CN115212356A - Compound for promoting regeneration of mesoderm-derived cell tissues, preparation method and application - Google Patents
Compound for promoting regeneration of mesoderm-derived cell tissues, preparation method and application Download PDFInfo
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- CN115212356A CN115212356A CN202210807379.1A CN202210807379A CN115212356A CN 115212356 A CN115212356 A CN 115212356A CN 202210807379 A CN202210807379 A CN 202210807379A CN 115212356 A CN115212356 A CN 115212356A
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Abstract
The invention discloses a compound for promoting regeneration of mesoderm-derived cell tissues, a preparation method and application thereof. The complex comprises mesenchymal stem cell factor, mesenchymal stem cell exosome, amino acid and albumin. The compound for promoting the regeneration of mesoderm-derived cell tissues can promote cell tissues differentiated from mesoderm to enter a cell division cycle and increase the synthesis effect of cell matrixes, and can be applied to the regeneration and repair of skin, fat, bone, cartilage, muscle and tendon. Through tests, the compound can lighten or even eliminate wrinkles in 4-5 weeks, can also promote skin tissue regeneration, and is suitable for removing wrinkles and activating and filling skin tissues.
Description
Technical Field
The invention belongs to the technical field of mesenchymal stem cells, and particularly relates to a compound for promoting regeneration of mesoderm-derived cell tissues, and a preparation method and application thereof.
Background
Mesenchymal Stem Cells (MSCs) are a class of adult stem cells derived from mesoderm, have self-renewal and multipotentiality, and can differentiate into various mesenchymal tissues, such as bone, cartilage, fat, bone marrow hematopoietic tissues, etc. Recent studies have shown that most of the roles of MSCs are through paracrine, with soluble protein secretion and exosomes being the most interesting. More and more have proposed the concept of using various factors and exosomes secreted by MSCs to replace MSCs cell therapy. The combination of the mesenchymal stem cell factor with different concentration ratios, the exosome and the amino acid can promote the growth of different germ layer cells, and the cell entering and exiting cell cycle time is different. However, the common combination of the ingredients has not been reported so far.
Disclosure of Invention
The invention mainly aims to provide a compound for promoting regeneration of mesoderm-derived cell tissues so as to overcome the defects in the prior art.
The invention also aims to provide a preparation method of the compound for promoting the regeneration of mesoderm-derived cell tissues.
The invention also aims to provide application of the compound for promoting regeneration of mesoderm-derived cell tissues.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
the embodiment of the invention provides a compound for promoting regeneration of mesoderm-derived cell tissues, which comprises a mesenchymal stem cell factor, a mesenchymal stem cell exosome, amino acids and albumin, and the compound is used for non-disease diagnosis and treatment purposes.
Further, the complex has a function of promoting cell tissues differentiated from mesoderm to enter a cell division cycle and increasing cell matrix synthesis.
Further, the complex has a function of promoting the regenerative repair of skin, fat, bone, cartilage, muscle or tendon.
The embodiment of the invention also provides a preparation method of the compound for promoting regeneration of mesoderm-derived cell tissues, which comprises the following steps:
mixing the mesenchymal stem cell factor, the mesenchymal stem cell exosome, the amino acid and the albumin to form the compound for promoting the regeneration of the mesoderm-derived cell tissue.
The embodiment of the invention also provides a functional product for non-disease diagnosis and treatment purposes, which comprises the compound for promoting the regeneration of mesoderm-derived cell tissues.
Compared with the prior art, the invention has the beneficial effects that at least:
the compound for promoting the regeneration of mesoderm-derived cell tissues can promote cell tissues differentiated from mesoderm to enter a cell division cycle and increase the synthesis effect of cell matrixes, and can be applied to the regeneration and repair of skin, fat, bone, cartilage, muscle and tendon. Through tests, the compound can lighten or even eliminate wrinkles in 4-5 weeks, can also promote skin tissue regeneration, and is suitable for removing wrinkles and activating and filling skin tissues.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the description of the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present invention, and it is also possible for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 is a schematic representation of the connection of a sterile bag to an inlet on the inner membrane of an aperture membrane-type plasma component separator in an exemplary embodiment of the invention;
FIG. 2 is a schematic representation of the connection of a sterile bag to an inlet on the outer membrane of an aperture membrane type plasma component separator in an exemplary embodiment of the invention;
FIG. 3 is a graph showing the result of experiment after injecting the compound for promoting regeneration of mesoderm-derived cell tissue in an exemplary embodiment of the present invention into the periocular wrinkles of volunteers;
FIG. 4 is a graph showing the results of experiments in which a composition for promoting regeneration of a mesoderm-derived cell tissue was subcutaneously injected into the temple of a human volunteer according to an exemplary embodiment of the present invention;
FIG. 5 is a graph showing the results of experiments on cervical striations before and after injection of a composition for promoting regeneration of mesoderm-derived cell tissue in an exemplary embodiment of the present invention;
fig. 6 is a graph of experimental results of cervical striations before and after injection of the complex in the absence of a mesenchymal stem cell factor concentrate in a control experiment;
fig. 7 is a graph of experimental results of cervical striations before and after injection of the complex in the absence of mesenchymal stem cell exosome concentrate in control experiments.
