CN114181899A - Method for obtaining mesenchymal stem cells from gum tissue of mice - Google Patents
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Abstract
The invention relates to the technical field of biomedicine, in particular to a method for obtaining mesenchymal stem cells from a gum tissue of a mouse. The method comprises the steps of collecting mouse gingival tissues, and digesting the mouse gingival tissues by adopting collagenase to obtain the mesenchymal stem cells. The mouse GMSCs obtained by the invention highly express CD29, CD44, CD73, CD90 and CD105, and hardly express CD34 and CD 45. After the mouse GMSCs are cultured for 21 days by the adipogenic induction liquid, the mouse GMSCs are differentiated into fat cells and secrete fat drops; after the culture of the osteogenic induction liquid, the cells gradually change from long spindle shape to polygon shape and grow in a multilayer covering manner, so that small calcification can be seen to appear in succession, and a new research method is provided for the autograft of GMSCs in animal models such as inflammation, autoimmune diseases, tissue repair and the like.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a method for obtaining mesenchymal stem cells from a gum tissue of a mouse.
Background
Mesenchymal Stem Cells (MSCs) are pluripotent stem cells, originally found in bone marrow, and have all the commonalities of stem cells, namely, the ability to self-renew, differentiate in multiple directions, and immunoregulation. The immune-modulating properties of MSCs are reflected in a number of aspects: when in vivo immune inflammation injury occurs and inflammatory signals (such as IL-6, TNF-alpha, IFN-gamma and the like) are released, the MSCs can be activated by immunoregulation to continuously inhibit the proliferation of immune abnormal B cells, T cells and NK cells and the production of cytokines. It has been reported that the use of umbilical cord blood mesenchymal stem cells (UC-MSCs) or bone marrow mesenchymal stem cells (BM-MSCs) has been effective in treating SLE and LN. MSCs have become one of its immunotherapeutic approaches for severe autoimmune diseases where conventional therapy is ineffective.
Gingiva-derived mesenchymal stem cells (GMSCs) are a mesenchymal stem cell subset obtained from gingival tissues, and in 2009, Zhang et al isolated GMSCs from human gingival tissues for the first time and confirmed their phenotypic and functional activities. Similar to other MSCs, GMSCs also have the effects of inhibiting inflammation and modulating immune response, and can be used to treat mouse and human animal models of various diseases, including rheumatoid arthritis, type I diabetes, systemic lupus erythematosus, atherosclerosis, etc., by decreasing inflammatory T cells, dendritic cells, macrophages, osteoclasts, and increasing regulatory T cells, etc.
In the establishment of a mouse disease model, the extraction of mouse GMSCs for autografting is more helpful for the deep exploration of the therapeutic action and mechanism research of GMSCs on the disease. The extraction methods of mouse GMSCs mentioned in the prior art use a variety of enzymes and complex culture medium conditions.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for obtaining mesenchymal stem cells from gingival tissues of mice.
The invention utilizes the mouse to carry out the test, establishes a simpler and more convenient and efficient in-vitro extraction and culture system, and provides a new research method for the autograft of GMSCs in animal models of inflammation, autoimmune diseases, tissue repair and the like.
In order to achieve the purpose, the invention adopts the technical scheme that: provided is a method for obtaining mesenchymal stem cells from a mouse gingival tissue, the method including collecting the mouse gingival tissue, and digesting the mouse gingival tissue with collagenase to obtain the mesenchymal stem cells.
After the method is used for culturing for 2-3 days, the primary cells can climb out of the tissue and grow adherent, the cells are in long spindle shapes and polygonal shapes, the primary cells are cultured until the cells are fully paved on 80% of the bottom of a dish for 10-14 days, and subculture can be carried out. The obtained mouse GMSCs highly express CD29, CD44, CD73, CD90 and CD105, and hardly express CD34 and CD 45.
