Use of tumor-derived acellular extracellular matrix in the preparation of a drug for promoting angiogenesis and tissue regeneration

Abstract

The application discloses application of tumor-derived acellular extracellular matrix in preparation of a drug for promoting angiogenesis and tissue regeneration. The application proposes a strategy for regulating tissue regeneration based on a tumor-derived acellular extracellular matrix microenvironment, combines the high similarity between a tumor microenvironment and a regeneration microenvironment such as angiogenesis, and constructs a novel angiogenesis-promoting biomaterial, so that high-efficiency regeneration is realized in various tissue defects. In addition, the characteristics of rapid proliferation of tumor tissues break through the production capacity bottleneck of a raw material source of a traditional acellular extracellular matrix, and provide sustainable biological raw materials for large-scale production. The application successfully solves the problem that a specific tissue-derived acellular extracellular matrix is difficult to regenerate in a complex damage microenvironment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to application of tumor-derived decellular extracellular matrix in preparation of drugs for promoting angiogenesis and tissue regeneration. BACKGROUND

[0002] The decellular extracellular matrix (dECM) retains the biological active components and three-dimensional topological structure of the natural extracellular matrix by selectively removing the immunogenic components in the tissue, and constructs a functionalized biological active scaffold. The dECM exhibits significant advantages in effectively reducing the risks of host immune rejection, inflammatory reaction and fibrosis. At present, the original material for preparing the dECM is mainly derived from a single specific tissue. Although the dECM derived from a specific tissue is beneficial to the construction of a biomimetic tissue microenvironment, it also leads to insufficient adaptability to a complex damage microenvironment (such as the bone-cartilage interface transition zone), and it is difficult to achieve coordinated regeneration of multiple lineages of tissues. The functional enhancement by compounding exogenous functional components or combining with other delivery systems also destroys the integrity of the intrinsic signal transduction network of the ECM, and increases the immunogenicity risk. SUMMARY

[0003] To solve at least part of the technical problems in the prior art, the present application provides application of tumor-derived decellular extracellular matrix in preparation of drugs for promoting angiogenesis and multi-directional tissue regeneration. Specifically, the present application includes the following contents.

[0004] In a first aspect of the present application, application of malignant tumor tissue-derived decellular extracellular matrix in preparation of drugs for promoting angiogenesis and / or tissue regeneration is provided.

[0005] In some embodiments, according to the application, the preparation of the decellular extracellular matrix comprises: obtaining the decellular extracellular matrix by removing immunogenic components from the malignant tumor tissue through treatment.

[0006] In some embodiments, according to the application, the malignant tumor tissue-derived decellular extracellular matrix has a collagen network scaffold structure.

[0007] In some embodiments, according to the application, the malignant tumor comprises an epithelial or neuroectodermal malignant tumor.

[0008] In some embodiments, according to the application, the promotion of angiogenesis and tissue regeneration comprises at least one of the following cases:

[0009] (1) promoting vascularization of endothelial cells;

[0010] (2) promoting vascular regeneration in a bone defect area;

[0011] (3) promoting bone regeneration in a bone defect region;

[0012] (4) promoting tissue healing.

[0013] In some embodiments, the use according to the present application, wherein the promoting is achieved by administering to the subject a therapeutically effective amount of the medicament.

[0014] In some embodiments, the use according to the present application, wherein the subject comprises a mammal.

[0015] In some embodiments, the use according to the present application, wherein the mammal comprises a human.

[0016] In some embodiments, the use according to the present application, wherein the therapeutically effective amount is 0.01-1000 mg / Kg.

[0017] In some embodiments, the use according to the present application, wherein the medicament further comprises a pharmaceutically acceptable carrier.

[0018] In some embodiments, the use according to the present application, wherein the pharmaceutically acceptable carrier comprises at least one of a diluent, a filler, an absorbent, a wetting agent, a binder, a lubricant, a preservative, and an antioxidant.

[0019] In a second aspect of the present application, there is provided a pharmaceutical composition for promoting angiogenesis and / or tissue regeneration, comprising a decellularized extracellular matrix derived from malignant tumor tissue.

