Rap1在大鼠睾丸发育中的表达以及在生精细胞凋亡中的研究

发布时间：2018-04-13 01:00

本文选题：Rap1 + 生精细胞　；参考：《第四军医大学》2012年博士论文

【摘要】：目的 Rap1是一种在真核生物广泛表达的GTP结合蛋白，参与着体内多种生物学活动。不同于其它GTP结合蛋白的是其在人胚肾以及部分肿瘤细胞中可被诱导入核。在之前研究中我们已经证实其在不育男性生精细胞中表达升高。推测其可能参与调节生精细胞的增殖和分化，调控精子的发生，为进一步明确Rap1在生精过程中的作用，我们研究了其在大鼠睾丸发育以及生精细胞凋亡模型中的表达，对其可能的作用机制进行了探讨。方法 1.使用不同固定试剂固定大鼠生精细胞各发育阶段（1，7，15，24，37，50和90天）的睾丸组织，进行免疫组织化学染色，了解Rap1在发育中的表达规律并分析固定试剂对其表达的影响。结合使用蛋白质免疫印迹研究Rap1在总体睾丸的表达水平。 2.使用单剂量MAA(650mg/kg)腹腔注射建立大鼠精母细胞凋亡模型，采用免疫组织化学、TUNEL、蛋白质免疫印迹杂交研究Rap1在生精细胞凋亡过程中表达的变化，并使用间接免疫荧光、免疫共沉淀研究Rap1与NF-κB的相互作用关系。结果 1.在Bouin’s液固定的大鼠睾丸组织中，P1和P7生精细胞有Rap1表达，从P15开始，Rap1表达于精母细胞核旁的高尔基体，，随着大鼠继续发育，Rap1在高尔基体表达逐渐增强，并在成年后大鼠精母及圆形精子细胞核旁出现Rap1表达。Rap1表达随着粗线期精母细胞高尔基体的增大而增高。 2.在多聚甲醛固定的大鼠睾丸组织中，P1生殖母细胞和P7精原细胞的胞核强表达Rap1，而P7每个生精小管的阳性细胞数显著低于P1(P 0.001)。Rap1在P15大鼠生精小管内主要表达于精原细胞胞浆。在P24、P37、P50表达在晚粗线期精母细胞核内。在P90大鼠睾丸中，Rap1在各种细胞无显著着色区域。在睾丸除生精细胞以外的其它细胞未发现Rap1表达。 3.在睾丸组织蛋白免疫印迹研究中，Rap1在P1仔鼠睾丸组织裂解物中的表达显著高于P7(P 0.01)。其它各时间点表达无显著差异。 4.在MAA腹腔注射6h大鼠睾丸中，Rap1、NF-κB以及TUNEL染色阳性的粗线期精母细胞在VIII-XIV期小管内开始出现，到12h及24h阳性细胞数明显增多。睾丸组织蛋白免疫印迹研究提示，各处理组与对照组间睾丸Rap1及NF-κB的表达量无显著差异。 5. Rap1间接免疫荧光加TUNEL染色提示：MAA处理后，各时间点在VIII-XIV期生精小管可见Rap1与TUNEL细胞核内共定位的细胞百分比分别为19.1%、57.7%、67.4%，各处理组Rap1+TUNEL阳性细胞数显著高于对照组(P 0.01)。 6. Rap1和NF-κB双标记间接免疫荧光提示在实验组，两种蛋白在VIII-XIV期粗线期精母细胞核内共定位细胞百分率分别为75.6%、91.8%、94.6%，高于RAP1与TUNEL共表达的百分比。对MAA处理24h及对照大鼠睾丸蛋白进行NF-κB与Rap1免疫共沉淀分析提示，MAA处理组NF-κB结合的蛋白中Rap1的表达明显强于对照组。结论 1. Rap1在大鼠睾丸发育过程中表达于生精细胞的胞核和高尔基体，可能参与了睾丸发育过程中生精细胞的基因调控。固定试剂的选择会影响Rap1的免疫组化显色结果。 2. Rap1和NF-κB在MAA诱导的凋亡的生精细胞中表达明显升高，并且均发生入核并共定位，免疫共沉淀结果提示两种蛋白存在相互作用，提示Rap1可能与NF-κB相互作用介导生精细胞凋亡。
[Abstract]:objective
Rap1 is a widely expressed in eukaryotic GTP binding protein involved in various biological activities. Different from other GTP binding proteins can be induced into the nucleus in human embryonic kidney cells and some tumor. In previous studies we have confirmed the increased expression of spermatogenic cells in infertile men. It is hypothesized to be involved in the regulation of proliferation and differentiation of spermatogenic cells, regulation of spermatogenesis, to further clarify the role of Rap1 in spermatogenesis, we studied its expression in rat testis development and apoptosis of spermatogenic cells in the model, the possible mechanism was discussed.
Method
The use of 1. different fixation reagent immobilized rats at different developmental stages of germ cells (1,7,15,24,37,50 and 90 days) of the testis tissue by immunohistochemical staining, understand the expression pattern of Rap1 in development and analysis of the impact of fixed reagents on the expression of binding protein. Immunoblotting studies using the Rap1 expression level in whole testis.
2. using a single dose of MAA (650mg/kg) on apoptosis of rat spermatocyte cell model was established by intraperitoneal injection, immunohistochemistry, TUNEL, Western blot the expression of Rap1 in apoptosis of spermatogenic cells in the process, and the use of indirect immunofluorescence and immunoprecipitation studies the interaction between Rap1 and NF- K B.
Result
