【作者】 张静；

【导师】 郑月慧；

【作者基本信息】 江西医学院 ， 生理学， 2004， 硕士

【摘要】 目的：本研究运用TRAP-ELISA法检测卵巢颗粒细胞中端粒酶活性的表达，以及在HCG、FSH、dbcAMP、Verapamil、反义c-myb ODN作用下端粒酶表达的变化，并探讨端粒酶活性与颗粒细胞E2、P0分泌量及颗粒细胞增殖变化之间的关系，为进一步了解卵巢的功能及其调控增添新资料，为开辟新的促生育尤其是治疗和预防卵巢功能早衰及抗生育途径提供新的方法。方法：分别用外源性激素已烯雌酚及孕马血清促性腺激素制备窦前期及排卵前期卵巢模型，收集颗粒细胞，并将其与HCG、FSH、dbcAMP、Verapamil、反义c-myb ODN共培养，最后再次收集颗粒细胞，采用TRAP-ELISA法及聚丙烯酰胺电泳银染法检测端粒酶活性及其水平的变化。用放射免疫分析法测定培养基中E2、P0含量并同步用MTT法测定颗粒细胞增殖指数，分析它们与端粒酶活性之间的相关关系。结果：（1） 正常卵巢组织中有端粒酶活性存在，并且窦前期卵巢颗粒细胞端粒酶活性明显强于排卵前期卵巢颗粒细胞。OD450－OD690值于排卵前卵巢颗粒细胞中为1.420± 0.4237、 窦前期卵巢颗粒细胞中为2.123±0.009，两组比较有统计学差异（P<0.05）。 （2）在各处理因素中FSH、 HCG、dbcAMP、Verapamil能显著增强端粒酶活性（P<0.01）；而反义c-myb ODN则明显抑制端粒酶活性（P<0.01）。（3）在对颗粒细胞增殖指数MTT的测定中我们发现窦前期卵巢颗粒细胞的增殖率高于排卵前期卵巢颗粒细胞组（P<0.05）。FSH颗粒细胞组出<WP=5>现明显的增殖（P<0.01）， Antisense-hTERT ODN，使颗粒细胞增殖受到明显的抑制（P<0.01）。同时在窦前期卵巢颗粒细胞组FSH可部分抵消Antisense-hTERT ODN的抑制作用，此组颗粒细胞增殖率与空白对照组无差异；而在排卵前期颗粒细胞组FSH可完全逆转Antisense-hTERT ODN的抑制作用，此组颗粒细胞仍然呈明显的增殖状态（P<0.01）。（4）窦前期卵巢颗粒细胞组在不同作用因素下的端粒酶活性与它相对应的E2分泌量呈正相关r=0.953, P<0.01。结论：（1）正常卵巢中表达有端粒酶活性，且窦前期卵巢颗粒细胞中端粒酶活性明显高于排卵前期卵巢颗粒细胞。（2）端粒酶活性受HCG、FSH、dbcAMP、Verapamil以及反义c-myb ODN的调控。（3）用MTT法测定表明反义hTERT ODN能明显抑制颗粒细胞的增殖。（4）端粒酶活性与窦前期卵巢颗粒细胞分泌E2功能密切相关，与窦前期卵巢颗粒细胞分泌P0功能及排卵前期卵巢颗粒细胞分泌E2、P0功能均无明显关系。更多还原

【Abstract】 Objectives: TRAP-ELISA method was used to analyze the activity of telomerase in ovarian granulosa cells and to study the effect of some important factors (such as HCG, FSH, dbcAMP, Verapamil and protooncogene ) that regulate ovarian function on telomerase activity. Furthermore, the relationship among the production of E2、P0 by ovarian granulosa cells, the proliferation of ovarian granulosa cells and the telomerase activity were also investigated. It may provide some important information about ovarian function and regulating mechanisms, provide new methods to cure and prevent premature senility of ovary.Methods: Preantral and preovulatory ovarian models were prepared by extrogenesis DES and PMSG, respectively, and then granulosa cells were collected. The activity of telomerase were analyzed by TRAP-ELISA and non-denaturing acrylamide gel silver electrophoresis. The level of E2, P0 in culture medium were determined by radioimmunoassay and the proliferation index of granulosa cells were determined by MTT.Results: (1) Telomerase activity were detected in normal ovary, and the telomerase activity in granulosa cells of preantral ovary was higher than of the preovulatory ovarian granulosa cells. OD450－OD690 value was 1.420±0.4237 in the preovulatory ovarian granulosa cells, <WP=7>and 2.123±0.009 in preantral ovarian granulosa cells, which indicated a significant difference between them( P<0.05).(2) The telomerase activity can be significantly enhanced by FSH、 HCG、dbcAMP and Verapamil treatments（P<0.01）, and be significantly reduced by antisense-c-myb ODN treatment（P<0.01）.(3)The results of MTT assay showed that the proliferation rate of granulosa cells in preantral ovary was significantly higher than that of in preantrory ovarian granulosa cells（P<0.05）. In both preantral ovarian granulosa cells and preovulatory ovarian granulosa cells, FSH may increase granulosa cells proliferation significantly（P<0.01）, while antisense-hTERT ODN inhibit granulosa cells proliferation significantly（P<0.01）. (4)Telomerase activity under different factors in preantral ovary are directly related with the serection of E2 r=0.953, P<0.01.Conclusions: (1)The telomerase activity can be detected in normal ovary, and the telomerase activity in granulosa cells of preantral ovary is higher than that of preovulation.(2) the activity of telomerase can be regulated by FSH, HCG, dbcAMP, Verapamil and antisense c-mybODN. (3)The antisense hTERT ODN can significantly inhibited the ovarian granulosa cells’ proliferation of indicated by MTT assay. (4)In preantral ovarian granulosa cells, telomerase activity was directly related with the secretion of E2, and none concerned with the secretion of P0. In preovulatory ovarian granulosa cells, telomerase activity had none concerned with the secretion of P0 and E2.更多还原