[0020] In a third aspect of the present application, there is provided a method for promoting tissue regeneration, comprising the step of contacting a decellularized extracellular matrix derived from a tumor with endothelial cells in vitro.

[0021] In a fourth aspect of the present application, there is provided a method for promoting endothelial cell vascularization, comprising the step of contacting a decellularized extracellular matrix derived from a tumor with endothelial cells in vitro.

[0022] The present application breaks through the traditional tissue source restriction, and innovatively proposes a strategy for regulating tissue regeneration based on the tumor-derived dECM microenvironment. Combined with the high similarity between tumor microenvironment and angiogenic microenvironment, a new type of pro-angiogenic biomaterial is constructed, which achieves high-efficiency regeneration in various tissue defects. In addition, the rapid proliferation characteristics of tumor tissue break through the production bottleneck of traditional dECM raw materials, providing sustainable biological raw materials for large-scale production. The present application successfully solves the problem of dECM from specific tissue sources for regeneration in complex damage microenvironment. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1Characterization of tumor tissue-derived decellularized extracellular matrix is shown.

[0024] Figure 2 Proteomic analysis results of decellularized extracellular matrix from different tumor tissue sources are shown.

[0025] Figure 3 Immunofluorescence and flow cytometry results of tumor tissue-derived decellularized extracellular matrix mixed with endothelial cells are shown.

[0026] Figure 4 Hepatocellular carcinoma tissue-derived dECM can promote vascular and bone regeneration in rat calvarial defect area is shown.

[0027] Figure 5 Hepatocellular carcinoma tissue-derived dECM can promote vascular and osteochondral regeneration in rat knee osteochondral defect area is shown.

[0028] Figure 6 Hepatocellular carcinoma tissue-derived dECM can promote vascular and skin healing in mouse skin defect area is shown.

[0029] Figure 7 HE and Masson tissue sections of skin defect area treated with hepatocellular carcinoma decellularized extracellular matrix are shown. DETAILED DESCRIPTION

[0030] Various illustrative embodiments of the present application are now described in detail below. The following description includes specific details for the purpose of providing a thorough understanding of the present application. However, it will be apparent to those skilled in the art that the present application can be practiced without these specific details. In some instances, well-known structures and functions have not been described in detail in order to avoid obscuring the concept of the present application.

[0031] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, for a range of values of a parameter, unless otherwise indicated, each intervening value and each smaller range between the stated values and intervening values in a stated range is also contemplated. Each smaller range that falls between a stated range, as well as each individual value, is incorporated into the present application as if expressly written therein. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and are also encompassed by the application, subject to any specifically excluded limit in the stated range. Values given are approximate, unless otherwise stated.

[0032] Unless defined otherwise, 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 application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the patents, patent applications, publications, and descriptions are cited. In case of conflict, the content of the present specification will control.

[0033] Application

[0034] In one aspect of the present application, there is provided a use of tumor-derived acellular extracellular matrix in the preparation of a medicament for promoting angiogenesis and / or tissue regeneration. In the present application, considering the high similarity between tumor microenvironment and regeneration microenvironment such as angiogenesis and the ability to retain more active ingredients, in a preferred embodiment, the acellular extracellular matrix of the present application is derived from tumor tissue rather than normal tissue or cell source, thereby increasing the adaptability to complex injury microenvironment and achieving multi-lineage tissue synergistic regeneration.

[0035] In the present application, the tumor includes solid tumor with high malignancy and fast growth, examples of which 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 tumor, esophageal cancer, thyroid cancer or brain cancer. The malignant tumor of the present application can be a primary tumor or a secondary tumor. In a preferred embodiment, the tumor includes an epithelial malignant tumor, such as liver cancer, colon cancer, lung cancer, etc. In another preferred embodiment, the tumor includes a neuroectodermal malignant tumor, such as melanoma. In yet another preferred embodiment, the tumor is a non-germ cell tumor. In a most preferred embodiment, the acellular extracellular matrix is derived from liver cancer tissue.

