原癌基因c-src在精原干细胞中表达及作用的研究

发布时间：2018-03-11 06:45

本文选题：精原干细胞　切入点：原癌基因c-src　出处：《南昌大学》2006年硕士论文　论文类型：学位论文

【摘要】：目的：探索及完善精原干细胞的分离、鉴定和体外培养技术；研究原癌基因c-src对体外培养的精原干细胞存活及与凋亡密切相关的caspase-3表达的影响。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和TERT细胞免疫组织化学鉴定精原干细胞；用MTT比色法检测精原干细胞的存活及增殖情况，观察AS-src对精原干细胞存活的剂量性影响及AS-src对精原干细胞存活的时间性影响；用RT-PCR检测c-src mRNA和caspase-3 mRNA的表达情况。结果：非连续密度梯度及差速贴壁法分离精原干细胞，台盼蓝染色计数细胞存活率分别为92.5％和92.1％；分离后所获取的细胞c-Kit和TERT免疫组织化学阳性细胞数平均值分别为90.8±1.0％和91.7±1.2％；精原干细胞在含10％NBS的DMEM中培养96h时增殖达高峰；10μmol／l AS-src作用12h后精原干细胞存活率下降(P＜0.05)，于24h，48h，，72h持续下降(P＜0.01)；与S-src和空白对照组相比，AS-src组c-src mRNA水平明显下调，而caspase-3 mRNA表达水平明显上调。结论：非连续密度梯度结合差速贴壁法是分离精原干细胞的有效方法；以c-Kit及TERT作为marker分子通过免疫组织化学检测为精原干细胞的鉴定提供较为充分的依据；精原干细胞可以在含10％NBS的DMEM培养基中短期增殖；c-Src可以促进精原干细胞的存活；c-Src可能通过PI-3K／PKB途径，导致Caspase-3磷酸化从而抑制精原干细胞的凋亡。
[Abstract]:Aim: to study the effects of proto-oncogene c-src on the survival of spermatogonial stem cells and the expression of caspase-3, which is closely related to apoptosis, and to explore and improve the isolation, identification and in vitro culture of spermatogonial stem cells. Methods: spermatogonial stem cells were isolated by discontinuous density gradient centrifugation and differential adherent method, c Kit and TERT cells were identified by immunohistochemistry, survival and proliferation of spermatogonial stem cells were detected by MTT colorimetry. To observe the dose effect of AS-src on the survival of spermatogonial stem cells and the effect of AS-src on the survival time of spermatogonial stem cells, and to detect the expression of c-src mRNA and caspase-3 mRNA by RT-PCR. Results: spermatogonial stem cells were isolated by discontinuous density gradient and differential adhesion. The cell survival rate of Trypan blue staining was 92.5% and 92.1, the average number of c-Kit and TERT immunocytochemical positive cells were 90.8 卤1.0% and 91.7 卤1.2, respectively. The proliferation of spermatogonial stem cells reached the peak after 96 h culture in DMEM containing 10NBS. After treated with 10 渭 mol/l AS-src for 12 h, the survival rate of spermatogonial stem cells decreased significantly (P < 0.05), and the level of c-src mRNA in AS-src group decreased significantly compared with S-src group and control group. The expression of caspase-3 mRNA was up-regulated. Conclusion: discontinuous density gradient method combined with differential adherent method is an effective method for isolation of spermatogonial stem cells, c-Kit and TERT as marker molecules provide sufficient basis for identification of spermatogonial stem cells by immunohistochemistry. The short-term proliferation of spermatogonial stem cells in DMEM medium containing 10NBS may promote the survival of spermatogonial stem cells through PI-3K/PKB pathway, which may lead to Caspase-3 phosphorylation and inhibit the 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】：2006
【分类号】：R321

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