Siva-1在猪卵巢颗粒细胞凋亡中的作用
毕业论文关键词：Siva-1；Bcl-2；卵巢颗粒细胞；细胞凋亡；
The role of Siva-1 in porcine ovarian granulosa cell apoptosis
Abstract： Siva-1 is one of the important components of the Siva proapoptotic protein family and plays an important role in a variety of cellular signaling pathways and apoptosis. There are few studies on the mechanism of Siva-1 gene on ovarian granulosa cells. We designed a specific siRNA according to the coding sequence of the pig Siva-1 gene, transfected porcine ovarian granulosa cells cultured in vitro. qRT-PCR and western blot were used to dectect the effect of Siva-1 knockdown, Flow cytometry was used to detect the apoptotic rate of ovarian granulosa cells after Siva-1 knockdown, qRT-PCR were used to detect Siva-1 target gene Bcl-2 expression level. The results showed that after Siva-1-siRNA transfected in, mRNA level of Siva-1 in porcine ovarian granulosa cells decreased significantly(p<0.05), protein level of Siva-1aslo decreased significantly(P<0.01), indicates that the Sivc-1-siRNA we designed can effectively decrease the endogenous Siva-1 expression level ; Flow cytometry showed that the apoptosis rate and early apoptosis rate of porcine ovarian granulosa cells were significantly decreased after Siva-1 knockdown (P <0.01), while no siginicant difference in late apoptotic rate(P>0.05);The expression level of Siva-1 target gene Bcl-2 in ovarian granulosa cells was significantly up-regulated after Siva-1 knockdown (P <0.01). Our results showed that Siva-1 was a pro-apoptotic factor of porcine ovarian granulosa cells, may play its role by inhibiting the key anti-apopotic factor Bcl-2.
Key words: Siva-1; Bcl-2; granulosa cells; apoptosis;
目 录
摘要3
关键词3
Abstract3
Key words3
引言3
1□材料与方法4
1.1□引物设计与合成4
1.2□干扰RNA的合成4
1.3□转染5
1.4□RNA提取5
1.5□RNA反转录5
1.6□荧光剂实时定量PCR检测5
1.7□Western blot5
1.8□AnnexinV-PI染色6
1.9□数据统计与分析6
2□结果与分析6
2.1□PCR产物检测6
2.2□Siva-1-siRNA对猪卵巢颗粒细胞中Siva-1基因mRNA表达的影响7
2.3□Siva-1敲减对猪卵巢颗粒细胞中Siva-1蛋白表达的影响7
2.4□Siva-1敲减对猪卵巢颗粒细胞中细胞凋亡率的影响8
2.5□Siva-1敲减对猪卵巢颗粒细胞中Bcl-2基因表达的影响9
3□讨论9
致谢10
参考文献10
Siva-1在猪卵巢颗粒细胞凋亡中的作用
引言
猪卵巢卵泡主要由卵母细胞、体细胞（如颗粒细胞和卵泡膜细胞等）和卵泡液等组成，其中颗粒细胞能够合成和分泌多种活性肽如抑制素、卵泡抑素和激活素等，这些活性蛋白不仅参与调节垂体释放卵泡刺激素（FSH），而且可以作为卵泡发育的局部调节因子，对卵泡的成熟发育起着至关重要的作用。在大多数哺乳动物体内，有70-99.9%的卵泡在排卵前就发生卵泡闭锁，只有少数卵泡细胞最终能够进行排卵。研究表明，在卵泡发生闭锁的过程中，颗粒细胞也产生相应变化，例如颗粒细胞核发生固缩，细胞质内出现空腔，颗粒细胞与周围细胞分离脱落，分解形成闭锁小体，即颗粒细胞凋亡。随着深入研究发现，颗粒细胞凋亡现象的发生早于卵泡闭锁，并进一步证实卵泡闭锁是由于颗粒细胞凋亡引起的。在猪卵巢卵泡闭锁过程中，促凋亡基因Caspase3、BAX和BIM的表达水平显着上调，而抗凋亡基因BCL-2和MCL-1显著下调[1]。卵巢颗粒细胞凋亡能够引发卵泡闭锁，同时卵泡的选择也依赖于颗粒细胞的凋亡。因此，探索颗粒细胞凋亡的作用机制对于调控猪卵泡发育和提高猪繁殖力等有着重大的意义。