CN116396929A - Composition for specifically inducing III-type collagen expression and application thereof - Google Patents
Composition for specifically inducing III-type collagen expression and application thereof Download PDFInfo
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- CN116396929A CN116396929A CN202310214451.4A CN202310214451A CN116396929A CN 116396929 A CN116396929 A CN 116396929A CN 202310214451 A CN202310214451 A CN 202310214451A CN 116396929 A CN116396929 A CN 116396929A
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- composition
- collagen
- type iii
- expression
- stem cells
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Abstract
The invention discloses a composition for specifically inducing III type collagen expression and application thereof; the main components of the composition are ammonium ions and TGF-beta factors. The composition can be used for stimulating cells to specifically induce the expression of type III collagen, namely, more type III collagen can be produced under the condition of the same amount of TGF-beta. Compared with the prior art, the invention can improve the induction efficiency of TGF-beta, thereby improving the production amount of III type collagen. It has also been found that the composition can obtain a natural, type III collagen-rich extracellular matrix and cell sheet. Can be used in the fields of burns, wounds, medical products, cosmetics, skin tissue repair, cell three-dimensional culture stent materials and the like.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a composition for specifically inducing III-type collagen expression and application thereof.
Background
Collagen is the main component of extracellular matrix, and is the most abundant and most widely distributed functional protein in mammals, and its common types are classified into type I, type ii, type iii, type iv and type V. Collagen is distributed in connective tissue such as mammal skin, cartilage, bone, ligament, viscera, blood vessel, iris, cornea, etc., and has functions of supporting organ and protecting organism. Collagen has strong biological activity and biological function, so that the connective tissue has good mechanical strength and can promote cell growth. Collagen has good biocompatibility, biodegradability and bioactivity, so that the collagen has wide application in fields such as foods, medicines, wound repair, cosmetics, tissue engineering, regenerative medicine and the like.
Type I and type III collagens are present in the dermis layer in amounts up to 90% and are the most important proteins supporting human skin tissue. Among them, type III collagen is called "infant collagen" because type III collagen is non-renewable and is contained in skin tissue up to 80% during infancy. Type III collagen is elastic and is an important protein for maintaining youthful and compact skin. The popular medical projects such as photoelectric projects, mesoderm, line carving and the like are to stimulate a great deal of regeneration of type I collagen in skin through wounds, but cannot stimulate the generation of type III collagen. Therefore, it is desired to find a low-cost method for effectively stimulating skin fibroblasts to express type III collagen, or a method for stimulating in vitro cultured cells to produce type III collagen in large quantity, and to produce type III collagen-rich extracellular matrix (ECM) in vitro, which is applied to the development of medical and aesthetic products.
At present, collagen used in the fields of medical science and tissue engineering is mostly extracted from animal tissues, however, animal-derived collagen is mostly type I collagen, has large protein sequence and three-dimensional structure, is greatly different from human bodies, has low activity, and has risks of allergy and animal-derived pollution. In recent years, with the mature application of genetic engineering and prokaryotic expression technologies, research has been initiated on the expression of human type III collagen peptide fragments by using yeast, that is, introducing a partial DNA sequence of human type III collagen into yeast by using a genetic recombination technology, so that the yeast expresses a partial amino acid polypeptide producing human type III collagen, and obtaining the human type III collagen peptide fragments by using a protein purification technology, for example, methods adopted in chinese patent nos. 202111598732.1 and 202110358113.9. However, human type III collagen obtained by recombinant protein expression methods differs significantly in sequence, structure and protein modification (including special modifications such as hydroxylation, acetylation, glycosylation, etc.) from natural type III collagen, resulting in a large difference in protein function. Therefore, how to effectively induce human skin cells or in vitro cultured cells to highly express type III collagen, so as to further promote and satisfy the pursuit of high quality in the medical industry, still requires more exploration.
TGF-beta factor is known to be the most effective protein molecule for inducing the expression of type I collagen, and it is also known to induce the expression of extracellular matrices such as type III collagen, fibronectin and laminin. However, the commercialized TGF-beta factor is expensive and it is expensive to induce type III collagen expression using TGF-beta alone.
Disclosure of Invention
The invention aims to provide a composition capable of effectively inducing III type collagen expression and application thereof.
