CN113789298A - Method for promoting regeneration capacity of oral mucosa mesenchymal stem cells and application thereof - Google Patents
Method for promoting regeneration capacity of oral mucosa mesenchymal stem cells and application thereof Download PDFInfo
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Abstract
The invention discloses a method for promoting regeneration capacity of mesenchymal stem cells of oral mucosa and application thereof, wherein the method comprises the following steps: (1) sucking 1mL of fibrin glue solution by using a syringe; (2) preparing a culture medium suspension of oral mucosa mesenchymal stem cells; (3) and (2) quickly injecting the fibrin glue solution sucked in the step (1) into a culture dish, quickly and uniformly dripping the culture medium suspension of the oral mucosa mesenchymal stem cells prepared in the step (2) onto the fibrin glue, quickly and uniformly mixing, uniformly solidifying and then placing into a culture solution for storage. The method for promoting the regeneration capacity of the oral mucosa mesenchymal stem cells solves the technical problem that the conventional matrix material is difficult to accurately regulate the fibroblast regeneration capacity of the stem cells, and can be widely applied to the field of regulating and controlling the regeneration of the three-dimensional mesenchymal stem cells by using gel fibers.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for promoting regeneration capacity of mesenchymal stem cells of oral mucosa and application thereof.
Background
The oral mucosa is a natural barrier of tissues, is sensitive to environmental change, has strong healing and repairing capabilities, contains mesenchymal stem cells in an inherent layer, can express stem cell related protein, has self-renewal, can realize cross-germ layer differentiation and the like, and plays an important role in maintaining dynamic balance of the tissues and repairing and regenerating the damaged tissues.
The mesenchymal stem cells can receive various differentiation signals in the surrounding environment through contacting with various tissue cells and the environment in vivo, and then differentiate, proliferate and migrate to the required cell types in the appropriate environment, thereby playing the functions of filling defects and reconstructing tissues. The proper mesenchymal stem cells are selected as seed cells, a proper cell proliferation and regeneration culture system is established, and the regeneration of the mesenchymal stem cells is promoted, and the function is not only related to the proliferation and differentiation of the mesenchymal stem cells, but also related to the secretion of cytokines beneficial to tissue regeneration or the formation of components such as extracellular matrix for promoting healing. However, tissue-specific proliferation and differentiation behavior of mesenchymal stem cells is of little concern without inducing differentiation by local specific host environments. Therefore, the function of the mesenchymal stem cells is researched in a specific microenvironment, and the method has important significance.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for promoting the regeneration capacity of mesenchymal stem cells of oral mucosa and application thereof, solves the technical problem that the prior matrix material is difficult to accurately regulate the fibroblast forming capacity of the stem cells, and can be widely applied to the field of regulating and controlling the regeneration of three-dimensional mesenchymal stem cells by gel fibers.
The technical problem to be solved by the invention is realized by the following technical scheme:
a method for promoting regeneration capacity of oral mucosa mesenchymal stem cells comprises the following steps:
(1) sucking 1mL of fibrin glue solution by using a syringe;
(2) preparing a culture medium suspension of oral mucosa mesenchymal stem cells;
(3) and (2) quickly injecting the fibrin glue solution sucked in the step (1) into a culture dish, quickly and uniformly dripping the culture medium suspension of the oral mucosa mesenchymal stem cells prepared in the step (2) onto the fibrin glue, quickly and uniformly mixing, uniformly solidifying and then placing into a culture solution for storage.
Preferably, the step (2) comprises the steps of:
(21) oral mucosal mesenchymal stem cells were cultured in a culture dish containing 10% FBS-containing alpha-MEM culture medium at 37 ℃ in 5% CO2Culturing in an incubator with saturated humidity;
(22) taking out the cell culture dish, and washing with PBS;
(23) adding trypsin to digest the cells cultured by adherent culture for 2-3min, removing the trypsin by aspiration, adding an alpha-MEM culture solution containing 10% FBS to terminate digestion, blowing the bottom layer of a culture dish, transferring to a centrifuge tube, centrifuging and removing the supernatant;
(24) collecting oral mucosa mesenchymal stem cells cultured in monolayer, mixing with 10% FBS-containing alpha-MEM culture solution, repeatedly beating, dispersing cell mass with mechanical force, and adjusting density to 2 × 106And (5) mixing the powder per ml for later use.
Preferably, the culture dish of step (21) is a culture dish with a diameter of 10cm, and the number of culture days is 3 days.