Detailed Description
In view of the deficiencies in the prior art, the inventors of the present invention have made extensive studies and practice to provide a technical solution of the present invention, which mainly aims at the growth regulation of mesoderm differentiated cells, and find that mesoderm differentiated cells can enter the cell division cycle by using a proper concentration range. The technical solution, its implementation and principles, etc. will be further explained as follows.
In one aspect of the embodiments of the present invention, the compound for promoting regeneration of mesoderm-derived cell tissue provided by the present invention comprises a certain concentration ratio of mesenchymal stem cell factor, mesenchymal stem cell exosome (also called extracellular membrane vesicle, outer vesicle, EVS, diameter of 30-150 nm), amino acid and albumin, and is for non-disease diagnosis and treatment purposes.
In some preferred embodiments, the compound for promoting regeneration of mesoderm-derived cell tissue comprises a mesenchymal stem cell factor concentrate, a mesenchymal stem cell exosome concentrate, an amino acid and human serum albumin.
In some preferred embodiments, the volume ratio of the mesenchymal stem cell factor concentrated solution, the mesenchymal stem cell exosome concentrated solution and the human albumin is (1-1.5) to (0.5-0.8) to (1.5-1.8) to (0.01-0.02).
The compound for promoting the regeneration of mesoderm-derived cell tissues provided by the invention is a concentrated solution of mesenchymal stem cell factors, a concentrated solution of mesenchymal stem cell exosomes, and the volume ratio of amino acid to human albumin is (1-1.5): (0.5-0.8): (1.5-1.8): (0.01-0.02); the mesenchymal stem cell factor concentrated solution contains various active ingredients such as various cell factors, growth factors, extracellular matrix proteins, tissue remodeling enzymes and the like, has the functions of chemotactic performance, immunoregulation, angiogenesis promotion, cell growth support, anti-apoptosis, stem cell activity maintenance and the like, and can promote the proliferation and/or migration of endothelial cells, keratinocytes and fibroblasts. Cytokines such as tumor necrosis factor alpha, basic fibroblast growth factor 2, transforming growth factor beta, interleukin 1, interleukin 6, etc., play an important role in collagen synthesis and angiogenesis.
The mesenchymal stem cell exosome concentrated solution is rich in exosome vesicles, and exosomes contain multiple miRNAs. Bioinformatics analysis shows that the miRNAs may participate in 161 biological processes including cell repair and immunity, aging resistance and the like. Wherein hsa-miR-181c-3p and hsa-miR-181c-5p have the function of relieving inflammatory reaction, and hsa-miR-125a-3p has the function of relieving scar tissue formation.
On one hand, the components for promoting cell regeneration and repair of the mesenchymal stem cell factor concentrated solution and the mesenchymal stem cell exosome concentrated solution can promote cell proliferation and extracellular matrix synthesis of mesoderm differentiation, such as proliferation of fibroblasts and synthesis of collagen; on the other hand, multiple miRNAs contained in the mesenchymal stem cell exosomes can reduce scar tissue formation in the tissue regeneration process, so that the tissue repair tendency is natural.
Further, the amino acids are derived from clinical grade MEM medium and comprise 20 amino acids, i.e., glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, and the like.
Further, the factor concentration multiple in the mesenchymal stem cell factor concentrated solution is 10-15 times of that in a conditioned medium (a medium when cell culture is completed).
Further, the concentration multiple of the exosomes in the mesenchymal stem cell exosome concentrated solution is 3.5-5 times of that in the conditioned medium.
