The Study Of MicroR-133 Induced Sheep Mesenchymal Stem Cells Differentiate Into Myoblasts

Individual miRNAs have been shown to regulate skeletal myogenesis in cell culture system as well as in developing and adult embryos.Mesenchymal stem cell is a kind of self-renewal and multi-directional differentiation potential of cells. In order to explore the method of miR-133 induce the sheep bone marrow and umbilical cord mesenchymal stem cells differentiate into myoblasts,this research did work as follows. miR-133 was transfected into sheep umbilical cord, bone marrow mesenchymal stem cells by liposome respectively, and then the cells was detected.Research results and conclusions shown as follows:1. Transfection of sheep umbilical cord and bone marrow mesenchymal stem cellSheep umbilical cord mesenchymal and bone marrow mesenchymal stem cells were transfected with pcDNA3.1-miR-133a2 expression vector and miR-133mimics by cationic liposome;2. Subculture and morphological observation of transfected cellsIn 28 days,after above four kinds of treatment,the cells were observed by the inverted fluorescence microscope,and cells shown fusiform or spindle; when the cells reached 80% confluence, cells grow orderly, and tended to fusion;3.The detection of transfected cells(1) qRT-PCR detection Before transfection, Skeletal muscle specific gene MyoD and MyoG couldn’t be detected using real-time quantitative PCR method, but both of them could be detected in transfected cells; Desmin, Tnni2, Myf5 expression was relative increased;(2) Immunofluorescence detection Compared with the control group, the transfected cells in 4 groups, MyoD and Desmin protein could be detected,but a-actin could not be detected;(3) Flow cytometry detection When the pcDNA3.1-miR-133 a2 expression vector and mimics were transfected into sheep umbilical cord and bone marrow mesenchymal stem cells respectively, using flow cytometry instrument to detect myoblast specific protein MyoD, Desmin and α-actin. The rate of MyoD positive cell is 12.2% and 29.6% in expression vector transfected group; The rate of Desmin positive cell is 99.3% and 99.5% in expression vector transfected group; The rate of a-actin positive cell is 0.917% and 9.24% in expression vector transfected group;The rate of MyoD positive cell is 99.3% and 43.7% in mimics transfected group; The rate of Desmin positive cell is 99% and 100% in mimics transfected group; The rate of a-actin positive cell is 1.07% and 1.68% in mimics transfected group;In this study, the results shown that sheep umbilical cord mesenchymal stem cells and sheep bone marrow mesenchymal stem cells could be induced into myoblast by miR-133 successfully, and the data provide a basis for the muscle cells and skeletal muscle cell differentiation and further explore the mechanism of inducing mesenchymal cells.