Preferably, the PBS wash in step (22) is repeated 3 times for 3min each time.
Preferably, the trypsin is added in an amount of 1mL and the 10% FBS-containing α -MEM culture medium is added in an amount of 2mL in step (23); the conditions of centrifugation were: centrifuge at 1000rpm for 5 min.
Preferably, step (3) is specifically:
quickly and uniformly injecting 1ml of fibrin solution sucked by the injector in the step (1)Injecting into a culture dish, sucking 1mL of the oral mucosa mesenchymal stem cell culture medium suspension prepared in the step (2), synchronously, rapidly and uniformly dripping onto a fibrin solution, rapidly and uniformly mixing in 10s, uniformly curing, standing for 2min, adding an alpha-MEM culture solution containing 10% FBS into the culture dish of a cell-fibrin glue complex, and adding the alpha-MEM culture solution into the culture dish at 37 ℃ and 5% CO2And culturing in an incubator with saturated humidity.
Preferably, the method further comprises the steps of:
(4) and (4) culturing the cell-fibrin glue complex block tissue in the step (3) for several days, fixing, embedding and slicing, and observing the cell morphology in the tissue after respectively adopting H & E staining and Masson staining.
A fibrin gel comprises a cell-fibrin glue complex obtained by a method for promoting regeneration capacity of mesenchymal stem cells of oral mucosa.
An application of a method for promoting the regeneration capacity of mesenchymal stem cells of oral mucosa.
Preferably, the method is applied to the fields of regeneration of mesenchymal stem cells and repair of mucosal tissues.
The technical scheme of the invention has the following beneficial effects:
(1) the collagen forming capacity of the oral mucosa mesenchymal stem cells in vivo is detected, the biomaterial scaffold can provide a favorable environment for the oral mucosa, the fibrin gel has a three-dimensional structure, is formed by fibrinogen under the cracking action of thrombin, has low immunogenicity, shows degradable performance after a series of coagulation reactions, and can be widely applied to the fields of surgical hemostasis, regenerative medicine and tissue engineering.
(2) The fibrin gel can be used as a carrier of mesenchymal stem cells, the mesenchymal stem cells can show natural biological behaviors on the mesenchymal stem cells, and the analysis of the oral mucosa mesenchymal stem cells shows that the oral mucosa mesenchymal stem cells keep fibrin forming capability in the gel, so the oral mucosa mesenchymal stem cells can be a new stem cell source for tissue engineering.
(3) The mesenchymal stem cells are combined with fibrin glue to form a fibrous tissue scaffold, and the collagen regenerated by the mesenchymal stem cells in the gel can be effectively used for tissue regeneration.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain the principles of the invention.
Fig. 1 is a schematic diagram of binding of oral mucosal mesenchymal stem cells to fibrin glue (cell-fibrin glue complex) according to the present application.
FIG. 2 is a graph of H & E staining of clumps of the cell-fibrin glue complex of the present application after several days of tissue culture.
FIG. 3 is a graph of Masson staining after several days of tissue culture of clumps of the cell-fibrin glue complex of the present application.
Detailed Description
Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that: the relative arrangement of the components and steps, the numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless specifically stated otherwise.
Fig. 1 is a schematic diagram of binding of oral mucosal mesenchymal stem cells to fibrin glue (cell-fibrin glue complex) according to the present application. FIG. 2 is a graph of H & E staining of clumps of the cell-fibrin glue complex of the present application after several days of tissue culture. FIG. 3 is a graph of Masson staining after several days of tissue culture of clumps of the cell-fibrin glue complex of the present application. As shown in fig. 1, 2 and 3:
a method for promoting regeneration capacity of oral mucosa mesenchymal stem cells comprises the following steps:
(1) the syringe aspirates 1mL of fibrin glue solution (commercially available);
(2) preparing a culture medium suspension of oral mucosa mesenchymal stem cells:
(21) oral mucosal mesenchymal stem cells were cultured in a culture dish containing 10% FBS-containing alpha-MEM (commercially available) medium at 37 ℃ in 5% CO2Culturing in an incubator with saturated humidity;
(22) taking out the cell culture dish, and washing with PBS;
(23) adding trypsin to digest the cells cultured by adherent culture for 2-3min, removing the trypsin by aspiration, adding an alpha-MEM culture solution containing 10% FBS to terminate digestion, blowing the bottom layer of a culture dish, transferring to a centrifuge tube, centrifuging and removing the supernatant;
(24) collecting oral mucosa mesenchymal stem cells cultured in monolayer, mixing with 10% FBS-containing alpha-MEM culture solution, repeatedly beating, dispersing cell mass with mechanical force, and adjusting density to 2 × 106Per ml for standby;
(3) quickly and uniformly injecting 1mL of fibrin solution sucked by the injector in the step (1) into a culture dish, sucking 1mL of oral mucosa mesenchymal stem cell culture medium suspension prepared in the step (2), synchronously, quickly and uniformly dripping the suspension onto the fibrin solution, quickly and uniformly mixing the suspension within 10s, uniformly curing the suspension, standing for 2min, adding alpha-MEM culture solution containing 10% FBS into the culture dish of the cell-fibrin glue complex, and adding the alpha-MEM culture solution into the culture dish of the cell-fibrin glue complex at 37 ℃ and 5% CO2And culturing in an incubator with saturated humidity.
