Experimental Research Of Bone Marrow Mesenchymal Stem Cells

Objective:1.To observe whether bone marrow mesenchymal stem cells can achieve directional migration and differentiation under electric field,and to observe whether electric field has influence on the survival and apoptosis of cochlear hair cells.2.Explore the conditions of electric field culture,and observe the state of cell migration and growth.Methods:1.Bone marrow mesenchymal stem cells(BMSCs)of SD rats were isolated and cultured by whole bone marrow direct adherence method,and the expressions of CD44 and CD90 on BMSCs surface markers and CD45 and CD34 on hematopoietic cell surface antigens were detected.Green fluorescent protein(GFP)was transfected into BMSCs,and GFP expression and GFP positive rate in BMSCs were observed under inverted fluorescence microscope.2.Ototoxic rat model:newborn SD rats were used,and the basement membrane was intervened by 1:100 mL neomycin for 10 hours.The hair cells were observed by Myosin ?a immunofluorescence.3.Electric field preparation and electric field migration experiment(the effect of electric field on the activity of bone marrow mesenchymal stem cells):the experimental group was divided into 4 groups,and the control group(a)was not exposed to electric field;Experimental group(b):exposed to electric:field for half an hour;Experimental group(c):exposed to electric field for an hour;Experimental group(d):exposed to electric field for 2 hours.4.Differentiation and identification of bone marrow mesenchymal stem cells:the basement membrane without electric field,the bone marrow mesenchymal stem cells trans-fected with GFP and the basement membrane after electric field were cultured in culture medium for 14 days respectively.Immunohistachemistry was used to identify whether the bone marrow mesenchyrnal stem cells differentiated into hair cells and expressed Myosin7A and Mathl.Results:1.The expression of CD90 and CD44 in bone marrow mesenchymal stem cells obtained by whole bone marrow culture was 99.79%and 99.1%,respectively.The expressions of CD45 and CD34 were 4.1%and 2.11%,which were consistent with the results of flow cytometry.Combined with cell morphology analysis,it is consistent with the characteristics of BMSCs.2.The BMSCs containing GFP and the damaged basement membrane were put into the electric field environment together.Under the real-time fluorescence microscope,BMSCs cells migrated to the anode,and some migrated to the basement membrane.Most BMSCs migrated to the basement membrane were located in the bottom circle,a few were found in the middle circle,and no obvious BMSCs migration was found in the top circle.After 14 days of co-culture,the differentiated cells were identified by immunocytochemistry to express specific markers Myosin7A and Mathl of hair cells.Conclusion:Bone marrow mesenchymal stem cells can migrate to the injured area and differentiate into hair cell-like cells with the molecular characteristics of inner ear hair cells under the electric field environment.Appropriate electric field environment provides a new way to treat deafness.