As a preferred embodiment of the method of the present invention, the gingival tissue is prepared by taking the mouse mandible, removing excess skin and muscle tissue, rinsing the mandible with PBS containing a double antibody, and isolating the gingival tissue.
In a preferred embodiment of the method of the present invention, the collagenase is collagenase type IV.
In a preferred embodiment of the method of the present invention, the collagenase type IV is present at a concentration of 0.8 to 1.2 mg/L.
In a preferred embodiment of the method of the present invention, the digestion is performed by adding collagenase to the gingival tissue and digesting the mixture at 35 to 37 ℃ for 20 to 30 minutes.
As a preferred embodiment of the method of the present invention, the method further comprises adding α -MEM containing FBS to terminate the digestion after completion of the digestion.
As a preferred embodiment of the method of the present invention, the method further comprises adding alpha-MEM containing 20% FBS to the gingival tissue after termination of the digestion, and then subjecting the mixture to 5% CO at 37 ℃2Culturing in an incubator.
The invention also provides the mouse gingiva mesenchymal stem cell prepared by the method.
The invention has the beneficial effects that:
the mouse GMSCs obtained by the invention highly express CD29, CD44, CD73, CD90 and CD105, and hardly express CD34 and CD 45. After the mouse GMSCs are cultured for 21 days by the adipogenic induction liquid, the mouse GMSCs are differentiated into fat cells and secrete fat drops, and after the fat cells are stained by oil red O, the formation of grape string-shaped red fat drops can be seen under an optical microscope; after culturing by osteogenic induction liquid, cells gradually change from long spindle shape to polygon and grow in a multilayer covering manner, small calcification can be seen to appear successively, alizarin red staining can be seen as reddish brown calcified nodules 28 days after osteogenic induction, and a new research method is provided for the autografting of GMSCs in animal models of inflammation, autoimmune diseases, tissue repair and the like.
Drawings
FIG. 1: showing that the primary cells climb out of the tissue and grow adherent after being cultured for 2-3 days, and the cells are in a long fusiform shape and a polygonal shape; wherein, the b and c show that the primary cells are cultured until the cells are paved on 80% of the bottom of the dish after 10-14 days, and subculture can be carried out.
FIG. 2: the CCK8 experiment plots the growth of mouse GMSCs; among them, GMSCs grow fastest on days 3-4, grow slower on days 5-8, and develop a plateau.
FIG. 3: flow cytometry results showed that mouse GMSCs highly expressed CD29, CD44, CD73, CD90, CD105, and hardly expressed CD34, CD 45.
FIG. 4: after the mouse GMSCs are cultured for 21 days by the adipogenic induction liquid, the mouse GMSCs are differentiated into fat cells and secrete fat drops, and after the fat cells are stained by oil red O, the formation of grape string-shaped red fat drops can be seen under an optical microscope.
FIG. 5: after the mouse GMSCs are cultured by osteogenic induction liquid, cells gradually change from long spindle shape to polygon shape and grow in a multilayer covering manner, small calcification can be seen in succession, and alizarin red staining can be seen as reddish brown calcified nodules after osteogenic induction for 28 days.
Detailed Description
To more clearly illustrate the technical solutions of the present invention, the following embodiments are further described, but the present invention is not limited thereto, and these embodiments are only some examples of the present invention.
Example 1 isolated culture of mouse GMSCs
C57 BL/6 mice were sacrificed and the mandible of the mice was removed, cleaned of excess skin and muscle tissue, and rinsed multiple times with PBS containing 5% double antibody. The gingiva in the molar region was gently separated with a syringe needle. The tissue was collected in a petri dish, 1mg/L collagenase type IV was added, digestion was performed at 37 ℃ for 30 minutes, and digestion was terminated by adding alpha-MEM containing FBSCentrifuging and discarding the supernatant. The tissue was plated in six well plates with 1ml of 20% FBS-containing alpha-MEM per well and placed at 37 ℃ in 5% CO2Culturing in an incubator. After 2-3 days, the cells are climbed out, the rest tissue blocks are washed out by changing the liquid, 2ml of alpha-MEM of 20% FBS is added into each hole, and the liquid is changed once in 3 days. After the cells grow to 80% of the plate bottom, the cells are passaged, and after the passage, the cells are replaced by complete culture solution containing 10% FBS for continuous culture.