Further, the culture dish of the step (21) is a culture dish having a diameter of 10cm, and the number of culture days is 3 days. In step (22), PBS washing is performed for 3min each time, and the washing is repeated for 3 times. In the step (23), the addition amount of trypsin is 1mL, and the addition amount of 10% FBS-containing alpha-MEM culture solution is 2 mL; the conditions of centrifugation were: centrifuge at 1000rpm for 5 min. Of course, the present application is not limited thereto, and the reaction conditions and the amounts of the reagents and drugs can be adjusted as required by those skilled in the art.
Further, the method comprises the following steps:
(4) culturing the cell-fibrin glue complex block tissue in the step (3) for 1 month, fixing, embedding and slicing, and observing the cell morphology in the tissue after respectively adopting H & E staining and Masson staining: h & E and Masson staining found that the cells in these tissues were in a spindle-shaped predominantly fibroblast-like morphology, abundant in extracellular matrix, with cells and collagen fibers running like the tissue of the lamina propria of the mucosa.
H & E staining procedure: slicing paraffin into xylene for 10min for 3 times; gradient alcohol 100%, 95%, 90%, 80%, 70% each for 5 min; rinsing with tap water for 5 min. The sections were stained in hematoxylin for about 2min and washed with running tap water to make the sections blue in color. Placing the slices into 1% hydrochloric acid alcohol solution to fade, wherein the slices turn red and the color is light, and the time is about several seconds to tens of seconds. And (3) carrying out contrast dyeing for 2-5 min by using 0.5% eosin alcohol solution. Washing with tap water for 5 min; gradient ethanol 70%, 80%, 90%, 95%, 100% dehydration, xylene 10min 3 times transparent, gum sealing, and observing under microscope.
Masson trichrome method step: slicing paraffin into xylene for 10min for 3 times; gradient alcohol 100%, 95%, 90%, 80%, 70% each for 5 min; chromizing or mercury salt removal precipitation. Washing with tap water and distilled water for 5 min. Staining the nuclei with Weigart hematoxylin liquid for 5-10 min. Washing with water, and differentiating with hydrochloric acid and ethanol. Washing with distilled water for 5 min. Adding Masson ponceau acid red recovering solution for 5-10 min. And (3) soaking and washing the fabric for a moment by using 2% glacial acetic acid aqueous solution. Differentiating with 1% phosphomolybdic acid water solution for 3-5 min. Directly dyeing with aniline blue or light green solution for 5min without washing with water. The plate was rinsed with 0.2% glacial acetic acid in water for a while. 95% alcohol, anhydrous alcohol, xylene transparent, and neutral gum sealing.
The cell-fibrin glue complex finally obtained by the method for promoting the regeneration capacity of the oral mucosa mesenchymal stem cells (as shown in figure 1). H & E staining shows that mesenchymal stem cells are in a spindle-shaped main fibroblast-like shape on a fibrin glue scaffold, and form a structure similar to the running of collagen fibers (as shown in figure 2); masson staining found that oral mucosal mesenchymal stem cells were abundant in extracellular matrix on the scaffold, forming a similar morphology of cell and collagen fiber trafficking (as shown in figure 3).
The fibrin gel comprises the cell-fibrin glue complex, and can be used in the field of mesenchymal stem cell regeneration and mucosal tissue repair.
The method for promoting the regeneration capacity of the oral mucosa mesenchymal stem cells or the fibrin gel can be applied to the regeneration field of the mesenchymal stem cells and the repair field of mucosa tissues, and has potential value.