[0036] In the present application, "promoting" refers to improving the condition after injury occurs. The degree of promotion or improvement is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100% compared with the reference group without treatment under the same conditions, as measured by any standard technique. "Injury" refers to the destruction of tissue or organ structure of skin, bone, cartilage, organs, etc. or the local and systemic reactions caused by various traumatic factors. Injury includes physical injury, chemical injury, biological injury, etc. In the present application, the medicament is particularly used for bone injury (or bone defect), cartilage injury (or cartilage defect) or skin injury (or skin defect).

[0037] In the present application, the beneficial or desirable clinical outcomes include but are not limited to the following results whether detectable or not, such as promoting angiogenesis and / or tissue regeneration. The promotion of angiogenesis and tissue regeneration includes at least one of the following: (1) promoting endothelial cell vascularization; (2) promoting vascular regeneration in the bone defect area; (3) promoting bone regeneration in the bone defect area; (4) promoting tissue (e.g. defect skin) healing.

[0038] In the present application, the promotion of angiogenesis and tissue regeneration is achieved by administering to a subject a therapeutically effective amount of the medicament. The subject includes, but is not limited to, a mammal, examples of which include, but are not limited to, a human, a mouse, a rabbit, a cat, a dog, a cow, a sheep, a pig, and the like.

[0039] In the present application, the medicament contains a therapeutically effective amount of tumor-derived acellular extracellular matrix, and optionally, a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the pharmaceutical agents from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

[0040] In the present application, the pharmaceutically acceptable carrier includes, but is not limited to, at least one of a diluent, a filler, an absorbent, a wetting agent, a binder, a lubricant, a preservative, and an antioxidant. Among them, examples of the diluent include, but are not limited to, physiological saline, an aqueous buffer solution, a solvent, a dispersion medium, and the like; the filler includes, but is not limited to, starch, lactose, mannitol, microcrystalline cellulose, and the like; the absorbent includes, but is not limited to, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, and the like; the wetting agent includes, but is not limited to, water, ethanol, and the like; the binder includes, but is not limited to, a synthetic material or a natural synthetic material, and the like; the lubricant includes, but is not limited to, magnesium stearate, talc, polyethylene glycol, sodium lauryl sulfate, colloidal silica, talc, and the like; the preservative includes, but is not limited to, a paraben, chlorobutanol, phenol, sorbic acid, and the like; the antioxidant includes, but is not limited to, ascorbic acid, methionine, and the like.

[0041] In certain embodiments, the synthetic biomaterials include, but are not limited to, polyethylene glycol, polyethylene glycol derivatives, polylactic acid, polylactic-glycolic acid copolymer, polyanhydrides, polyesters, polyamino acids, polyethylene oxide, polyesters, polymethyl methacrylate, polycarbonates, polyurethanes, polycaprolactone, polyhydroxyalkanoates, polysiloxanes, polyethylene, polyvinyl chloride, polytetrafluoroethylene, polystyrene, polypropylene, N-(3-aminopropyl)methacrylamide hydrochloride (APMA), N-[tris(hydroxymethyl)methyl]acrylamide (THMA), maleic anhydride grafted copolymer, polyacrylamide, polyacetals, polypyrrole, or any combination thereof. In certain embodiments, the natural biomaterials include, but are not limited to, natural proteins, collagen and collagen derivatives, gelatin and gelatin derivatives, agar and agar derivatives, proteoglycans, alginate and alginate derivatives thereof, Matrigel, propolis, cellulose and cellulose derivatives, chitin and chitin derivatives, silk fibroin and derivatives thereof, laminin and derivatives thereof, fibronectin and derivatives thereof, sodium hyaluronate and hyaluronic acid derivatives, agarose and derivatives thereof, dextran and derivatives thereof, sucrose and sucrose derivatives, starch, chitosan and chitosan derivatives, or any combination thereof, preferably gelatin and gelatin derivatives, chitosan and chitosan derivatives.

[0042] In a preferred embodiment, the carrier is selected from at least one of collagen, gelatin, polyvinyl alcohol, hydroxyapatite.

