CN114350603B - Mesenchymal stem cell extracellular matrix containing exosome, preparation method thereof and application thereof in cell repair - Google Patents
Mesenchymal stem cell extracellular matrix containing exosome, preparation method thereof and application thereof in cell repair Download PDFInfo
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Abstract
The invention relates to a mesenchymal stem cell conditioned medium, and preparation and application thereof, in particular to a human umbilical cord mesenchymal stem cell extracellular matrix containing exosome, and preparation and application thereof in repairing corneal epithelial cells. The preparation is culture supernatant produced by culturing human umbilical cord mesenchymal stem cells in a basal medium containing low-concentration serum under the condition of low oxygen content. According to the invention, the culture supernatant obtained by culturing the mesenchymal stem cells under the in vitro simulated hypoxia condition and controlling the serum concentration shows the capability of remarkably promoting the injury repair of corneal epithelial cells, and compared with the culture supernatant obtained under the normoxic condition, the culture supernatant has high expression of PDGF and IV collagen.
Description
Technical Field
The invention relates to a mesenchymal stem cell conditioned medium, and preparation and application thereof, in particular to a human umbilical cord mesenchymal stem cell extracellular matrix containing exosomes, and preparation and application thereof in repairing corneal epithelial cells.
Background
The mesenchymal stem cells can secrete various cytokines, such as immune factors (HGF, LIF), chemotactic factors (RANTES, SDF-1 alpha, fractalkine, MIP-1 alpha, MCP-1, MCP-2), vascular regeneration factors (VEGF165, FGF-2, PDGF-AA, PDGF, EGF), scar inhibition factors (HGF, FGF-2), various types of collagen (collagen type I, II, III, IV) and various lysozyme, and the like. These cytokines can be involved in the treatment and repair of a variety of diseases.
The corneal epithelium is a physical barrier against the invasion of external pathogenic agents, and the maintenance of its integrity depends on the intercellular, tight and anchored junctions between cells and basement membrane and the constant self-renewal of epithelial cells. The damage of the corneal epithelial layer can affect the connection between cells, cause the change of the permeability and selectivity of cell membranes, thereby affecting the barrier function of the cell membranes, and lead the cornea to be easily attacked by external pathogenic factors to cause inflammation, cause corneal opacity and even cause blindness. The clinical treatment method for corneal epithelial injury comprises the treatment by adopting autologous serum and nutrient factor eye drops, but the treatment effect is poor.
The hypoxia is widely applied to stem cell proliferation, differentiation and stress response researches, and most of the researches show that the hypoxia does not influence the phenotype and the dryness of stem cells, can promote the proliferation, differentiation, migration and anti-apoptosis of the stem cells and the tolerance of ischemia and hypoxia, and can also improve the anti-inflammatory effect, the immunoregulation effect and the paracrine effect of the stem cells. As discovered by research of Horie and the like, 2% hypoxia can promote the proliferation of mouse Neural Stem Cells (NSCs) compared with normoxic, and simultaneously does not influence the differentiation of the NSCs to astrocytes; in the review of the influence of the hypoxia microenvironment on the biological functions of the mesenchymal stem cells, such as cinnabar and the like, the mesenchymal stem cells can be inhibited from apoptosis and promoted to proliferate under the hypoxia environment with the oxygen integration number not less than 1%, but the promotion mechanism may have certain time dependence; ohnishi et al found that 139 and 49 genes were expressed in the mesenchymal stem cells and the mononuclear cells, respectively, in two types of cells, which were cultured under 1% hypoxia condition. However, the role of extracellular matrix secreted by stem cells cultured in a hypoxic environment in diseases is rarely studied, and at present, no report on the use of extracellular matrix of human umbilical cord mesenchymal stem cells containing exosomes for repairing corneal epithelial cell injury is found.
Disclosure of Invention
The inventor creatively finds that the mesenchymal stem cells cultured in the culture medium with low serum concentration and containing polyvinyl alcohol can produce more factors and collagen which are favorable for promoting the proliferation and damage repair of corneal epithelial cells under the condition of hypoxia, such as platelet-derived growth factor (PDGF) and type IV collagen, and the factors and the collagen are multiplied in the culture supernatant obtained by the invention and show obviously higher than the extracellular matrix composition obtained by culturing under the condition of normal oxygen content and serum content.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a mesenchymal stem cell extracellular matrix containing exosome, and the preparation is culture supernatant produced by culturing human umbilical cord mesenchymal stem cells in a basal medium containing low-concentration serum under the condition of low oxygen content.
In most studies, mesenchymal stem cells are cultured under the normoxic condition (oxygen concentration is 21%), while the inventors found that the PDGF and type IV collagen in culture supernatant obtained by combining the culture of polyvinyl alcohol with lower serum concentration under the hypoxic condition are highly expressed, and an ELISA detection kit shows that the PDGF and type IV collagen positive expression rate is increased in a fold manner and has very significant difference (P is less than 0.01) compared with the normoxic 10% serogroup.
