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.

5. Process for the preparation of a pharmaceutical composition according to any one of claims 1 to 4, characterized in that, Further comprising a step of isolating the cysteine-rich acidic secretory protein, the biglycan and the collagen type XV alpha 1 chain from a decellularized extracellular matrix.

6. The production method according to claim 5, wherein Further comprising a step of preparing the decellularized extracellular matrix.

7. A non-therapeutic method for promoting tissue regeneration in vitro, characterized in that, Further comprising a step of contacting the pharmaceutical composition according to any one of claims 1-4 with cells in vitro, wherein the promoting tissue regeneration is at least one of the following: (1) promoting endothelial cell vascularization; (2) promoting vascular regeneration in a tissue defect area.

8. Use of the pharmaceutical composition according to any one of claims 1-4 in the preparation of a product for promoting angiogenesis.

9. A method for promoting tissue regeneration, comprising a step of contacting a pharmaceutical composition according to any one of claims 1-4 with cells in vitro, wherein the promoting tissue regeneration is at least one of the following: (1) promoting endothelial cell vascularization; (2) promoting vascular regeneration in a tissue defect area.

10. Use of the pharmaceutical composition according to any one of claims 1-4 in the preparation of a product for promoting tissue regeneration.

11. A method for promoting tissue regeneration, comprising a step of contacting a pharmaceutical composition according to any one of claims 1-4 with cells in vitro, wherein the promoting tissue regeneration is at least one of the following: (1) promoting endothelial cell vascularization; (2) promoting vascular regeneration in a tissue defect area.

12. Use of the pharmaceutical composition according to any one of claims 1-4 in the preparation of a product for promoting vascularization.

13. A method for promoting tissue regeneration, comprising a step of contacting a pharmaceutical composition according to any one of claims 1-4 with cells in vitro, wherein the promoting tissue regeneration is at least one of the following: (1) promoting endothelial cell vascularization; (2) promoting vascular regeneration in a tissue defect area.

14. Use of the pharmaceutical composition according to any one of claims 1-4 in the preparation of a product for promoting vascularization.

15. A method for promoting tissue regeneration, comprising a step of contacting a pharmaceutical composition according to any one of claims 1-4 with cells in vitro, wherein the promoting tissue regeneration is at least one of the following: (1) promoting endothelial cell vascularization; (2) promoting vascular regeneration in a tissue defect area.

16. Use of the pharmaceutical composition according to any one of claims 1-4 in the preparation of a product for promoting vascularization.

17. A method for promoting tissue regeneration, comprising a step of contacting a pharmaceutical composition according to any one of claims 1-4 with cells in vitro, wherein the promoting tissue regeneration is at least one

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