[0043] In the present application, the administration of the drug is not particularly limited, and representative administration methods include, but are not limited to, parenteral (intravenous, intramuscular, or subcutaneous) and topical administration. Accordingly, the drug of the present application can be prepared into various dosage forms acceptable in the clinic, including injection dosage forms, topical administration dosage forms, or external use dosage forms, etc. In certain embodiments, the dosage form of the drug comprising the tumor-derived decellularized extracellular matrix can be a composite film, a patch, a gel, a microneedle, a porous sponge, a fiber scaffold, a 3D printed structure, an injection, etc.

[0044] The therapeutically effective amount according to the present application means a pharmaceutically effective administration dose, i.e. the amount of tumor-derived acellular extracellular matrix sufficient to significantly improve the condition without causing serious side effects. The daily administration dose of tumor-derived acellular extracellular matrix is usually 0.01-1000 mg / Kg, preferably 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, most preferably 0.01-20 mg / Kg. Exemplary effective administration doses are, for example, 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. The administration can be performed as a single dose once a day, as multiple doses per day, or with intervals.

[0045] The person skilled in the art will understand that the tumor-derived acellular extracellular matrix can also be used in combination with other drugs to prepare a combined drug for promoting angiogenesis and / or tissue regeneration. The other drugs are not particularly limited and can be any suitable drug for promoting angiogenesis and / or tissue regeneration.

[0046] In the present application, the preparation method of tumor-derived acellular extracellular matrix is not particularly limited, and the known preparation method of acellular matrix in the art can be used. In a preferred embodiment, the acellular extracellular matrix is obtained by removing immunogenic components (including tumor cells, various cytokines) from malignant tumor tissue. The malignant tumor tissue can be from a tumor-bearing animal model or from a tumor cell primary three-dimensional culture to obtain an organoid.

[0047] An exemplary preparation method of acellular extracellular matrix comprises the following steps: (1) obtaining tumor tissue; (2) chopping the tumor tissue and immersing it in double distilled water, and oscillating 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 buffers for continuous stirring treatment: first, reacting in a low-osmotic Tris buffer (1-50 mM Tris, 1-10 mM EDTA) at 50-300 rpm overnight; second, washing with ddH2O at 50-300 rpm for 0.5-5 hours; then, reacting in a high-osmotic 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, washing with ddH2O at 100-300 rpm for 0.5-5 hours; subsequently, treating with 0.5-5% (v / v) Triton X-100 at 30-40°C and 50-300 rpm for 12-24 hours; washing with ddH2O at 50-300 rpm for 0.5-5 hours; treating with DNase I at 30-40°C and 50-300 rpm for 3-6 hours; and finally, washing with ddH2O at 50-300 rpm overnight; (4) using supercritical carbon dioxide extraction with the following operating conditions: pressure 200-400 bar, temperature 30-40°C, ethanol as an assistant (concentration 1-10%), total extraction flow rate 20-40 g / L, extraction time 1-5 hours, and after extraction, grinding the obtained acellular extracellular matrix into powder for use.

[0048] Methods of promoting tissue regeneration or endothelial cell vascularization

[0049] Another aspect of the present application also provides a method for promoting tissue regeneration or endothelial cell vascularization, comprising the step of contacting tumor-derived acellular extracellular matrix with endothelial cells in vitro. The method of the present application can be used for non-diagnostic and therapeutic purposes (such as but not limited to disease mechanism research, drug screening and evaluation, organoid model construction, organ chip construction, artificial blood vessel development, etc.), which can be used to promote the expression of markers of tip cells by endothelial cells or to promote the transformation of endothelial cells into tip cells.

[0050] Example 1

[0051] 1. Preparation and characterization of acellular extracellular matrix from different tumor tissues, the preparation method is as follows:

[0052] (1) After the negative tumor nude mice and Balb / c mice were euthanized in the carbon dioxide anesthesia chamber, dissection was performed to remove the tumor and liver.

[0053] (2) The above tissues were cut into pieces and immersed in double distilled water (ddH2O), and shaken in a shaker at 4°C and 150 rpm for 12 hours to achieve fat separation.