Further, the concentration of human blood albumin in the complex is 0.25-0.5 wt%.
Further, the complex has the functions of promoting cell tissues differentiated from mesoderm to enter a cell division cycle and increasing cell matrix synthesis.
Further, the complex has a function of promoting the regenerative repair of skin, fat, bone, cartilage, muscle or tendon.
As another aspect of the embodiments of the present invention, the present invention also provides a method for preparing a complex for promoting regeneration of mesoderm-derived cell tissue, comprising:
mixing the mesenchymal stem cell factor, the mesenchymal stem cell exosome, the amino acid and the albumin to form the compound for promoting the regeneration of the mesoderm-derived cell tissue.
In some embodiments, the method of making comprises:
providing a mesenchymal stem cell supernatant;
connecting a first aperture membrane type separation unit, a second aperture membrane type separation unit and a third aperture membrane type separation unit in series to form a serial graded membrane type separator, wherein the membrane aperture of the first aperture membrane type separation unit, the membrane aperture of the second aperture membrane type separation unit and the membrane aperture of the third aperture membrane type separation unit are sequentially decreased progressively;
and enabling the mesenchymal stem cell supernatant to sequentially pass through the first aperture membrane type separation unit, the second aperture membrane type separation unit and the third aperture membrane type separation unit to respectively obtain the mesenchymal stem cell factor and the mesenchymal stem cell exosome.
Further, the membrane aperture of the first aperture membrane type separation unit is 150 to 180nm, the membrane aperture of the second aperture membrane type separation unit is 20 to 30nm, and the membrane aperture of the third aperture membrane type separation unit is 5 to 10nm.
Further, the adopted series connection mode is as follows: the lower outlet of the outer membrane of the first aperture membrane type separation unit is connected with the upper inlet of the inner membrane of the second aperture membrane type separation unit, and the lower outlet of the outer membrane of the second aperture membrane type separation unit is connected with the upper inlet of the inner membrane of the third aperture membrane type separation unit. Alternatively, the lower inner membrane outlet of the first aperture membrane type separation unit may be connected to the upper outer membrane inlet of the second aperture membrane type separation unit, and the lower inner membrane outlet of the second aperture membrane type separation unit may be connected to the upper outer membrane inlet of the third aperture membrane type separation unit.
In some embodiments, the method for separating and preparing the mesenchymal stem cell factor and the mesenchymal stem cell exosome is a serial graded membrane separator separation method, and the respective concentration ratios of the mesenchymal stem cell factor, the mesenchymal stem cell exosome, the clinical grade MEM culture medium and the human serum albumin are separated by the method. Namely specifically comprising: separating 2L of mesenchymal stem cell supernatant through a serial graded membrane type separator, and respectively collecting 100-120 mL of exosome concentrated solution and 50-70 mL of mesenchymal stem cell factor concentrated solution. The two concentrates were combined with clinical grade MEM medium, human serum albumin (20% concentration specification). The volume concentration ratio of the components is mesenchymal stem cell factor concentrated solution: exosome concentrate: the clinical grade MEM culture medium and the human serum albumin are (1-1.5): (0.5-0.8): (1.5-1.8): (0.01-0.02).
Accordingly, another aspect of the embodiments of the present invention also provides a functional product for non-disease diagnosis and treatment purposes, comprising the aforementioned complex for promoting regeneration of mesoderm-derived cellular tissue.
Furthermore, the functional product has the functions of promoting cell tissues differentiated from mesoderm to enter a cell division cycle and increasing cell matrix synthesis.
Further, the functional product has a function of promoting the regeneration and repair of skin, fat, bone, cartilage, muscle or tendon.
In conclusion, by the technical scheme, the compound can promote cell tissues obtained by mesoderm differentiation to enter a cell division cycle, increases the function of cell matrix synthesis, and can be applied to regeneration and repair of skin, fat, bone, cartilage, muscle and tendon. Through tests, the compound can lighten or even eliminate wrinkles in 4-5 weeks, can also promote skin tissue regeneration, and is suitable for removing wrinkles and activating and filling skin tissues.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention are further described in detail below with reference to the accompanying drawings and several preferred embodiments. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the manufacturers. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example 1
The invention mainly aims at the growth regulation of the cells differentiated from the mesoderm, and the appropriate concentration range can ensure that the cells differentiated from the mesoderm enter a cell division cycle, exit the cell division cycle in 3-5 weeks and return to G 0 The specific operation steps are as follows:
1. culturing the human mesenchymal stem cells under the conditions of no serum and no double antibody.
