Proteomics Analysis Of Human Embryonic Germ Cells Induced To Differentiate Into Cardiomyocytes

Objectives:1. To induce hEGCs to differentiate into cardiomyocytes by ascorbicacid.2. To detect the gene expressions of GATA-4、ACTC1and others on hEGCs andcardiomyocytes induced from hEGCs,using the semi-quantitative RT-PCR method.3.To analysis the interaction relationship between the proteins related to the process ofdifferentiation with the method of bioinformatics.Methods: Through tissue culture of the gonadal ridge taken from human embryosaged5-10weeks, with the human embryo fibroblast as feeder cells, hEGCs wereobtained. hEGCs which grew well and steadily were seeded in the petri dish forsuspension culture. After7days, embryoid bodies (EBs) derived from hEGCs wereplaced in gelatin-treated24-well plate, and incubated with differentiation mediumcontaining0.1mg/ml retinoic acid. Immunofluorescence microscopy was used to detectthe expression of connexin43(Cx43) on cardiomyocytes differentiated for2weeks, andthrough the CCK-8method to detect the cell proliferation rate. Classified asexperimental group (the cardiomyocytes induced from hEGs) and control group (thehEGCs without differentiation), the RNA of the different groups were extracted todetect the quality and concentration of RNA with the uv spectrophotometery. TheRT-PCR (RT-polymerase chain reaction) were performed on GATA-4、ACTC1、nkx2.5、KRIT1、Anxa2、VDAC1and CSRP3,and8μL PCR products were take to carryout the1%agarose gel electrophoresis and gel camera to save up images. Thedifferences of space structure of the proteins from different groups, and their interactionrelationship were researched through the "NCBI" and related website.Results: hEGCs of subculture proliferated strongly on human embryo fibroblastfeeder cells，and the shape of the clone was nest-like. Cx43was positive expression insome polygonal differentiated cells induced by ascorbic acid for2weeks，the shape ofthe cells was fusiformis mostly，and the differentiated cells proliferated constantly. The results of semi-quantitative RT-PCR showed that, the cardiomyocytes derived fromhEGCs differentiation expressed myocardial special genes: GATA-4, ACTC1andNkx2.5; both groups of cells expressed Anxa2,KRIT1,which were not myocardialspecific gene,however, the expression intensities were different from each other, whichwere consistent with the mass spectrometry analysis results. There were intimaterelationships between GATA-4, Nkx2.5and the varion kinds of cardiac troponin.Conclusions: Ascorbic acid can induce hEGCs to differentiate intocardiomyocytes. Through screening to identify some of the important proteins related todiversification, and study their structure and function which considered to be thefoundation for us to find out the process and regulation mechanism of hEGCs inducedto differentiate into cardiomyocytes.