[0054] (3) Subsequently, the tissue samples were sequentially placed in the following designated buffers for continuous stirring treatment: first, reaction in hypotonic Tris buffer (10 mM Tris, 5 mM EDTA, pH 8.0) at 150 rpm overnight; second, washed with ddH2O at 150 rpm for 1 hour; then, reaction in hypertonic Tris buffer (50 mM Tris, 1 M NaCl, 10 mM EDTA, pH 8.0) at 37°C and 150 rpm for 24 hours; then, washed with ddH2O at 150 rpm for 1 hour; then, treated with 1% (v / v) Triton X-100 at 37°C and 150 rpm for 12-24 hours; then, washed with ddH2O at 150 rpm for 1 hour; finally, treated with 20 U / ml DNase I at 37°C and 150 rpm for 3-6 hours. Finally, washed with ddH2O at 150 rpm overnight. Unless otherwise specified, all steps were performed at 4°C.

[0055] (4) In the final treatment stage, supercritical carbon dioxide (scCO2) extraction was used, with the following operating conditions: pressure 300 bar, temperature 37°C, ethanol as an assistant (concentration 3%), total extraction flow rate 30 g / L, and extraction time 3 hours. After extraction, the obtained acellular extracellular matrix (dECM) was ground into powder.

[0056] The HE DAPI results showed that after the above decellularization method, the cells in the liver, hepatocarcinoma, colon cancer, lung cancer and melanoma dECM were completely removed, and the collagen network scaffold was relatively complete. The safranin staining results showed that the proteoglycans in the dECM were also retained. In summary, this preparation method not only removes the cell immunogenic components in the tissue, but also completely retains the biological active components and three-dimensional topological structure of the natural extracellular matrix, so this embodiment successfully constructs a functionalized acellular extracellular matrix Figure 1 ).

[0057] 2. Proteomic study of dECM

[0058] Proteomic analysis was performed on four kinds of dECM obtained by decellularizing liver cancer, colon cancer, lung cancer and melanoma tissues, respectively. The results are shown in Figure 2 Figure 2 The left chart GSVA results show that liver cancer dECM is more closely related to stem cell life activities, angiogenesis, immune response and histogenesis than the other three kinds of tumors. Figure 2 The right chart of the regeneration potential score results further shows that liver cancer dECM is more related to regeneration.

[0059] Example 2

[0060] This example shows that dECM derived from liver cancer tissue can promote endothelial cells to express markers of tip cells.

[0061] 1 ml of gelatin was added to 10 mg of liver cancer dECM and human umbilical vein endothelial cells, and after photocuring for 30 s, endothelial cell culture medium was added. As shown in Figure 3 After 24 h of culture, the results of immunofluorescence and flow cytometry showed that the addition of liver cancer dECM gelatin (GelMA+H-dECM) could significantly promote endothelial cells to express markers of tip cells (CD34) compared to the addition of liver dECM (GelMA+L-dECM). Figure 3 The quantitative results of the fluorescence intensity of CD34-positive endothelial cells are as follows: liver 6.293±1.464; liver cancer 22.713±3.651 P <0.05). The percentage of CD34-positive endothelial cells in endothelial cells: liver 39.875%±0.386, liver cancer 48.1%±0.917 P <0.05).

[0062] Example 3

[0063] This example shows that dECM derived from liver cancer tissue can promote vascular regeneration and bone regeneration in the rat skull defect area.

[0064] A rat animal model with a skull defect area was constructed, and collagen-hydroxyapatite loaded with liver cancer dECM (COL+HA+H) was applied at the defect site for 2 weeks and 4 weeks, and the vascular regeneration and bone regeneration in the defect area were determined.

[0065] Figure 4 ​Real-time blood perfusion and CT photos of collagen loaded liver cancer dECM at 2 weeks and 4 weeks at rat calvarial defect sites are shown, and the control is collagen-hydroxyapatite loaded liver dECM (COL+HA+L). The 4-week vascular area coefficient results show: liver 0.213±0.028; liver cancer 0.281±0.041( P <0.05). The 4-week bone / tissue volume results show: liver 32.607%±6.589; liver cancer 49.175%±1.082( P <0.05). The results show that liver cancer tissue-derived dECM can significantly promote the vascular regeneration and bone regeneration of the rat calvarial defect region.

