c-raf在精原干细胞中的表达及作用的研究

发布时间：2018-04-27 05:35

本文选题：大鼠精原干细胞 + c-raf　；参考：《南昌大学》2007年硕士论文

【摘要】： 目的:完善大鼠精原干细胞的分离和体外培养技术;研究c-raf对精原干细胞体外培养的存活作用及机制。方法:非连续密度梯度离心及差速贴壁法分离精原干细胞;c-kit细胞免疫组织化学鉴定精原干细胞;基因测序和SeqMan、检验精原干细胞扩增DNA和c-raf的同源性;MTT比色法检测体外培养精原干细胞的存活及增殖情况,观察c-raf AON对精原干细胞体外培养存活的量效关系及c-raf AON对精原干细胞体外培养存活的时效关系;RT-PCR检测c-raf mRNA和caspase-3 mRNA的表达情况;TUNEL检测c-raf AON对精原干细胞凋亡的影响。结果:非连续密度梯度离心及差速贴壁法分离的精原干细胞,经台盼蓝染色计数其存活率分别为92.3%和91.5%;分离后的精原干细胞经c-kit鉴定其纯度为90.1%;精原干细胞在含10%NBS的DMEM中培养120h时增殖达高峰,在无10%NBS的DMEM中培养其存活率持续下降;精原干细胞扩增DNA和c-raf的同源性为98%;15μmol/L c-raf AON作用12h后精原干细胞存活率下降(P0.05),于18h后存活率持续下降(P0.05);与对照组相比c-raf AON组的c-raf mRNA水平明显下调(P0.05),而caspase-3 mRNA表达水平明显上调(P0.05);c-raf AON组凋亡指数(40.5%)明显大于对照组(30.2%)和错义组(29.4%)的凋亡指数(P0.05)。结论:非连续密度梯度离心结合差速贴壁法是分离精原干细胞的有效方法;c-kit可作为鉴定精原干细胞的分子标志物;精原干细胞在含10%NBS的DMEM培养基中短期增殖;c-raf在九天的雄性SD大鼠的精原干细胞上表达;c-raf可能通过抑制凋亡而促进精原干细胞存活;c-raf可能通过下调capspase-3的表达而抑制精原干细胞凋亡。
[Abstract]:Aim: to improve the isolation and in vitro culture of rat spermatogonial stem cells and to study the survival effect and mechanism of c-raf on spermatogonial stem cells in vitro. Methods: spermatogonial stem cells were isolated by discontinuous density gradient centrifugation and differential adherent method. The survival and proliferation of spermatogonial stem cells in vitro were detected by DNA sequencing and SeqMan. the homology of DNA and c-raf was detected by MTT colorimetry. To observe the dose-effect relationship of c-raf AON on the survival of spermatogonial stem cells in vitro and the time-dependent relationship of c-raf AON to the survival of spermatogonial stem cells in vitro, the expressions of c-raf mRNA and caspase-3 mRNA were detected by RT-PCR. Tunel was used to detect the effect of c-raf AON on apoptosis of spermatogonial stem cells. Results: spermatogonial stem cells were isolated by discontinuous density gradient centrifugation and differential adhesion. The survival rate of spermatogonial stem cells was 92.3% and 91.5% by trypan blue staining, the purity of spermatogonial stem cells was 90.1% by c-kit, the proliferation of spermatogonial stem cells reached the peak after 120 h culture in DMEM containing 10%NBS, and the survival rate of spermatogonial stem cells cultured in DMEM without 10%NBS continued to decline. The homology of amplified DNA and c-raf of spermatogonial stem cells was 980.15 渭 mol/L c-raf AON, the survival rate of spermatogonial stem cells decreased after 12h, the survival rate of spermatogonial stem cells continued to decrease after 18h, and the c-raf mRNA level of c-raf AON group was significantly lower than that of control group, but the expression level of caspase-3 mRNA was lower than that of control group. The apoptotic index of P0.05 AON group was significantly higher than that of control group (30.2%) and missense group (29.444). Conclusion: discontinuous density gradient centrifugation combined with differential adhesion is an effective method for isolation of spermatogonial stem cells. C-kit can be used as a molecular marker for identification of spermatogonial stem cells. Short-term proliferation of spermatogonial stem cells on DMEM medium containing 10%NBS c-raf expression of c-raf on spermatogonial stem cells of nine-day male SD rats may inhibit the survival of spermatogonial stem cells by inhibiting apoptosis. C-raf may inhibit the expression of capspase-3 by down-regulating the expression of capspase-3. Apoptosis of spermatogonial stem cells.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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