The technical scheme adopted by the invention is as follows:
in a first aspect of the invention, there is provided a composition comprising ammonium ions and TGF- β.
In some embodiments of the invention, the ammonium ion source comprises ammonium chloride and/or ammonium sulfate.
In some embodiments of the invention, the working concentration of ammonium chloride or ammonium sulfate is 50 to 1000 μm.
In some preferred embodiments of the invention, the working concentration of ammonium chloride or ammonium sulfate is 250 to 1000 μm.
In some preferred embodiments of the invention, the working concentration of ammonium chloride or ammonium sulfate is 500 to 1000. Mu.M.
In some embodiments of the invention, the TGF- β concentration is 0.5 to 10ng/mL.
In some preferred embodiments of the invention, the TGF- β concentration is 1 to 3ng/mL.
In a second aspect of the invention there is provided a culture medium comprising a composition according to the first aspect of the invention.
In some embodiments of the invention, the basal medium in the culture medium comprises high sugar DMEM and/or alpha-MEM medium.
In some embodiments of the invention, the basal medium comprises 5-15% (v/v) fetal bovine serum.
In some embodiments of the invention, the basal medium further comprises 1% (v/v) penicillin and streptomycin.
In a third aspect of the invention there is provided the use of a composition according to the first aspect of the invention or a medium according to the second aspect of the invention in at least one of:
(a1) Promoting type III collagen expression;
(a2) Preparing a product for promoting the expression of type III collagen;
(a3) Promoting extracellular matrix expression;
(a4) Preparing a product that promotes expression of extracellular matrix;
(a5) Promoting the formation of a cell film;
(a6) A product is prepared that promotes cell film formation.
In a fourth aspect of the invention, there is provided a method comprising the step of employing a composition according to the first aspect of the invention or a medium according to the second aspect of the invention; the methods are useful for promoting the expression of type III collagen, extracellular matrix, or the formation of a cell membrane.
In some embodiments of the invention, the cells comprise mesenchymal stem cells, fibroblasts, or cells suitable for inducing production of large amounts of extracellular matrix (ECM).
In some preferred embodiments of the present invention, the mesenchymal stem cells include at least one of gingival mesenchymal stem cells, umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, amniotic fluid mesenchymal stem cells, and placental mesenchymal stem cells.
In some preferred embodiments of the invention, the mesenchymal stem cells are human mesenchymal stem cells.
In some preferred embodiments of the invention, the fibroblast is a human fibroblast.
In some embodiments of the invention, the method for promoting type III collagen comprises: inoculating the cells into a culture dish, culturing until the cell confluence is 80-100%, sucking the culture solution, and adding ammonium chloride (NH) with working concentration 4 Cl) or ammonium sulfate (NH) 4 ) 2 SO 4 And fresh cell culture solution of TGF-beta factor, and culturing for 2-7 days to obtain the induced cell with specific high expression of III type collagen.
In a fifth aspect, the invention provides a type III collagen or extracellular matrix or cell membrane prepared by the method of the fourth aspect of the invention.
In a sixth aspect, the invention provides an application of the type III collagen according to the fifth aspect in preparing skin care products, repairing materials, implants, artificial skin, medical instruments, medicines, foods, bionic tissues, medical products and scaffold materials for three-dimensional cell culture.
The skin care product can be used for skin moisture retention, scar repair, whitening and the like.
In some preferred embodiments of the present invention, the repair material includes a repair material applied to the fields of burn, wound, beauty, skin tissue repair, and the like.
The beneficial effects of the invention are as follows:
the invention provides a composition for specifically inducing III type collagen expression, the main component of the composition is ammonium chloride (NH) 4 Cl) and TGF-beta factor. The invention discovers that the cell stimulated by the composition can specifically induce the expression of the type III collagen, namely, more type III collagen can be produced under the condition of the same amount of TGF-beta. Compared with the prior art, the invention can improve the induction efficiency of TGF-beta and further improve the production amount of III type collagen. The invention also discovers that the cells induced by the composition specifically improve the expression level of the III type collagen, and can further undergo decellularization treatment to obtain natural extracellular matrix rich in the III type collagen; the cell slice rich in III type collagen can also be prepared; can be widely used in the fields of burns, wounds, medical products, cosmetics, skin tissue repair, cell three-dimensional culture stent materials and the like.