[0066] Example 4

[0067] This example shows that liver cancer tissue-derived dECM can promote the vascular regeneration and osteochondral regeneration of the rat knee osteochondral defect region.

[0068] A rat animal model with a knee osteochondral defect region is constructed, and gelatin loaded liver cancer dECM (GelMA+H) is applied at the defect site. The vascular regeneration and osteochondral regeneration of the osteochondral defect region are determined at 2 weeks and 4 weeks.

[0069] Figure 5 Vascularization tissue sections and gross observation at 2 weeks and 4 weeks when gelatin loaded liver cancer dECM is applied at the rat knee osteochondral defect are shown. The CD31 positive endothelial cell fluorescence intensity quantitative results show: liver 66.368±19.198; 134.165±20.696( P <0.05). The 4-week ICRS cartilage gross observation score results (full score 12) show: liver 5.6±0.894; liver cancer 9.4±0.548( P <0.05). The results show that liver cancer tissue-derived dECM can significantly promote the vascular regeneration and osteochondral regeneration of the rat knee osteochondral defect region compared to liver dECM (GelMA+L).

[0070] Example 5

[0071] This example shows that liver cancer tissue-derived dECM can promote the vascular regeneration and skin healing of the mouse skin defect region.

[0072] A mouse animal model with a skin defect region is constructed, and PVA loaded liver cancer dECM (PVA+H) is applied at the defect site for 10 days. The vascular regeneration and skin healing of the mouse skin defect region are determined.

[0073] Figure 6The tissue sections and healing rate of angiogenesis at 10 days after applying PVA loaded liver cancer dECM to mouse skin defects are shown. The blood vessel density results ( / mm 2 ) show: liver 103.242±11.510; liver cancer 140.987±9.288 P <0.05). The skin wound healing rate (%) results at 10 days show: liver 89.892±3.245; liver cancer 96.533±0.501 P <0.05). The results show that compared with liver dECM (PVA+L), liver cancer tissue derived dECM can significantly promote the vascular regeneration and skin healing in the mouse skin defect area.

[0074] Example 6

[0075] This example shows the safety evaluation of liver cancer decellularized extracellular matrix. After treatment with liver cancer decellularized extracellular matrix, the skin defect area is observed by HE and Masson staining, as shown in Figure 7 Figure 7 The HE and Masson tissue section results of the skin defect area after treatment with liver cancer decellularized extracellular matrix are shown. The section results show that compared with the liver decellularized extracellular matrix group, the liver cancer decellularized extracellular matrix treatment group forms an epidermal layer, a dermal layer and skin appendage structure closer to healthy tissue, accelerates the healing of skin defects, and no abnormal tumor cells are produced.

[0076] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and not to limit it; although the present application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacements for part of the technical features. 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 application.​

Claims

1. The use of liver cancer tissue-derived acellular extracellular matrix in the preparation of a medicament for skin defects and / or bone defects, characterized in that, The decellularized extracellular matrix is obtained by treating a liver cancer tissue to remove immunogenic components, and its preparation comprises the following steps: (1) obtaining a tumor tissue; (2) chopping the tumor tissue and immersing it in double-distilled water, and oscillating at 50-300 rpm at 0-10°C for 5-20 hours to obtain a tissue sample; (3) sequentially placing the tissue sample in the following buffers for continuous stirring treatment: first, reacting overnight in a low-osmotic Tris buffer containing 1-10 mM Tris and 1-10 mM EDTA at 50-300 rpm, second, washing with ddH2O at 50-300 rpm for 0.5-5 hours, then, reacting in a high-osmotic Tris buffer containing 50-80 mM Tris, 0.5-2 M NaCl and 1-20 mM EDTA at 30-40°C and 50-300 rpm for 20-30 hours, and then washing with ddH2O at 100-300 rpm for 0.5-5 hours, followed by treating with 0.5-5% Triton X-100 at 30-40°C and 50-300 rpm for 12-24 hours, washing with ddH2O at 50-300 rpm for 0.5-5 hours, treating with DNase I at 30-40°C and 50-300 rpm for 3-6 hours, and finally washing with ddH2O at 50-300 rpm overnight; (4) using supercritical carbon dioxide extraction, and the operation conditions are as follows: pressure 200-400 bar, temperature 30-40°C, ethanol as an assistant, total extraction flow rate 20-40 g / L, extraction time 1-5 hours, and after the extraction is completed, grinding the obtained decellularized extracellular matrix into powder for standby.

