Induction Of Myoblasts From Sheep Umbilical Cord Mesnchymal Stem Cells By Mouse MyoD

It has been confirmed that umbilical cord mesenchymal stem cells have potential to differentiate into osteoblasts, chondrocytes, adipocytes, cardiomyocytes, nerve cells, liver cells, and myoblasts. In order to establish a method that how to induce umbilical cord mesenchymal stem cells of sheep into myoblasts, mouse MyoD-pcDNA3.1 eukaryotic expression vector was constructed, which was transfected into umbilical cord mesenchymal stem cells of sheep. At last, cell morphology, protein expression and gene expression were detected, and sheep myoblasts were obtained eventually. Main results and conclusions were showed as follows:1.Cloning of mouse MyoD gene Refering to GenBank MyoD gene CDS （NM₀10866）, mouse MyoD gene CDS was amplified by RT-PCR and then it was linked into the pcDNA3.1（+） vector to obtain MyoD-pcDNA3.1 eukaryotic expression vector; mouse embryonic fibroblasts cells were transfected with MyoD-pcDNA3.1, Real-Time PCR, immunofluorescence, flow cytometry were used to detect whether the vector had biological activity. The results showed that MyoD-pcDNA3.1 had biological activity in mouse embryonic fibroblasts;2.Umbilical cord mesenchymal stem cells of sheep were transfected by MyoD-pcDNA3.1 Thawing umbilical cord mesenchymal stem cells of sheep which was cryopreserved, the following treatment were performed respectively,when the cells reached approximately 80% confluence:1) cells were transfected with MyoD-pcDNA3.1 endofree plasmid into by lipofect transfection reagent, and added DMSO into the medium （group A）; 2) cells were transfected with MyoD-pcDNA3.1 endofree plasmid into by lipofect transfection reagent（group B）; 3) Cells was cultured by the medium which was supplemented with 0.5% DMSO （group C）. After these treatment, cells were detected respectively as follows:（1） Detection of cell morphology In 12 days, after three kinds of treatment,the cells were observed by the inverted fluorescence microscope, All three groups of cells changed from spindle like into myoblasts state, that is an elongated tubular, spiral-shaped disappearing.（2） Immunofluorescence In 8 days, MyoD and Desmin were detected,in spite of the three kinds of cells showed positive expression of MyoD, compared with group A and B, group C had lower relative expression levels;All groups were expressed Desmin, and no significant differences in the relative expression level; In 16 days, MyoD,MyoG and Desmin were detected, compared with the 8 days time,all groups had lower relative expression levels of MyoD, but the expression of Desmin was no significant differences.however, the expression of MyoG was very low.（3） Flow cytometry The cells were detected by flow cytometry, Three groups of cells express myoblast-specific factors MyoD, Desmin and MyoG, and rate of positive cell of three proteins was more than 86.6%（group A:98.6%,99.0% and 99.0%; group B:93.5%,99.5% and 97.4%; group C:99.3%,99.5% and 86.6%）.（4） Real-Time PCR Using Real-Time PCR method to detect the relative expression of MyoD, MyoG and Desmin of three group cells, compared with non-transfected cells （normalized to 1）, the relative expression of MyoD,MyoG and Desmin were all increased （group A:3.217 ± 0.01 times,4.345 ± 0.01 times and 5.107 ± 0.01 times; group B:2.046 ± 0.01 times,2.389 ± 0.01 times and 5.489 ± 0.01 times; group C:3.713 ± 0.01 times,1.861 ± 0.01 times and 5.271 ± 0.01 times）.In summary, the results showed that MyoD-pcDNA3.1 eukaryotic expression vector had the function of inducing umbilical cord mesenchymal stem cells of sheep into myoblasts.This study laid the foundation for further research into mesenchymal stem cells during muscle damage repair.