Example 2 detection of proliferative Capacity of mouse GMSCs
Operating according to the instruction of a CCK8 cell proliferation and cytotoxicity detection kit, continuously measuring the absorbance at 450nm by using a microplate reader at the same time for 8 days, and drawing a GMSCs growth curve according to the result.
Example 3 surface marker flow cytometry detection of mouse GMSCs
The 2 nd generation GMSCs (about 1X 10)6Individual cells) cell suspension was washed 2 times with PBS, resuspended in 100 μ l PBS, and then added with flow antibodies CD29, CD34, CD45, CD44, CD73, CD90, and CD105, respectively, incubated at 4 ℃ in the dark for 20min, washed 3 times with PBS, and the expression of the above surface markers was detected by flow cytometry.
Example 4 Induction of the multipotentiality of mouse GMSCs
(1) Adipogenic induced differentiation
The composition of the adipogenic induction liquid is as follows: alpha-MEM medium + 10% FBS + 200. mu. mol/L indomethacin +10mg/L insulin + 1. mu. mol/L dexamethasone + 500. mu. mol/L IBMX.
The 2 nd generation GMSCs were seeded in six well plates at approximately 3X 10 per well3And (3) replacing the culture solution into a adipogenesis induction solution when the cells are 70% of the bottom of the plate after the cells are paved, and replacing the culture solution once every 3 days. After 21 days of induction, cells were washed once with PBS, fixed with 4% paraformaldehyde for 30 minutes, washed 2 times with deionized water, stained with oil red O stain for 20 minutes, washed 3 times with distilled water, dried, observed under an inverted microscope and photographed.
(2) Osteogenic induced differentiation
The osteogenesis inducing liquid comprises the following components: alpha-MEM medium + 10% FBS +10nmol/L dexamethasone +50mg/L ascorbic acid +10mmol/L sodium beta-glycerophosphate.
Inoculating 2 nd generation GMSCs to six-well plate with each well3×103And (3) replacing the cells with osteogenesis inducing liquid once every 3 days when the cells are 70% of the bottom of the plate to be paved. After 28 days of induction, cells were washed once with PBS, fixed with 4% paraformaldehyde for 30 minutes, washed 2 times with deionized water, stained with alizarin red stain for 30 minutes, washed 3 times with distilled water, dried, observed under an inverted microscope and photographed.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (8)
1. A method for obtaining mesenchymal stem cells from a mouse gingival tissue is characterized by comprising the steps of collecting the mouse gingival tissue, and digesting the mouse gingival tissue by adopting collagenase to obtain the mesenchymal stem cells.
2. The method of claim 1, wherein the gingival tissue is a mouse mandible, the excess skin and muscle tissue is removed, the mandible is washed with PBS containing a double antibody, and then the gingival tissue is isolated.
3. The method of claim 1, wherein the collagenase is collagenase type IV.
4. The method according to claim 3, wherein the collagenase type IV is present at a concentration of 0.8 to 1.2 mg/L.
5. The method of claim 1, wherein the digestion is performed by adding collagenase to the gingival tissue and digesting the mixture at 35-37 ℃ for 20-30 minutes.
6. The method of claim 5, further comprising adding α -MEM containing FBS to terminate digestion after digestion is complete.
7. The method of claim 6, further comprising, after terminating the digestion, adding α -MEM containing 20% FBS to the gingival tissue and incubating the gingival tissue at 37 ℃ and 5% CO2Culturing in an incubator.
8. Mouse gingiva mesenchymal stem cell prepared according to the method of any one of claims 1-7.
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