2. Use according to claim 1, characterized in that, The liver cancer tissue-derived decellularized extracellular matrix has a collagen network scaffold structure.

3. A pharmaceutical composition for skin defects and / or bone defects, characterized by, The liver cancer tissue-derived decellularized extracellular matrix is obtained by treating a liver cancer tissue to remove immunogenic components, and its preparation comprises the following steps: (1) obtaining a tumor tissue; (2) chopping the tumor tissue and immersing it in double-distilled water, and oscillating at 50-300 rpm at 0-10°C for 5-20 hours to obtain a tissue sample; (3) sequentially placing the tissue sample in the following buffers for continuous stirring treatment: first, reacting overnight in a low-osmotic Tris buffer containing 1-10 mM Tris and 1-10 mM EDTA at 50-300 rpm, second, washing with ddH2O at 50-300 rpm for 0.5-5 hours, then, reacting in a high-osmotic Tris buffer containing 50-80 mM Tris, 0.5-2 M NaCl and 1-20 mM EDTA at 30-40°C and 50-300 rpm for 20-30 hours, and then washing with ddH2O at 100-300 rpm for 0.5-5 hours, followed by treating with 0.5-5% Triton X-100 at 30-40°C and 50-300 rpm for 12-24 hours, washing with ddH2O at 50-300 rpm for 0.5-5 hours, treating with DNase I at 30-40°C and 50-300 rpm for 3-6 hours, and finally washing with ddH2O at 50-300 rpm overnight; (4) using supercritical carbon dioxide extraction, and the operation conditions are as follows: pressure 200-400 bar, temperature 30-40°C, ethanol as an assistant, total extraction flow rate 20-40 g / L, extraction time 1-5 hours, and after the extraction is completed, grinding the obtained decellularized extracellular matrix into powder for standby. (3) the tissue sample is sequentially placed in the following buffers for continuous stirring treatment: first, reaction in a low-osmotic Tris buffer containing 1-10 mM Tris, 1-10 mM EDTA at 50-300 rpm for overnight; second, washing with ddH2O at 50-300 rpm for 0.5-5 hours; then, reaction in a high-osmotic Tris buffer containing 50-80 mM Tris, 0.5-2 M NaCl, 1-20 mM EDTA at 30-40°C and 50-300 rpm for 20-30 hours; then, washing with ddH2O at 100-300 rpm for 0.5-5 hours; then, treatment with 0.5-5% Triton X-100 at 30-40°C and 50-300 rpm for 12-24 hours; washing with ddH2O at 50-300 rpm for 0.5-5 hours; treatment with DNase I at 30-40°C and 50-300 rpm for 3-6 hours; finally, washing with ddH2O at 50-300 rpm for overnight; (4) supercritical carbon dioxide extraction is used, and the operating conditions are: pressure 200-400 bar, temperature 30-40°C, ethanol as an auxiliary agent, total extraction flow rate 20-40 g / L, extraction time 1-5 hours; after the extraction is completed, the obtained decellularized extracellular matrix is ground into powder for standby.

4. The pharmaceutical composition according to claim 3, characterized by The amount of the liver cancer tissue-derived decellularized extracellular matrix can be administered in a therapeutically effective amount of 0.01-1000 mg / Kg.

5. The pharmaceutical composition of claim 3, wherein, The medicine further comprises a pharmaceutically acceptable carrier.

6. The pharmaceutical composition of claim 5, wherein, The pharmaceutically acceptable carrier comprises at least one of a diluent, a filler, an absorbent, a wetting agent, a binder, a lubricant, a preservative and an antioxidant.

7. The pharmaceutical composition of claim 5, wherein, The carrier is selected from at least one of collagen, gelatin, polyvinyl alcohol, hydroxyapatite.

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