2. The culture supernatant was collected into a sterile centrifuge tube and accumulated to 2L. The cell debris was removed by centrifugation at 1200g for 5 min.
3. The supernatant from which the cell debris was removed was filtered through a bacteria filter and transferred to a sterile bag.
4. From top to bottom, the membrane type plasma component separators with the membrane aperture ranges of 150-180 nm, 20-30 nm and 5-10 nm are connected in series to form a series membrane type separator unit. As shown in FIG. 1, the series connection mode can be that the lower outer membrane outlet of the first aperture membrane type separation unit with the aperture of 150-180 nm is connected with the upper inner membrane inlet of the second aperture membrane type separation unit with the aperture of 20-30 nm; the lower outlet of the outer membrane of the second aperture membrane type separation unit with the aperture of 20-30 nm is connected with the upper inlet of the inner membrane of the third aperture membrane type separation unit with the aperture of 5-10 nm. Alternatively, as shown in FIG. 2, the series connection mode may be that the lower inner membrane outlet of the first pore diameter membrane type separation unit with the pore diameter of 150-180 nm is connected with the upper outer membrane inlet of the second pore diameter membrane type separation unit with the pore diameter of 20-30 nm; the lower outlet of the inner membrane of the second aperture membrane type separation unit with the aperture of 20-30 nm is connected with the upper inlet of the outer membrane of the third aperture membrane type separation unit with the aperture of 5-10 nm.
5. If the series unit in figure 1 is connected, the sterile bag is connected with an upper inlet of an inner membrane of a first aperture membrane type separation unit with the aperture of 150-180 nm; if the series unit of fig. 2 is connected, the sterile bag is connected to the inlet on the outer membrane of the 150-180 nm first-pore membrane-type separation unit.
The supernatant is passed under gravity through a first pore size membrane type separation unit having a pore size of 150-180 nm, where particles >150nm are removed, i.e. where residual cell debris is removed. The supernatant fluid flows into a second aperture membrane type separation unit with the aperture of 20-30 nm, and particles with the particle size of 20-30 nm are intercepted in a separator by the separation unit, namely the mesenchymal stem cell exosome is obtained in the second aperture membrane type separation unit with the aperture of 20-30 nm. The supernatant fluid continuously flows into a third aperture membrane type separation unit with the aperture of 5-10 nm, particles with the particle size of 5-20 nm are intercepted in the separation unit, namely, the mesenchymal stem cell factor and polypeptide chain are separated, and finally the residual supernatant fluid flows out from the lower outlet of a second aperture membrane type separation unit with the aperture of 20-30 nm until the residual supernatant fluid is almost clean.
6. And (3) flushing the membrane type separator by using physiological saline with different volumes to obtain the mesenchymal stem cell factors and the mesenchymal stem cell exosomes with various concentrations.
7. Combining the mesenchymal stem cell factor, the mesenchymal stem cell exosome and clinical-grade MEM culture medium and human serum albumin according to a certain concentration proportion to form a regenerated compound. Through experimental tests, 100-120 mL of physiological mediumFlushing a second aperture membrane type separation unit with the aperture of 20-30 nm by using saline, and collecting 100-120 mL of mesenchymal stem cell exosome concentrated solution; flushing the third aperture membrane type separation unit with the aperture of 5-10 nm by 50-70 mL of physiological saline, and collecting 50-70 mL of mesenchymal stem cell factor concentrated solution most suitably; the two concentrates were combined with clinical grade MEM medium, human serum albumin (20% concentration specification). The volume concentration ratio is mesenchymal stem cell factor concentrated solution: mesenchymal stem cell exosome concentrated solution: the clinical grade MEM culture medium and the human serum albumin are equal to 1-1.5: 0.5-0.8: 1.5-1.8: 0.01-0.02. The compound with the concentration ratio can perform growth regulation on cells differentiated from mesoderm, and the compound with the concentration ratio can make the cells differentiated from mesoderm enter a cell division cycle and exit the cell division cycle at the later 3-5 weeks and return to G 0 And (4) period. The composition can lighten or even disappear wrinkles after 4-5 weeks by volunteer tests, can also promote skin tissue regeneration, and is suitable for removing wrinkles and activating and filling skin tissues.