Particularly, the low oxygen content in the invention means that the oxygen content is 0.1-5%. In another aspect, the oxygen content is at least > 1%; in another aspect, the oxygen content may be any value between 1 to 5%, 1 to 4%, 1 to 3%, 1 to 2%. On the other hand, the oxygen content is preferably any value between 2 and 3%. During culture, in the condition of simulating low-oxygen environment, a culture dish or a culture bottle is placed in a low-oxygen device, and 95% nitrogen and 5% CO are introduced 2 To achieve the desired hypoxic environment, e.g., 1%. In contrast, an normoxic environment refers to 95% air + 5% CO 2 。
Particularly, the concentration of the serum in the serum-containing basic culture medium is 1-5%. In another aspect, the serum concentration is preferably > 3%; in another aspect, the serum concentration may be in the range of 3-4%, 3-5%; in another aspect, the serum concentration is 4% or 5%. It is to be noted that, in a hypoxic environment, the concentration of serum is particularly important for the growth and proliferation of mesenchymal stem cells, and at a concentration lower than 1%, good growth and proliferation of mesenchymal stem cells cannot be maintained, but higher than 5%, the expression of PDGF and type IV collagen in the obtained culture supernatant is reduced, and the higher the serum concentration is, the more unfavorable the high expression of PDGF and type IV collagen is. When the oxygen content of the system is maintained at 1-5%, the serum concentration in the growth medium is maintained within the range of 3-5%, and the PDGF and IV type collagen content is remarkably increased compared with that under the normoxic condition, which shows that the effect is only reflected between the lower serum concentration and the polyvinyl alcohol, but the synergistic effect does not exist between the high-concentration serum and the polyvinyl alcohol.
Particularly, the concentration of polyvinyl alcohol in the basic culture medium containing the serum is 1-3%.
PDGF plays an important role in tissue injury repair, while type IV collagen is an important component of the cornea, and in the test of promoting corneal epithelial cells, the culture supernatant obtained by culturing mesenchymal stem cells by adopting the culture medium under the condition of low oxygen concentration shows a more obvious role in promoting the proliferation of the corneal epithelial cells.
In particular, the basic medium in the invention is one of DMEM medium, low-sugar DMEM medium or DMEM/F12 medium. In particular, the basic medium is preferably DMEM/F12 medium.
The invention also provides a preparation method of the mesenchymal stem cell extracellular matrix, which comprises the following steps:
selecting P4-substituted mesenchymal stem cells with good proliferation capacity and growth state, inoculating the cells in a basic culture medium containing 1-5% of serum and 1-3% of polyvinyl alcohol, and adding 1-5% of O 2 、5％CO 2 Culturing at 37 deg.C for 12-48h, collecting culture medium supernatant, centrifuging, and filtering.
In the preparation method, the P4 generation mesenchymal stem cells can be obtained by using a conventional subculture method, and for example, the method can comprise the following steps:
collecting fresh umbilical cord tissue, cleaning, cutting into small pieces, removing umbilical artery, umbilical vein and epidermis, transferring into DMEM medium containing streptomycin and serum at 37 deg.C and 5% CO 2 Culturing in a saturated humidity box, changing the liquid for 1 time every 3 days, and digesting by pancreatin when the umbilical cord mesenchymal stem cells climb out more densely, and carrying out subculture.
In the above preparation method, the filtration treatment part may be performed by using an apparatus having filtration performance in the prior art, and a microporous membrane and an ultrafiltration tube are exemplified.
The invention also provides application of the mesenchymal stem cell extracellular matrix in preparing a preparation for promoting cell repair and regeneration. The cells referred to in the present invention are corneal epithelial cells.
The invention also provides a preparation with the function of repairing corneal epithelial cell injury, which contains the mesenchymal stem cell extracellular matrix. The extracellular matrix obtained under specific conditions contains more cytokines, such as PDGF and type IV collagen, so that the extracellular matrix can be prepared into preparations aiming at corneal injury, and particularly can be applied to repairing corneal epithelial cells. Such a formulation may be in the form of an eye drop formulation, and when formulated into an eye drop formulation, may further comprise various pharmaceutically acceptable adjuvants, and the formulation may theoretically further comprise various preservatives, cryoprotectants and stabilizers for maintaining the activity of the cytokine at low temperatures. The various preservatives, cryoprotectants and stabilizers involved can generally be of the kind conventional in the art and can be selected according to the actual needs.