Drawings
FIG. 1 shows the effect of specifically inducing the expression of type III collagen through Western blot experiment after the composition of the invention is used for stimulating human gingival mesenchymal stem cells; wherein 50, 100, 250, 500, 1000 respectively represent NH 4 The working concentration of Cl is in micromoles per liter (μm). Col1 represents type I collagen; col3 represents type III collagen; col4 represents type IV collagenThe method comprises the steps of carrying out a first treatment on the surface of the Beta-actin represents cytoskeletal protein as an internal reference.
FIG. 2 shows the effect of inducing type III collagen expression by Western blot after stimulating human skin fibroblasts with the composition of the present invention; wherein 50, 100, 250, 500, 1000 respectively represent NH 4 The working concentration of Cl is in micromoles per liter (μm). Col3 represents type III collagen, and beta-actin represents cytoskeletal protein as an internal reference.
FIG. 3 shows the effect of specifically inducing the expression of type III collagen by Western blot after stimulating cells with the composition of the present invention, wherein the cells are cultured by mixing two cells, and H represents HaCAT: i.e., human skin keratinocyte cell lines; n represents NHDF: i.e. human skin fibroblasts; h: N represents the ratio of 1:1, mixing and culturing in proportion; col1 represents type I collagen; col3 represents type III collagen; col4 represents type IV collagen; FN stands for fibronectin, and beta-actin stands for cytoskeletal protein as an internal reference.
FIG. 4 shows the effect of stimulating extracellular matrix protein expression by immunofluorescence experiments after stimulating cells with the composition of the present invention; the cells were identical to fig. 3; anti-Col1/3/4/FN represents an antibody mixture capable of recognizing type I collagen, type III collagen, type IV collagen and fibronectin. Wherein the signals seen are all signals of N.
FIG. 5 shows that the composition of the present invention induces fibroblasts to form a cell film rich in type III collagen by a cell film-forming experiment after culturing human skin fibroblasts for 7 days using a culture solution containing the composition of the present invention.
FIG. 6 shows the effect of specifically inducing the expression of type III collagen through Western blot experiment after the composition of the invention is used for stimulating the mesenchymal stem cells of the gum; wherein 50, 100, 250, 500, 1000 respectively represent (NH) 4 ) 2 SO 4 In micromoles per liter (μm). Col3 represents type III collagen, and beta-actin represents cytoskeletal protein as an internal reference.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
Extracellular matrix (Extracellular Matrix, ECM): refers to a complex network of multiple macromolecules surrounding cells in multicellular organisms, wherein Collagen (Collagen) is the major component of the extracellular matrix, and is spread throughout organs and tissues.
Transforming growth factor beta ((Transforming Growth Factor beta, TGF-beta): is a secreted protein belonging to the transforming growth factor-beta family, transforming growth factor beta-1 is expressed in large amounts in bone, articular cartilage and chondrocytes, and its expression is increased in Osteoarthritis (OA). Transforming growth factor beta-1 has various cell functions including regulation of cell growth, cell proliferation, cell differentiation and apoptosis. The basal medium used in the examples of the present invention is commercial products, alpha-MEM (cat# 10-022-CV, corning Co.) and DMEM (C11995500 BT, gibco Co.).
Example 1
A culture solution: comprising ammonium chloride (NH) 4 Cl) and TGF- β, wherein the basal medium is an alpha-MEM medium or DMEM high sugar medium comprising 10% fetal bovine serum, 1% penicillin.
Wherein ammonium chloride (NH) 4 Cl) was 50. Mu.M, 100. Mu.M, 250. Mu.M, 500. Mu.M, 1000. Mu.M, and TGF-. Beta.was 3ng/mL in the culture medium.
