Isolation&Purification Of Myoblasts And Construction Of Flrg Transgenic Cell Lines

Myopathy, ischemic cardiomyopathy and type II diabetes are all caused by musclecell defect. At present there are no effective treatments. Myoblasts transplantation is ableto alleviate or cure these diseases recently. As a kind of unipotent stem cell, myoblasts candirectional differentiate into muscle cells and fuse with each other to form new myofiber.After transplantation, myoblasts will produce contractive albumen which can enhance thesystolic function of skeletal muscle and myocardial cell. Thus they can be used to treatmyopathy and myocardial infarction effectively. Besides, myoblasts are capable of naturalfusion and achieving genome therapy with nuclear transferring. Thus they can be used totreat type II diabetes. At present, the method of isolating and purifying myoblasts is still inresearch stage. In order to apply it into clinic, amounts of research are needed. Thus, thepurpose of this thesis is using Wistar rats as model to establish the method of isolating andpurifying myoblasts which can be used in clinic. The main research results are as follows.1． With two enzymes mixture digestion method, primary myoblasts have beensuccessfully isolated from Wistar rats and purified with differential attachmenttechnique. Purification rate are as high as90%above. Then, the in vitro culturesystem of primary myoblasts has been established.2． Myoblasts have been identified both on gene and immune level with desminwhich is a gene specifically expresses in myoblasts.3． flrg-pcDNA3.1(-) plasmid has been successfully constructed. After transfectingit into myoblasts, the results of qRT-PCR and western blotting assays haveproved that over-expressing worked, which also has claimed the successfulconstruction of flrg transgenic cell lines.4． The results of counting cell number have proved that over-expressing flrg genecould promote myoblasts’ capacity of proliferation. Then the result of flowcytometry assay showed higher proportion of S phase myoblasts in transfectedmyoblasts than that of control. The results indicated the protein FLRG furthermyoblasts’ growth through accelerating the process of transition from G0/G1 phase to S phase.