The invention has the beneficial effects that:
(1) according to the invention, the serum concentration is controlled to be kept in a lower range by simulating the hypoxia condition in vitro, and a certain amount of polyvinyl alcohol is added to culture the mesenchymal stem cells, so that PDGF and IV type collagen can be highly expressed.
(2) The extracellular matrix can also be prepared into a preparation for repairing corneal injury, and has more remarkable treatment effect compared with the traditional autologous serum and trophic factor eye drops.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.
Example one, Single factor test
1.1 Effect of oxygen concentration
1.1.1 test methods
1.1.2 obtaining culture supernatants of human umbilical cord mesenchymal stem cells cultured in different oxygen concentrations: taking identified P4 generation human umbilical cord mesenchymal stem cells (hUCMSCs), and adjusting the cell concentration to 1 × 10 6 Inoculating into DMEM/F12 culture medium containing 5% fetal calf serum and 2% polyvinyl alcohol, placing the culture bottle in a low oxygen box, and introducing mixed gas (95% N/min) at a flow rate of 1L/min 2 +5％CO 2 ) To control the environment with different oxygen concentration, the culture bottle is placed under the condition of different oxygen concentration and 5% CO 2 Culturing at 37 deg.C for 48h, collecting culture medium supernatant, centrifuging, and filtering with microporous membrane.
1.1.3 Positive expression Rate assay for PDGF and type IV collagen: the culture supernatants obtained were tested for positive expression of PDGF and type IV collagen by ELISA kits and the results are shown in Table 1.
2 results
Table 1: comparison of Positive rates of PDGF and type IV collagen expression in culture supernatants of human umbilical cord mesenchymal stem cells in hypoxic and normoxic culture ((for example))
％)
Note: * p < 0.05, and normoxic group (21% O) 2 ) And (6) comparing.
As can be seen from the above tables 1 and 2, compared with the normoxic group, in the culture supernatant obtained by culturing in the hypoxic environment, the expression positive rate of PDGF is higher, and when the oxygen content is 2-5%, the PDGF expression is significantly different from the normoxic group (P < 0.05); the positive expression rate and the highest expression rate of PDGF and IV type collagen when the oxygen content is 2 percent are adopted, so that the serum concentration test is continued by selecting the oxygen concentration of 2 percent.
1.2 Effect of serum concentration
Taking identified P4 generation human umbilical cord mesenchymal stem cells (hUCMSCs), and adjusting the cell concentration to 1 × 10 6 Each of the flasks was filled with DMEM/F12 medium containing 1% by weight of serum, 2% by weight of serum, 3% by weight of serum, 4% by weight of serum, 5% by weight of serum, and 10% by weight of serum (the concentration of polyvinyl alcohol in each medium was controlled to 2%), and the flasks were placed in the same oxygen concentration (2% O) 2、 95％N 2 、 5％CO 2 ) Culturing at 37 deg.C for 48h, collecting culture medium supernatant, centrifuging, and filtering with microporous membrane. The positive expression rates of PDGF and type IV collagen in the collected culture supernatants were determined using the kit, and the results are shown in Table 2.
Table 2: comparison of Positive rates of PDGF and type IV collagen expression in culture supernatants obtained by culture at different serum concentrations (for example:)
％)
Note: p < 0.05, P < 0.01 compared to 10% serogroup.
The results show that at 2% O 2 Under the hypoxic environment, the positive expression rates of PDGF and IV type collagen in culture supernatant are gradually improved along with the increase of serum concentration within the range of 1-5%, but when the serum concentration reaches 10%, the positive expression rates of PDGF and IV type collagen are obviously reduced, and the result shows that under the hypoxic environment, the expression of PDGF and IV type collagen can be effectively promoted by polyvinyl alcohol and serum with lower concentration, and the effect is improved along with the increase of serum concentration within the range of 3-5% of serum concentration, but the effect does not exist under the condition of higher serum concentration (10%). When the serum concentration is 3-5%, the PDGF and IV type collagen expressions are very different from 10% of the serum, wherein,supernatants from 5% FBS cultures were selected to contain the most PDGF and type IV collagen in hypoxic conditions, with 5% serum concentrations, 2% O 2 Proceed to the next test.
1.3 Effect of containing and not containing polyvinyl alcohol
Taking identified P4 generation human umbilical cord mesenchymal stem cells (hUCMSCs), adjusting cell concentration to 1 × 10 6 Separately, the culture flasks were placed in DMEM/F12 medium containing 5% serum, 2% polyvinyl alcohol and 5% serum, and the flasks were placed in the same oxygen concentration (2% O) 2、 95％N 2 、5％CO 2 ) Culturing at 37 deg.C for 48h, collecting culture medium supernatant, centrifuging, and filtering with microporous membrane. The positive expression rates of PDGF and type IV collagen in the collected culture supernatants were determined using the kit, and the results are shown in Table 3.
