【作者】 余荣娇；

【导师】 徐斯凡；

【作者基本信息】 江西医学院 ， 生理学， 2005， 硕士

【摘要】 目的:摸索完善精原干细胞分离、纯化、培养的方法与技术;研究EGF 对体外培养的精原干细胞自我更新﹑增殖过程中所起的调控作用;研究EGF 对体外培养的精原干细胞的影响是否特异性地通过表皮生长因子受体介导;以及探讨EGF 影响精原干细胞增殖中的可能的作用机制。建立完善的精原干细胞体外培养体系,为精原干细胞的体外大量扩增提供技术和方法,为治疗男性不育症等提供相关技术。方法:不连续Percoll 梯度液分离纯化精原干细胞,并应用选择性贴壁法进一步纯化精原干细胞;台盼蓝排斥实验确定精原干细胞的活力;c-kit 细胞免疫组化鉴定细胞类型;HE 染色法,根据细胞形态镜下随机取10 个视野细胞记数进行细胞纯度分析;细胞计数法研究不同浓度的表皮生长因子对精原干细胞增殖的效应;MTT 法研究表皮生长因子对精原干细胞增殖的效应:1、不同浓度表皮生长因子对精原干细胞增殖的效应;2、表皮生长因子对精原干细胞增殖的时间性效应;3、加入表皮生长因子受体（epidermal growth factor receptor,EGFR）抑制剂AG1478 观察表皮生长因子受体的可能性作用;4、加入MAPK-ERK 信号通路特异性抑制剂PD98095﹑JAK-STAT 信号通路特异性抑制剂AG490 及细胞内Ca²⁺螯合剂探讨表皮生长因子对精原干细胞增殖作用的可能机制。结果:（1）台盼蓝实验显示细胞活力维持于88.73%±0.85%。（2）c-kit 细胞免疫组化结果显示分离得到细胞为精原干细胞。（3）细胞纯度为90.48%±1.78%。（4）细胞计数法结果显示与对照组相比表皮生长因子各剂量组对精原干细胞均有增殖作用（p﹤0.01）, 20ng/ml 剂量组增殖作用最显著。（5）MTT 结果也显示各实验组比对照组细胞数量均有显著增多（p﹤0.01）,并且20ng/ml 剂量组的增殖作用最明显;（6）20ng/ml 表皮生长因子对精原干细胞的增殖效应呈时间依赖性,在加入表皮生长因子的第2d 到第4d 增殖作用最明显;（7）加入表皮生长因更多还原

【Abstract】 Objectives: To study the stimulating effects of epidermal growth factor （EGF） on the self-renew and/or proliferation of the type A Spermatogonial stem cells and its mechanisms. Furthermore,to explore the effect of EGF on the type A Spermatogonial stem cells is whether or not induced by epidermal growth factor receptor（EGFR）.Finally,to establish the culture system of spermatogonial stem cells in vitro and to offer the therapy technique for the male sterility. Methods: Type A spermatogonial stem cells were isolated from 9 day-old SD rat testes by discontinuous Percoll gradient centrifugation. The cell viability was determined by Trypan blue dye exclusion test.Anti c-kit antibody was applied to identify spermatogonial stem cells. The percentage of spermatogonial stem cells were determined by the cells shape through Hematoxylin-eosin coloration.The spermatogonial stem cells were cultured with EGF at different concentrations in vitro and the quantity of live cells was detected by MTT assay at different time. EGF and AG1478（EGF receptor inhibitor）,PD98059（mitogen-activated protein kinase inhibitor, MAPK）,AG490（janus kinase inhibitor, JAK-STAT） and BAPTA(intracellular Ca²⁺ chelator) were added respectively to the medium and the percentage of the live cells was determined by MTT assay after incubation. Results: The cell viability of stermatogonial stem cells in the medium was 88.73%±0.85%. 90.48%±1.78% of the cells which we harvested was spermatogonial stem cells. EGF significantly enhanced the number and percentage of cell survival in a dose-dependent and time-dependent manner （P<0.01）.AG1478 could reduce the effects of EGF significantly（P﹤0.5）.Compared with EGF group, the percentage of cell survival in PD98059 group,AG490 group and BAPTA group reduced markedly（P﹤0.01）.Conclusions: EGF could promote spermatogonial stem cells proliferation obviously. It suggests that EGF play a role in the regulation of survival and /or proliferation of spermatogonial stem cells. Activation of epidermal growth factor receptor may play a very important role in EGF induced the spermatogonial stem cells proliferation. The effect of EGF on the spermatogonial stem cells may be mediated by the activations of intracellular MAPK system and/or STAT system. In addition, intracellular Ca2+ may also play a role.更多还原