The preparation method of the culture solution comprises the following steps:
1.1 preparation of ammonium chloride (NH) at 250mM concentration 4 Cl) initial solution: taking 133.7mg of ammonium chloride solid, adding 10mL of double distilled water or PBS solvent, sufficiently shaking for dissolution, filtering and sterilizing by a 0.22 mu m sterile filter membrane to obtain an initial concentration solution with the concentration of 250mM, and storing at the temperature of 4 ℃;
1.2 preparation of TGF-beta initial solution at 10. Mu.g/mL concentration: dissolving 100 mug of TGF-beta protein powder with 10mL of 10mM citric acid solution to obtain 10 mug/mL initial concentration solution, subpackaging, and storing at-80 ℃;
1.3 preparing working concentration culture solution: respectively taking corresponding ammonium chloride (NH) 4 Cl) initial solution and the TGF-beta initial solution are added into alpha-MEM culture medium or DMEM high-sugar culture medium containing 10% of fetal calf serum and 1% of green streptomycin to obtain the culture solution with corresponding working concentration.
Example 2
A culture medium comprises ammonium sulfate (NH) 4 ) 2 SO 4 And a basal medium of TGF-beta, wherein the basal medium is an alpha-MEM medium or a DMEM high-sugar medium comprising 10% fetal bovine serum, 1% penicillin.
Wherein ammonium sulfate (NH) 4 ) 2 SO 4 The concentration of TGF-. Beta.in the culture medium was 50. Mu.M, 100. Mu.M, 250. Mu.M, 500. Mu.M, 1000. Mu.M, and 3ng/mL.
The preparation method of the culture solution comprises the following steps:
1.1 preparation of ammonium sulfate (NH) at 250mM concentration 4 ) 2 SO 4 Initial solution: 330.3mg of ammonium sulfate (NH) 4 ) 2 SO 4 Adding 10mL of double distilled water or PBS solvent into the solid, sufficiently oscillating and dissolving, filtering and sterilizing by a 0.22 mu m sterile filter membrane to obtain an initial concentration solution with the concentration of 250mM, and storing at the temperature of 4 ℃;
1.2 preparation of TGF-beta initial solution at 10. Mu.g/mL concentration: dissolving 100 mug of TGF-beta protein powder with 10mL of 10mM citric acid solution to obtain 10 mug/mL initial concentration solution, subpackaging, and storing at-80 ℃;
1.3 preparing working concentration culture solution: respectively taking corresponding ammonium sulfate (NH) 4 ) 2 SO 4 The initial solution and the TGF-beta initial solution are added into alpha-MEM culture medium or DMEM high-sugar culture medium containing 10% of fetal calf serum and 1% of green streptomycin to obtain the culture solution with corresponding working concentration.
Comparative example 1
A culture broth comprising 10% fetal bovine serum, 1% green streptomycin alpha-MEM medium or DMEM high sugar medium.
Comparative example 2
A culture medium comprises ammonium chloride (NH) 4 Cl), wherein the basal medium is an alpha-MEM medium or DMEM high sugar medium comprising 10% fetal bovine serum, 1% penicillin.
Wherein ammonium chloride (NH) 4 Cl) was 50. Mu.M, 100. Mu.M, 250. Mu.M, 500. Mu.M, 1000. Mu.M in the culture broth.
The preparation method of the culture solution comprises the following steps:
2.1 preparation of ammonium chloride (NH) at 250mM concentration 4 Cl) initial solution: taking 133.7mg of ammonium chloride solid, adding 10mL of double distilled water or PBS solvent, sufficiently shaking for dissolution, filtering and sterilizing by a 0.22 mu m sterile filter membrane to obtain an initial concentration solution with the concentration of 250mM, and storing at the temperature of 4 ℃;
2.2 preparing working concentration culture solution: respectively taking corresponding ammonium chloride (NH) 4 Cl) initial solution, and adding the initial solution to an alpha-MEM medium or DMEM high sugar medium containing 10% fetal bovine serum and 1% penicillin streptomycin to obtain the culture solution with the corresponding working concentration.
Comparative example 3
A culture broth comprising a basal medium of TGF- β, wherein the basal medium is an alpha-MEM medium or DMEM high sugar medium comprising 10% fetal bovine serum, 1% penicillin. The concentration of TGF-beta in the culture broth was 3ng/mL.
The preparation method of the culture solution comprises the following steps:
2.1 preparation of TGF-beta initial solution at 10. Mu.g/mL concentration: dissolving 100 mug of TGF-beta protein powder with 10mL of 10mM citric acid solution to obtain 10 mug/mL initial concentration solution;
2.2 preparing 50mL of the above culture solution: 15.1. Mu.L of the TGF-beta initial solution was added to an alpha-MEM medium or DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin, to obtain a culture broth.
