The Research Of Using Myf5 Inducing Bone Marrow Mesenchymal Stem Cells Of Sheep To Myoblast

Myogenic factor5 belongs to myogenic determining factor gene family.The gene family include MyoD、 Myogenin、Myf5 and Mrf4, which control the proliferation and differentiation of muscle cells, the number and size of muscle fiber lonely or synergically.In order to estiblash the method that induce bone mesenchymal stem cells of sheep with Myf5 gene to myoblasts, this study did the work as followed.Myf5 gene was amplified from mouse muscle,and expression vector pcDNA3.1-Myf5 successfully constructed. BMSCs of sheep were transfected with pcDNA3.1-Myf5 by liposome,and the ability of the cell differentiation was observed. The following results obtained from the research:1. Myf5 gene eukaryotic expression vector constructionReferring to the sequence in GenBank (AK044894),primers were designed, and then total RNA was extracted from muscle tissue in mouse. Myf5 gene was ampified by RT-PCR.The fragment was linked with cloning vector pMD18-T Simple and expression vector pcDNA3.1 (+) respectively.Double enzyme digestion and sequencing showed that contained the eukaryotic expression vector pcDNA3.1-Myf5 successfully.2. Transfection of BMSCsThawing out cryopreserved BMSCs,the cell morphology and cell growth curve were similar with BMSCs.When the cells confluented about 80%, processing cell respectively as follow:Group A was transfected with pcDNA3.1-Myf5 endotoxin free plasmid,added F12 medium containing 0.5% DMSO; Group B was transfected with pcDNA3.1-Myf5 endotoxin free plasmid, added F12 medium;Group C added F12 medium containing 0.5% DMSO;d12 later, under inverted microscope, three groups cells were present myoblast like with fine tubular shape.3. Testing transfection cell(1)Immunofluorescence analysis After 17 days transfection cell, the above three groups of cells and BMSCs were analyzed by immunofluorescence with MyoD,MyoG and Desmin antibody.The results showed that three groups of cells were present green fluorescence.(2) Flow cytometry After 21 days transfection cell, three groups of transfection cell were tested by flow cytometry. The result was that all groups of gene expression (Desmin,MyoD,MyoG) reached above 96.4%(Group A,98.6%,99.9%,96.9%; Group B,99.9%,98.0%,99.1%;Group C,97.4%,96.4%,99.9%)(3)Real-Time PCR detection On the 28th day after transfection cell subculture, the above three groups of cells were analysed by real time quantitative PCR (three pairs of primers was MyoG, Myf5 and Desmin,and reference gene was GAPDH).The results showed that compared with BMSCs (normalized to 1), Myf5,MyoD and Desmin relative transcript level of the treatment groups increased(Group A,5.126±0.01,4.315±0.013.099;Group B, 4.484±0.01,3.124±0.01,2.889±0.01;Group C3.321,2.557±0.01,2.712±0.01)The research connstructed the eukaryotic expression vector pcDNA3.1-Myf5,it could induce BMSC of sheep differentiate into myoblast.The research provided a theoretical basis and technical support for the further application of BMSCs in tissue engineering.