The Expression And Effect Of Proto-oncogene C-src On Spermatogonial Stem Cells In Vitro

Objectives: To establish the culture system of spermatogonial stem cells in vitro; To study the effect of proto-oncogene c-src and the expression of caspase-3 gene on spermatogonial stem cells. 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 and TERT protein; MTT assay was used to detect to survival and proliferation of spermatogonial stem cells; RT-PCR was utilized to observe the expressions of c-src mRNA and caspase~Z mRNA.Results: Viabilities of cells separated by discontinuous Percoll gradient centrifugation and purified by the different cells' adhering speeds to dish were respectively 92. 5% and 92. 1% determinated by Trypan blue dye exclusion test. The percentages of spermatogonial stem cells expressing c-Kit and TERT were 90.8 ± 1.0% and 91.7 ± 1.2% respectively; Spermatogonial stem cells proliferated and selfrenewed significantly in the DMEM containing 10% NBS at culture 96h;The survival rate of spermatogonial stem cells decreased significantly at culture 12h in 10μmol/l AS-src group (P<0. 05); The expression of c-src mRNA decreased while the expression of caspase-3 mRNA increased in AS-src group.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; Identification...