中文摘要：

【目的】探讨牛卵泡颗粒细胞中可卡因-苯丙胺调节转录肽（cocaine-and amphetamine-regulated transcript,CART）的表达对牛不同阶段卵泡发育的影响。【方法】通过对牛卵泡转录组ODF1（the largest onset of deviation follicle）和ODF2（the second largest onset of deviation follicle）颗粒细胞（granulesa cells,GCs）构建RNA文库,Illumina Hi Seq 2000平台测序;采集屠宰牛双侧卵巢,通过黄体形态特征筛选处于第一卵泡波阶段的最大卵泡和第二大卵泡;分别抽取卵泡液,竞争性ELISA法测定卵泡液雌激素（estrodiol,E2）和孕酮（progestin,P）浓度,确定优势卵泡（dominant follicles,DF）和从属卵泡（subordinate follicles,SF）;分别分离DF和SF颗粒细胞（granulosa cells,GCs）,提取总RNA;设定3个重复样本,以RPLP0作为内参基因,设计引物在牛DF和SF进行q RT-PCR表达量检测,Graph Pad Prism 5.0作图并进行显著性分析;兔抗CART一抗检测CART在牛卵泡的表达。【结果】转录组测序结果显示,CART在ODF1的表达量是ODF2的38.2倍;通过黄体形态特征及激素测定共筛选出3头牛的卵泡发育处于第一卵泡波,并获得DF和SF;q RT-PCR结果显示CART在DF与SF的表达量差异极显著（P〈0.01）,高达2 310倍,与转录组测序结果的表达差异趋势一致;免疫组化结果表明,CART在牛DF和SF中均有表达,且DF表达量明显高于SF,同时,CART主要在卵泡颗粒细胞层表达。【结论】在牛卵泡发育过程中,卵泡发育选择期有其它抑制因子抑制了大多数卵泡的发育,阻止其成为DF;当出现偏差后,CART的高表达抑制了DF进入排卵期,推测CART可能是通过促进卵泡颗粒细胞的凋亡,进一步抑制了颗粒细胞层E2的分泌,最终导致DF闭锁。

英文摘要：

【Objective】The objective of this paper is to investigate the effect of CART（cocaine-and amphetamineregulated transcript） expression in granulosa cells on bovine follicular development at different stages.【Method】 Two RNA libraries from ODF1（the largest onset of deviation follicle） and ODF2（the second largest onset of deviation follicle） granulosa cells were prepared and sequenced using Illumina Hi Seq 2000 platform.Cows were slaughtered,and both ovaries were collected,then the largest and the second largest follicles were screened through the morphology of corpus luteum in the first follicular wave.Follicular fluid was extracted for the concentration of E2 and P measurement using competitive ELISA method to determine DF（dominant follicles） and SF（subordinate follicles）.Granulesa cells were isolated from DF and SF,and total RNA were extracted,respectively.Using RPLP0 as a reference gene,primers were designed.q RT-PCR was performed to detect the expression of CART in DF and SF,Graph Pad Prism 5.0 was used for construction and significant analysis.Rabbit anti-CART antibody was used to detect the CART expression in cow follicles.【Result】Deep sequencing results showed that CART amounts in ODF1 were 38.2 times more than that in ODF2.Ovaries of 3 cows were screened out which were in the first follicular wave according to the observation of the morphology of corpus luteum,and the DF and SF were obtained.Real-time PCR results showed that CART expressed in DF was extremely significantly higher than that in SF,up to 2310-fold,which is consistent with the deep sequencing results.Immunohistochemistry results showed that CART expressed both in DF and SF,and the amounts in DF was significantly greater than that in SF,at the same time,CART was mainly expressed in granulosa cell layer.【Conclusion】During the process of follicular development in cattle,other inhibitors have suppressed most of the follicles,and prevented them to develop into DF;when the deviation appears,CART inhibited DF to furth