The Expression And Effects Of Proto-oncogene C-raf In Spermatogonial Stem Cells In Vitro

Objective:To establish the culture system of rat spermatogonial stem cells in vitro;To study the effects and the mechanism of c-raf on spermatogonial stem cells culture in vitro.Methods:Spermatogonial stem cells were separated by discontinuous Percoll gradient centrifugation from 9-day-old SD rat testes and purified by the different cells'adhering speeds to dish;The spermatogonial stem cells were identified by immunohistochemistry of expressing c-Kit protein;To identify the homology between the amplified spermatogonial stem cell'DNA and c-raf by sequencing and SeqMan;MTT assay was used to detect to survival and proliferation of spermatogonial stem cells; RT-PCR was utilized to observe the expressions of c-raf mRNA and caspase-3 mRNA in spermatogonial stem cells;TUNEL was used to assay the apoptosis index of spermatogonial stem cells.Results: The survival rate of cells separated by discontinuous Percoll gradient centrifugation and purified by the different cells' adhering speeds to dish was 92.3% and 91.5% separately;The percentages of spermatogonial stem cells expressing c-Kit was 90.1%;Spermatogonial stem cells proliferate increased significantly in the DMEM containing 10% NBS and decreased continously in the DMEM without NBS;The highest proliferation rate of spermatogonial stem cells was at culture 120h;The homology between the amplified spermatogonial stem cell'DNA and the c-raf is 98%;The survival rate of spermatogonial stem cells decreased significantly at culture 12h in 15μmol/L c-raf AON group(P<0.05);The expression of c-raf mRNA decreased and the expression of caspase-3 mRNA increased in c-raf AON group(P<0.05);The apoptotic index of c-raf AON group(40.5%) was much higher than that of the c-raf MON group(29.4%) and control group(30.2%)(P<0.05).Conclusions: Discontinuous Percoll gradient centrifugation combined with the different cells'adhering speeds to dish is an effective method for isolating and purifying spermatognial stem cells;C-Kit could be used as a molecular marker in identifying the spermatogonial stem cells;The spermatogonial stem cells proliferate significantly at culture 120h in DMEM containing 10%NBS in vitro and the survival rate of spermatogonial stem cells decreased continously in the DMEM without NBS;The c-raf is expressed in the rat's spermatogonial stem cells;The survival of spermatogonial stem cells could be prolonged by c-raf;C-raf mightly inhibit the apoptosis of spermatogonial stem cells by decreasing the expression of the caspase-3.