Research On Crystallization And Cell Biological Function Of Muscular Atrophy-related Protein MEF2C

Amyotrophy caused by weightlessness enviroment is hamfule to the healthy of astronaut.Studying the treatment and prevention of amyotrophy caused by weightless is critically important for the development of manned spacecraft engineering in our country.MEF2C(Myocyte enhancer factor 2C)was found widely expressed in many types of cells and tissues,including skeletal muscle cells,smooth muscle cells,cardiomyocytes,neurons,chondrocytes,neural crest cells,endothelial cells,spleen,brain,liver and so on.After knocking down MEF2C in mouse,muscle fibers were deteriorated rapidly after birth,resulting in myocardial,skeletal muscle and smooth muscle development disorders.For mouse,after space flight,expression level of MEF2C was decreased signficatly,indicating that MEF2C is correlative with myotrophy under weightlessness enviroment.Therefore,we studied the crystallization and function of MEF2C,and it will be helpful to reveal the molecular mechanism of MEF2C signal pathway,and also provided the theoretical basis and assistance for the treatment of MEF2C-related diseases.Base on these information,we have carried out a series of work as followed:(1)Prokaryotic and eukaryotic expression reconbinated vectors of MEF2C-pET30a(+)and MEF2C-pcDNA3.1(+)were constructed;(2)Expression,purification and crystallization of MEF2C.The optimal expression condition was obtained(temperature was 37℃,1 mM IPTG,and induction time was 6 h,the more than 90% purity proteinwas obtained by nickel ion affinity and gel filtration chromatography,and the complex crystal of MEF2C and DNA fragment was screened out,and its resolution was 8 ?;(3)Constructed the C2C12 cells with different MEF2C expression level.The effect of MEF2C expression level on cell morphology was detected.The morphological growth rates of the overexpression group was increased by 79%,and decreased by 6% siRNA group for siRNA group,indicating that the expression of MEF2C protein can promote cell expansion;(4)The effect of MEF2C expreesion level on cell proliferation and cell cycle was detected.Cell proliferation was increased by 33% for overexpression group,and decreased by 67% for siRNA group,indicating that MEF2C expression is positively correlated with cell proliferation.Cell transfection was performed 3 days later using flow cytometry for cell cycle detection.The S phase of the cells were increased by 26% for overexpression group,and decreased by 11% for siRNA group;(5)The effect of MEF2C expreesion level on cell diffraction was detected.In the early stage of differentiation,the variation expression level of Myo G was most significant,MyHC for the middle stage,and Atrogin for the late stage;(6)The effect of MEF2C expreesion level on its directly-interacting protein was detected.The most significant directly-interacting protein is MyoD,HDAC4,Notch2,TEF1,and ERK5;(7)The effect wightlessness condition on the expression of MEF2C and its directly-interacting protein was detected.Random three-dimensional rotation technique and mouse tail suspension technique was used to simulate the weightlessness condition,the most significant protein is MEF2C,MEF2C,MyoD,and HDAC4.