1. in Bouin 's was fixed in rat testicular tissue, P1 and P7 in spermatogenic cells with expression of Rap1, P15 from the beginning, Golgi Rap1 expressed in spermatocytes by the rats with continued development, the expression of Rap1 in the Golgi gradually increased, and in the adult rat spermatocytes and round sperm near Rap1 daughter nucleus.Rap1 expression increases with the increase of pachytene spermatocytes Golgi body.
In 2. paraformaldehyde fixed rat testis, P1 germ cells and P7 spermatogonia nucleus strong expression of Rap1 and P7, the number of positive cells per seminiferous tubules was significantly lower than that of P1 (P 0.001).Rap1 P15 in rat seminiferous tubule mainly expressed in spermatogonia cytoplasm. In P24, P37, P50 expression in late pachytene spermatocytes in the nucleus. In the testis of P90 rats, Rap1 had no significant color regions in a variety of cells. Other cells in testis except spermatogenic cells showed Rap1 expression.
3. in the Western blot study of testicular tissue, the expression of Rap1 in lysates of P1 offspring was significantly higher than that of P7 (P 0.01). No significant difference was found in other time points.
4. in the testis of 6h rats by intraperitoneal injection of MAA, Rap1, NF- kappa B and TUNEL positive pachytene spermatocytes appeared in VIII-XIV tubules, 12h and 24h positive cells increased significantly. The Western blot of testicular tissue suggest that there was no significant difference between the expression of each treatment group and control group testis Rap1 and NF- K B.
5. Rap1 indirect immunofluorescence and TUNEL staining indicated that after MAA treatment at different time points in the percentage of VIII-XIV phase cells of seminiferous tubules and TUNEL Rap1 visible nucleus co localization were 19.1%, 57.7%, 67.4%, the number of Rap1+TUNEL positive cells in each treatment group was significantly higher than the control group (P 0.01).
6. Rap1 and NF- K B double labeling immunofluorescence indicated in the experimental group, two proteins in the VIII-XIV phase of pachytene spermatocytes in the co localization of cell percentage were 75.6%, 91.8%, 94.6%, the percentage is higher than that of RAP1 and TUNEL co expression of MAA and 24h. The control rat testis protein NF- kappa B and Rap1 analysis showed that the expression of immune co precipitation, MAA treatment group Rap1 NF- B binding protein is significantly stronger in the control group.
conclusion
1., Rap1 is expressed in the nuclei and Golgi bodies of spermatogenic cells during the development of rat testis. It may be involved in the gene regulation of spermatogenic cells during testicular development. The selection of immobilized agents will affect the immunohistochemical results of Rap1.
2., the expression of Rap1 and NF- kappa B increased significantly in MAA induced apoptotic spermatogenic cells, and all of them were nucleated and Co located. The results of immunoprecipitation indicated that there existed interaction between the two proteins, suggesting that Rap1 may interact with NF- kappa B to mediate apoptosis of spermatogenic cells.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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