Study On The In Vitro Culture Of Murine SSCs And SCs And The Toxic Effect Of Busulfan On Them

Spermatogonial stem cells(SSCs) are the source of the spermatogenesis that plays an important role in the process of genetic information transfer of male animals. Sertoli cells(SCs)is the only somatic cell in testis which is closely linked with the spermatogenic cells. SCs and SSCs are tightly fitted and interacted together to mantain a stable microenvironment that ensures a series of physiological functions concerning SSCs proliferation, differentiation and apoptosis in testis. Recently, the recipient preparation for murine SSCs transplantation has drawn increased attention. Busulfan could act on G0/G1 phase cells damaging cells by alkylated DNA,and finally resulting in depletion of endogenous spermatogneic cells. Therefore, busulfan has been used widely in the preparation of murine SSCs transplantation recipient and the sperm regeneration model commonly. However, till now little information is available about the effect of busulfan on SSCs and SCs. In this study, we successively used the method of two-step enzymatic digestion, Magnetic Activated Cell Sorting(MACS) and differential adhesion to separated SSCs and SCs. Meanwhile,we chose vimentin and Factor associated suicie ligand(FasL) protein immunofluorescence staining and salkaline phosphatase selection(AP) staining to identify SCs and SSCs respectively. 0.01 mg/mL Mitomycin C(MMC) was used to pretreat SCs,and then the SSCs isolated by MACS was transferred to coculture with the SCs feeder layer pretreated by MMC. Moreover, in order to optimize the density of SCs feeder layer pretreated with MMC for SSCs in vitro, we also studied the effects of three different densities(1?104cells/m L, 2?104cells/mL and 1?105cells/mL) of SCs feeder layer on the proliferation of SSCs. Tthiazolyl blue tetrazolium bromide(MTT) was also ulilized to curve the growth of SSCs with 2?104 cells/mL SCs feeder or free-feeder to study the effect of SCs feeder on the SSCs proliferation in vitro. To study the adverse effect of busulfan on the proliferation and apoptosis of SCs and SSCs in vitro, SCs was treated with(2.46 mg/L(10 μmol/L)、12.3 mg/L(50 μmol/L)�'� 24.6 mg/L(100 μmol/L)) three different concentrations of busulfan, SSCs with(10 ng/L, 1μg/L) two concentration. The results showed that:(1) Vimentin and FasL IF staining, and AP staining were positive demonstrating that the SCs and SSCs was successfully isolated;(2) The proliferation of SCs feeder layer was successfully inhibited with 0.01 mg/mL MMC treatment;(3) 2?104 cells/mL density of SCs feeder layer is optimal to coculture with SSCs in vitro, which was evaluated further by MTT effectively;(4) The proliferation of SCs was inhibited after24.60mg/L(100 μmol/L) busulfan treatment, with the obvious decrease of the cell body. SCs were damaged by Busulfan in a dose-dependent manner. Furthermore, the proliferation of theSSCs were obviously inhibited after exposure to 10 ng/L and 1 μg/L busulfan, and the adverse effect was also altered in a dose-dependent manner. Meanwhile, after busulfan(10ng/L, 1μg/L)treatment, the refraction of SSCs was weakened and cells represented a loose state with the descreasing number. This result indicated that busulfan inhibit the proliferation of SSCs. The study of busulfan toxicity mechanism to SSCs is significant for the preparation of SSCs transplant recipients and the research of spermatogenesis mechanism providing a new research method to settle the proble of male infertility.