Table 3: comparison of Positive rates of PDGF and type IV collagen expression in culture supernatants obtained by culture at different serum concentrations (for example:)
％)
Note: p < 0.05, compared to 5% serogroup.
The results show that the culture with the addition of the polyvinyl alcohol can obviously improve the expression of the type IV collagen, and compared with the culture without the polyvinyl alcohol, the culture with the addition of the polyvinyl alcohol has very obvious difference.
In conclusion, in the process of culturing the mesenchymal stem cells, on the basis of a single-factor test, the optimal conditions of culture are determined as follows: oxygen concentration 2% O 2 (volume fraction), the serum concentration of the medium is 5%, and the polyvinyl alcohol is preferably contained at a concentration of 2%. Under the condition, the PDGF positive expression rate in the culture supernatant reaches 62.35%, and the collagen type IV positive expression rate reaches 51.24%. The result shows that the PDGF and IV collagen can be highly expressed by the mesenchymal stem cells by adopting low-concentration serum and polyvinyl alcohol culture in a hypoxia environment. Lower partThe obtained culture supernatants were subjected to a test for promoting proliferation of corneal epithelial cells.
Test for promoting proliferation of corneal epithelial cells
S1, primary isolation and subculture amplification culture of human umbilical cord mesenchymal stem cells: collecting fresh umbilical cord tissue, cleaning, cutting into small pieces, removing umbilical artery, umbilical vein and epidermis, transferring into DMEM medium containing streptomycin and serum, and culturing at 37 deg.C with 5% CO 2 Culturing in a saturated humidity box, changing the liquid for 1 time every 3 days, and digesting by pancreatin when the umbilical cord mesenchymal stem cells climb out more densely, and carrying out subculture;
s2, obtaining culture supernatant of human umbilical cord mesenchymal stem cells: taking identified P4 generation human umbilical cord mesenchymal stem cells (hUCMSCs), adjusting cell concentration to 1 × 10 6 Inoculating into DMEM/F12 culture medium containing 5% fetal calf serum and 2% polyvinyl alcohol, placing the culture bottle in a low oxygen box, and introducing mixed gas (95% N/min) at a flow rate of 1L/min 2 +5％CO 2 ) Controlling the oxygen concentration in the hypoxia chamber to be 2%, culturing at the constant temperature of 37 ℃ for 48h, collecting culture medium supernatant, centrifuging, and filtering with a microporous filter membrane to obtain the product.
Corneal epithelial cells were seeded in a 96-well plate at a density of 1000 cells/well, and the culture supernatants obtained above were added for culture, and proliferation of corneal epithelial cells was measured by CCK-8 reagent at 12 hours, 24 hours, and 48 hours, and the results are shown in Table 4.
Table 4: the proliferation of corneal epithelial cells (OD value,
)
corneal epithelial injury repair test
Corneal epithelial cells were cultured at 2X 10 5 Inoculating to 96-well plate at a density of one well, adding DMEM medium containing 10% fetal calf serum to culture, washing with DPBS solution for 2 times when cell fusion degree reaches 100%, and washing with 10% serumScratching and molding muL gun head every 1cm, washing with DPBS solution, adding the obtained culture supernatant at 37 deg.C and 5% CO 2 Culturing under the condition of (3), observing in an inverted microscope at 12h, 24 h and 48h, photographing, and performing statistical analysis.
Table 5: test results of corneal epithelial cell injury repair
The results in tables 4 and 5 show that the culture supernatants prepared by the method of the invention have significant promotion effect on the proliferation of corneal epithelial cells; the wound surface of the damaged corneal epithelial cells is gradually reduced, and the excellent function of promoting the repair of the corneal epithelial cell damage is shown.
Finally, it should be noted that the above embodiments are intended to illustrate the technical solutions of the present invention and not to limit the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (2)
1. The preparation method of the mesenchymal stem cell extracellular matrix containing the exosome is characterized by comprising the following steps of:
selecting P4-substituted secondary human umbilical cord mesenchymal stem cells with good proliferation capacity and growth state, and adjusting the cell concentration to 1 × 10 6 Inoculating into DMEM/F12 culture medium containing 5% fetal calf serum and 2% polyvinyl alcohol, placing the culture medium in a low oxygen box, and introducing mixed gas 95% N at flow rate of 1L/min 2 +5%CO 2 The oxygen concentration in the low oxygen box is controlled to be 2 percent and is controlled to be 2 percent O 2 、5%CO 2 Culturing at 37 deg.C for 48h, collecting culture medium supernatant, centrifuging, and filtering with microporous membrane.
2. Use of extracellular matrix of mesenchymal stem cells containing exosomes prepared by the preparation method according to claim 1, in preparation of a preparation for promoting cell repair and regeneration, wherein the cells are corneal epithelial cells.
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