表皮生长因子在精原干细胞中的作用及其机理研究

发布时间：2018-06-16 12:30

本文选题：精原干细胞 + 表皮生长因子　；参考：《江西医学院》2005年硕士论文

【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Ca~(2+)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㩳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㩳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
[Abstract]:Objective: to explore the methods and techniques of isolation, purification and culture of spermatogonial stem cells, and to study the self-renewal of EGF on spermatogonial stem cells cultured in vitro. The effects of EGF on the proliferation of spermatogonial stem cells in vitro were studied. The effects of EGF on the proliferation of spermatogonial stem cells were investigated by epidermal growth factor receptor (EGF), and the possible mechanism of EGF on the proliferation of spermatogonial stem cells was discussed. To establish a perfect culture system of spermatogonial stem cells in vitro, to provide techniques and methods for the in vitro expansion of spermatogonial stem cells, and to provide related techniques for the treatment of male infertility. Methods: spermatogonial stem cells were isolated and purified by discontinuous Percoll gradient solution and further purified by selective adherent method, and the viability of spermatogonial stem cells was determined by trypan blue rejection assay. According to cell morphology, 10 visual field cells were randomly selected for cell purity analysis. Effects of different concentrations of EGF on proliferation of spermatogonial stem cells; MTT assay on proliferation of spermatogonial stem cells; effects of EGF on proliferation of spermatogonial stem cells: 1; EGF at different concentrations on proliferation of spermatogonial stem cells The temporal effect of EGF on the proliferation of spermatogonial stem cells was 3. The possibility of EG1478, an inhibitor of epidermal growth factor receptor, was used to observe the possibility of EGF receptor, and MAPK-ERK signaling pathway was added specifically to inhibit the proliferation of spermatogonial stem cells. Preparation of PD98095 / JAK-STAT signaling pathway specific inhibitor AG490 and intracellular CaFU 2) chelating agent to explore the possible mechanism of EGF on proliferation of spermatogonial stem cells. Results the cell viability was maintained at 88.73% 卤0.85%.(2)c-kit cells by trypan blue assay. The results of immunohistochemistry showed that the cells isolated were spermatogonial stem cells. The purity of the cells was 90.48% 卤1.78%. The proliferation of spermatogonial stem cells in the dose group was significantly higher than that in the control group, and the proliferative effect in the 20ng/ml group was the most significant. The results showed that the number of cells in each experimental group was significantly higher than that in the control group, and the proliferative effect of the 20ng/ml group was the most obvious in the 20ng/ml group with 20 ng / ml epidermal growth. The proliferation of spermatogonial stem cells was time-dependent. Epidermal growth factor was added on the 2nd to 4th day after adding epidermal growth factor (EGF) and epidermal growth factor (EGF) was added to the epidermal growth factor (EGF).
【学位授予单位】：江西医学院
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R321

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