哺乳动物雷帕霉素靶蛋白（mTOR）在SD大鼠睾丸中的表达及功能初探

发布时间：2018-09-19 14:05

【摘要】：目的：精子发生是男性生殖中的主要过程，精原细胞的不断分裂增殖又保证了精子发生的顺利进行。随着年龄的不断增长，男性精子的数量、质量出现下降趋势。mTOR信号转导通路在细胞增殖分化中发挥着中心调控作用，因此mTOR信号通路可能在精子发生过程中有着重要的地位。通过检测mTOR在大鼠睾丸组织中的表达规律，探讨其与精子发生的关系，为进一步研究具体机制打下重要基础。方法：采用免疫组化方法对mTOR进行组织学定位，RT-PCR方法研究mTOR在SD性成熟雄性大鼠心脏、肝脏、肾脏、脑、睾丸组织中的表达水平。利用荧光定量PCR、western blot方法检测mTOR在不同发育阶段的SD雄性大鼠睾丸中的表达情况。用HE染色观察不同发育阶段过程中SD大鼠睾丸组织形态学上的改变。构建雷帕霉素SD大鼠模型，用荧光定量PCR检测mTOR mRNA在正常组、雷帕霉素组、对照组中的表达情况。再采用western blot方法检测mTOR蛋白以及mTOR下游靶蛋白P-P70S6K/P70S6K的表达情况。结果： 1、免疫组织化学结果显示mTOR阳性免疫染色主要位于睾丸生精小管的精母细胞及少量初级精母细胞的胞质中。 2、与性成熟SD大鼠的其他各个主要器官相比，睾丸组织中mTOR的表达相对较高。随着SD大鼠年龄的增长，，mTOR在睾丸中的表达逐渐降低，并且伴随着生精小管空泡化的逐渐产生；mTOR下游靶蛋白P70S6K的磷酸化效率也逐渐降低。 3、雷帕霉素处理SD雄性大鼠后，睾丸中生精小管出现类似老龄鼠的空泡化以及mTOR下游靶蛋白P70S6K的磷酸化效率降低。结论 1、我们首次发现mTOR在大鼠生精小管精母细胞表达。 2、随着年龄的增长，由于mTOR表达下降及其对下游靶蛋白P70S6K的磷酸化能力下降，导致大鼠精子数量减少、生精小管空泡化增多。 3、mTOR通过对P70S6K的磷酸化，调控促进精原细胞增殖的蛋白合成，从而促进精子发生。
[Abstract]:Objective: spermatogenesis is the main process in male reproduction. The continuous division and proliferation of spermatogonia ensure the smooth progress of spermatogenesis. With the increasing of age, the quantity and quality of male spermatozoa decrease. The signal transduction pathway of mTOR plays a central role in cell proliferation and differentiation. Therefore, mTOR signaling pathway may play an important role in spermatogenesis. The relationship between mTOR and spermatogenesis was studied by detecting the expression of mTOR in rat testis, which laid an important foundation for further study of the specific mechanism. Methods: the expression of mTOR in the heart, liver, kidney, brain and testis of male SD rats was studied by immunohistochemical method. Fluorescence quantitative PCR,western blot was used to detect the expression of mTOR in the testis of SD male rats at different developmental stages. HE staining was used to observe the morphological changes of testis in SD rats during different developmental stages. The rat model of rapamycin SD was established, and the expression of mTOR mRNA in normal group, rapamycin group and control group was detected by fluorescence quantitative PCR. Western blot was used to detect the expression of mTOR protein and mTOR downstream target protein P-P70S6K/P70S6K. Results: 1. Immunohistochemical results showed that mTOR immunoreactive staining was mainly located in spermatocytes of testicular seminiferous tubules and in the cytoplasm of a small number of primary spermatocytes. 2. Compared with the major organs, The expression of mTOR was relatively high in testis. With the age of SD rats, the expression of mTOR in testis gradually decreased, and accompanied by the production of seminiferous tubule vacuolation. The phosphorylation efficiency of P70S6K, the downstream target protein of mTOR, was also decreased. 3After rapamycin was treated with rapamycin, vacuolation of seminiferous tubules in testis and phosphorylation of mTOR downstream target protein P70S6K were observed in testis of male rats. Conclusion 1. We found that mTOR was expressed in spermatocytes of rat seminiferous tubules for the first time. With the increase of age, the expression of mTOR decreased and its phosphorylation ability to downstream target protein P70S6K decreased. The result showed that the number of spermatozoa decreased and the vacuolation of seminiferous tubules increased. 3mTOR promoted spermatogenesis by phosphorylation of P70S6K and regulation of protein synthesis of spermatogonia proliferation.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R321

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