Comparative example 4
A specific formulation of a culture solution can be found in example 2 of patent document CN115369079A, which is a culture solution that has been confirmed to induce fibroblasts to form a cell film.
Comparative example 5
A culture medium comprises ammonium sulfate (NH) 4 ) 2 SO 4 Wherein the basal medium is an alpha-MEM medium or a DMEM high-sugar medium containing 10% fetal bovine serum, 1% penicillin. Wherein ammonium sulfate (NH) 4 ) 2 SO 4 The concentration in the culture medium was 50. Mu.M, 100. Mu.M, 250. Mu.M, 500. Mu.M, 1000. Mu.M.
The preparation method of the culture solution comprises the following steps:
2.1 preparation of ammonium sulfate (NH) at 250mM concentration 4 ) 2 SO 4 Initial solution: 330.3mg of ammonium sulfate (NH) 4 ) 2 SO 4 Adding 10mL of double distilled water or PBS solvent into the solid, sufficiently oscillating and dissolving, filtering and sterilizing by a 0.22 mu m sterile filter membrane to obtain an initial concentration solution with the concentration of 250mM, and storing at the temperature of 4 ℃;
2.2 preparing working concentration culture solution: respectively taking corresponding ammonium sulfate (NH) 4 ) 2 SO 4 The initial solution was added to an alpha-MEM medium or DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin, to obtain the above culture solution at the corresponding working concentration.
Effect example 1
1) Inducing mesenchymal stem cells to express type III collagen by using the culture solutions of comparative example 1, comparative example 2, comparative example 3 and example 1, wherein the basal medium used for the mesenchymal stem cells is alpha-MEM:
gingiva mesenchymal stem cells were inoculated into 24-well culture plates, after stable culture for 24 hours, the culture supernatants were aspirated, and culture solutions described in comparative example 1, comparative example 2, comparative example 3 and example 1 were added respectively for further culture for 3 days, and then extracellular matrix protein expression was analyzed by total cell protein extraction and Western blot experiments, and the results are shown in fig. 1: it can be seen that the culture solution in example 1 can significantly and specifically increase the expression level of type III collagen compared to comparative example 1, comparative example 2 and comparative example 3, and in particular, the induction of expression of type III collagen is most remarkable in example 1 at the concentrations of 250 μm, 500 μm and 1000 μm of ammonium chloride.
2) The culture solutions in comparative example 1, comparative example 2, comparative example 3 and example 1 were used to induce human skin fibroblasts to express type III collagen, and the basal medium used for the fibroblasts was high-sugar DMEM:
human skin fibroblasts were inoculated into 24-well plates, after stable culture for 24 hours, the culture supernatants were aspirated, and culture solutions in comparative example 1, comparative example 2, comparative example 3 and example 1 were added, respectively, and further culture was continued for 3 days, and then extracellular matrix protein expression was analyzed by total cell protein extraction and Western blot experiments, and the results are shown in fig. 2: example 1 significantly increased the expression level of type III collagen compared to comparative examples 1, 2 and 3, and in particular, three concentrations of 250 μm, 500 μm and 1000 μm in example 1 were evident in inducing expression of type III collagen.
Effect example 2
1) Human skin fibroblasts were induced to express type III collagen using the culture solutions of comparative example 1, comparative example 2, comparative example 3 and example 1 with an ammonium chloride concentration of 250. Mu.M:
human skin keratinocytes (H) and human skin fibroblasts (N) were individually seeded, or at 1:1 were inoculated in 24-well plates, after stable culture for 24 hours, the culture supernatant was aspirated, and the culture solutions in comparative example 1, comparative example 2, comparative example 3 and example 1 were added for further culture for 3 days, and then extracellular matrix protein expression was analyzed by total cell protein extraction and Western blot experiments, and the results are shown in fig. 3: the culture solution in example 1 was able to significantly and specifically increase the expression level of type III collagen in human skin fibroblasts without inducing the expression levels of type I collagen, type III collagen, type IV collagen and fibronectin, compared to comparative example 1, comparative example 2 and comparative example 3.