The test results are as follows:
please refer to fig. 3, the injection of wrinkles around eyes of the volunteer was performed 3 times, and once in 3 weeks, the wrinkles were significantly reduced, and the effect was satisfactory. The depression of temple was significantly improved and the effect was satisfactory 3 times, 2 to 3 weeks after the temples were subcutaneously injected, as shown in fig. 4.
Fig. 5 shows that the neck striae basically disappear after 3 times of injection and once in 3 weeks, and the effect is obvious.
In addition, the inventors also performed a control experiment in the absence of the concentrated solution of mesenchymal stem cell factor or the concentrated solution of mesenchymal stem cell exosome, and tested the effect of wrinkle removal, and the results are shown in fig. 6 and 7. The results in fig. 6 show that the wrinkle-removing ability is decreased in the absence of the mesenchymal stem cell factor concentrate. The results of fig. 7 show that the wrinkle-removing ability is decreased and the skin growth is not uniform in the absence of the mesenchymal stem cell exosome concentrate.
While the invention has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions and/or additions may be made and substantial equivalents may be substituted for elements thereof without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims.
Claims (10)
1. A compound for promoting regeneration of mesoderm-derived cell tissues, which comprises mesenchymal stem cell factor, mesenchymal stem cell exosome, amino acid and albumin, and is used for non-disease diagnosis and treatment purposes.
2. The compound for promoting regeneration of mesoderm-derived cell tissues according to claim 1, which comprises a mesenchymal stem cell factor concentrated solution, a mesenchymal stem cell exosome concentrated solution, an amino acid and human serum albumin.
3. The compound for promoting regeneration of mesoderm-derived cellular tissue according to claim 2, wherein: the volume ratio of the mesenchymal stem cell factor concentrated solution to the mesenchymal stem cell exosome concentrated solution to the amino acid to the human serum albumin is (1-1.5) to (0.5-0.8) to (1.5-1.8) to (0.01-0.02);
and/or the concentration of human serum albumin in the compound is 0.25-0.5 wt%.
4. A complex for promoting regeneration of a cell tissue of mesoderm origin according to any one of claims 1 to 3, wherein: the compound has the functions of promoting cell tissues differentiated from mesoderm to enter a cell division cycle and increasing cell matrix synthesis.
5. A complex for promoting regeneration of a cell tissue of mesoderm origin according to any one of claims 1 to 3, wherein: the compound has the function of promoting the regeneration and repair of skin, fat, bone, cartilage, muscle or tendon.
6. A method for preparing a composition for promoting regeneration of a cell tissue of mesoderm origin according to any one of claims 1 to 5, comprising:
mixing the mesenchymal stem cell factor, the mesenchymal stem cell exosome, the amino acid and the albumin to form the compound for promoting the regeneration of the mesoderm-derived cell tissue.
7. The method of claim 6, comprising:
providing a mesenchymal stem cell supernatant;
connecting a first aperture membrane type separation unit, a second aperture membrane type separation unit and a third aperture membrane type separation unit in series to form a series connection grading membrane type separator, wherein the membrane apertures of the first aperture membrane type separation unit, the second aperture membrane type separation unit and the third aperture membrane type separation unit are sequentially reduced;
and enabling the mesenchymal stem cell supernatant to sequentially pass through the first aperture membrane type separation unit, the second aperture membrane type separation unit and the third aperture membrane type separation unit to respectively obtain the mesenchymal stem cell factor and the mesenchymal stem cell exosome.
8. The method for producing according to claim 7, characterized in that: the membrane aperture of the first aperture membrane type separation unit is 150-180 nm, the membrane aperture of the second aperture membrane type separation unit is 20-30 nm, and the membrane aperture of the third aperture membrane type separation unit is 5-10 nm.
9. A functional product for non-disease diagnostic and therapeutic purposes comprising a complex for promoting regeneration of mesoderm-derived cellular tissue according to any one of claims 1 to 5.
10. The functional product according to claim 9, characterized in that: the functional product has the functions of promoting cell tissues differentiated from mesoderm to enter a cell division cycle and increasing cell matrix synthesis; and/or, the functional product has the function of promoting the regeneration and repair of skin, fat, bone, cartilage, muscle or tendon.
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Application publication date: 20221021 |