Studies On The Mechanism Of Proto-oncogene C-src On The Proliferation Of Rat Spermatogonial Stem Cells In Vitro

Objectives:To establish the culture system of spermatogonial stem cells in vitro;To study the mechanism of proto-oncogene c-src on the proliferation of rat spermatogonial stem cells.Methods:The percoll discontinue density gradient centrifugation, followed by removal of contaminating somatic cells through adhesion to plastic dishes, was used to purify the spermatogonial stem cells. The special antibody of telomerase-reverse transcriptase ( TERT) was used to identify the spermatogonial stem cells. The MTT was used to observe the growth of the spermatogonial stem cells in vitro.; RT-PCR was utilized to observe the expressions of c-src mRNA and western blot was used to observe the c-Src,p-STAT3 and p-ERK1/2 contents.Results:The percentage of cells expressing TERT was 90.5 % after isolated the rat spermatogonial stem cells; The proliferation inhibition rate of spermatogonial stem cells was 12.6% after being treated with 10μmol/L antisense-src ODNs for 12 h(P<0.05);The expression of c-src mRNA decreased significantly(P<0.05).Compared with the control group,The c-Src, p-STAT3 and p-ERK1/2 contents decreased 33.8 %(P<0.01),45.3 %(P<0.01) and 38.4 %(P<0.01) respectively after transfected antisense c-src ODNs.Conclusions: The percoll discontinue density gradient centrifugation, followed by removal of contaminating somatic cells through adhesion to plastic dishes is an efficiency method for isolation of rat spermatogonial stem cells.The immunocytochemistry of TERT could be used to identify spermatogonial stem cells.Proto-oncogene c-src can proliferate the spermatogonia stem cells and the effect of c-Src on spermatogonial stem cells may correlate with the p-STAT3 and p-ERK1/2 protein.