2) Comparative example 1, comparative example 2, comparative example 3 and example 1 the culture medium with an ammonium chloride concentration of 250 μm promotes the extracellular matrix protein content of human skin fibroblasts:
human skin keratinocytes (H) and human skin fibroblasts (N) were individually seeded, or at 1:1 were inoculated in 24-well plates, after stable culture for 24 hours, the culture supernatants were aspirated, and the culture solutions of comparative example 1, comparative example 2, comparative example 3 and example 1 were added for further culture for 3 days, and extracellular matrix protein expression was analyzed by immunofluorescence experiments, and as a result, as shown in FIG. 4, the culture solution of example 1 showed a strong positive signal for extracellular matrix protein in H: N mixed cells compared to those of comparative example 1, comparative example 2 and comparative example 3. Example 1 was shown to have a greater extracellular matrix-promoting capacity.
3) Comparative example 4 and example 1 with a 250 μm ammonium chloride concentration, the human skin fibroblasts were promoted to form a cell film:
human fibroblasts were cultured in the culture solution of example 1 or comparative example 4 for 7 days, and then the cells were taken out, an appropriate amount of the culture medium was sucked with a pipette, the bottom edge of the dish was gently blown, and the cell film was obtained by peeling, as shown in fig. 5: the culture solutions of example 1 and comparative example 4 both induced cell film formation, wherein the cell film obtained in example 1 was intact, larger in volume and thinner in thickness.
Effect example 3
1) Comparative example 1, comparative example 3, comparative example 5 and example 2 culture solutions induce mesenchymal stem cells to express type III collagen:
gingiva mesenchymal stem cells were inoculated into 24-well culture plates, after stable culture for 24 hours, the culture supernatants were aspirated, and culture solutions in comparative example 1, comparative example 3, comparative example 5 and example 2 were added, respectively, and further culture was continued for 3 days, and then extracellular matrix protein expression was analyzed by total cell protein extraction and Western blot experiments, and the results are shown in fig. 6: it can be seen that the culture solution of example 2 was able to weakly induce the expression of type III collagen, compared to comparative example 1, comparative example 3 and comparative example 5, wherein the concentration of 1000 μm ammonium sulfate of example 2 was most significant for the induction of the expression of type III collagen.
In conclusion, the culture medium composition provided by the embodiment of the invention can obviously and specifically improve the expression level of the type III collagen in cells such as mesenchymal stem cells, fibroblasts and the like.
The present invention has been described in detail in the above embodiments, but the present invention is not limited to the above examples, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. A composition comprising an ammonium ion and TGF- β.
2. The composition of claim 1, wherein the source of ammonium ions comprises ammonium chloride and/or ammonium sulfate.
3. The composition according to claim 2, wherein the working concentration of ammonium chloride or ammonium sulphate is 50 to 1000 μΜ;
preferably, the working concentration of TGF-beta is 0.5-10 ng/mL.
4. A culture medium comprising the composition of any one of claims 1-3.
5. The medium according to claim 4, wherein the basal medium in the medium is a high sugar DMEM and/or alpha-MEM medium.
6. Use of a composition according to any one of claims 1 to 3 or a medium according to any one of claims 4 to 5 in at least one of the following:
(a1) Promoting type III collagen expression;
(a2) Preparing a product for promoting the expression of type III collagen;
(a3) Promoting extracellular matrix expression;
(a4) Preparing a product that promotes expression of extracellular matrix;
(a5) Promoting the formation of a cell film;
(a6) A product is prepared that promotes cell film formation.
7. A method for promoting expression of type III collagen, extracellular matrix or cell film formation, comprising the step of employing the composition of any one of claims 1 to 3 or the culture medium of any one of claims 4 to 5.
8. The method of claim 7, wherein the cells comprise mesenchymal stem cells, skin fibroblasts; preferably, the mesenchymal stem cells include at least one of gingival mesenchymal stem cells, umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, amniotic fluid mesenchymal stem cells, and placenta mesenchymal stem cells.
9. A type III collagen or extracellular matrix or cell membrane prepared by the method of claim 7 or 8.
10. Use of collagen type III or extracellular matrix or cell membrane according to claim 9 for the preparation of skin care products, repair materials, implants, artificial skin, medical devices, pharmaceutical products, food products, biomimetic tissues.
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