The oral mucosa mesenchymal stem cells can be expressed as a cell population with cell therapy potential, and are a new cell source for oral mucosa tissue engineering. In addition, the fibrin gel can provide a carrier for regenerating collagen for the mesenchymal stem cells, and the regenerated microenvironment can promote the regeneration capacity of the mesenchymal stem cells of the oral mucosa.
Although the present invention has been described with reference to the above embodiments, it should be understood that the present invention is not limited thereto, and various changes and modifications may be made by those skilled in the art without departing from the spirit and scope of the present invention.
Claims (10)
1. A method for promoting regeneration capacity of mesenchymal stem cells of oral mucosa is characterized by comprising the following steps:
(1) sucking 1mL of fibrin glue solution by using a syringe;
(2) preparing a culture medium suspension of oral mucosa mesenchymal stem cells;
(3) and (2) quickly injecting the fibrin glue solution sucked in the step (1) into a culture dish, quickly and uniformly dripping the culture medium suspension of the oral mucosa mesenchymal stem cells prepared in the step (2) onto the fibrin glue, quickly and uniformly mixing, uniformly solidifying and then placing into a culture solution for storage.
2. The method for promoting regeneration capacity of mesenchymal stem cells of oral mucosa according to claim 1, wherein the step (2) comprises the following steps:
(21) oral mucosal mesenchymal stem cells were cultured in a culture dish containing 10% FBS-containing alpha-MEM culture medium at 37 ℃ in 5% CO2Culturing in an incubator with saturated humidity;
(22) taking out the cell culture dish, and washing with PBS;
(23) adding trypsin to digest the cells cultured by adherent culture for 2-3min, removing the trypsin by aspiration, adding an alpha-MEM culture solution containing 10% FBS to terminate digestion, blowing the bottom layer of a culture dish, transferring to a centrifuge tube, centrifuging and removing the supernatant;
(24) collecting oral mucosa mesenchymal stem cells cultured in monolayer, mixing with 10% FBS-containing alpha-MEM culture solution, repeatedly blowing, and dispersing with mechanical forceCell mass, density adjusted to 2X 106And (5) mixing the powder per ml for later use.
3. The method for promoting regeneration capacity of mesenchymal stem cells of oral mucosa according to claim 2, wherein the culture dish of step (21) is a culture dish with a diameter of 10cm, and the number of culture days is 3 days.
4. The method for promoting regeneration of mesenchymal stem cells of oral mucosa according to claim 2, wherein the PBS washing in the step (22) is 3min for 3 times.
5. The method for promoting regeneration capacity of mesenchymal stem cells of oral mucosa according to claim 2, wherein the amount of trypsin added in step (23) is 1mL, and the amount of α -MEM culture solution containing 10% FBS is 2 mL; the conditions of centrifugation were: centrifuge at 1000rpm for 5 min.
6. The method for promoting regeneration capacity of mesenchymal stem cells of oral mucosa according to claim 1, wherein the step (3) is specifically as follows:
quickly and uniformly injecting 1mL of fibrin solution sucked by the injector in the step (1) into a culture dish, sucking 1mL of the oral cavity mucosa mesenchymal stem cell culture medium suspension prepared in the step (2), synchronously, quickly and uniformly dripping the suspension onto the fibrin solution, quickly and uniformly mixing the suspension within 10s, uniformly curing the suspension, standing for 2min, adding alpha-MEM culture solution containing 10% FBS into the culture dish of the cell-fibrin glue complex, and adding the alpha-MEM culture solution into the culture dish of the cell-fibrin glue complex at 37 ℃ and 5% CO2Culturing in an incubator with saturated humidity.
7. The method for promoting regeneration capacity of mesenchymal stem cells of oral mucosa according to claim 6, further comprising the following steps:
(4) and (4) culturing the cell-fibrin glue complex block tissue in the step (3) for several days, fixing, embedding and slicing, and observing the cell morphology in the tissue after respectively adopting H & E staining and Masson staining.
8. A fibrin gel comprising the cell-fibrin glue complex obtained by the method for promoting the regeneration capacity of mesenchymal stem cells of oral mucosa according to claim 6 or 7.
9. Use of a method according to any one of claims 1 to 7 for promoting regenerative capacity of mesenchymal stem cells of oral mucosa.
10. The application of the method for promoting the regeneration capacity of the mesenchymal stem cells of the oral mucosa according to claim 9 is characterized by being applied to the field of regeneration of the mesenchymal stem cells and the field of repair of mucosal tissues.
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