摘要
为了研究胞质视黄酸结合蛋白2(cytoplasm retinoic acid binding proteins 2,Crabp2)对猪卵泡发育的调控作用及机制,笔者通过构建猪Crabp2过表达载体并设计Crabp2 siRNA干扰片段,揭示它们对猪卵泡颗粒细胞凋亡的影响。通过从屠宰场回收猪卵巢,应用TRIzol法提取组织总RNA,并反转录成cDNA。设计Crabp2基因全长引物,利用PCR克隆Crabp2基因,构建Crabp2过表达载体。同时,设计并筛选出干扰效率最高的Crabp2 siRNA。将Crabp2过表达载体和siRNA片段分别转染至猪卵泡颗粒细胞中24 h,利用实时荧光定量PCR(qRT-PCR)和蛋白质免疫印迹(Western-blot)检测颗粒细胞Crabp2的表达水平,流式细胞术检测细胞凋亡率的变化,同时检测线粒体凋亡通路和死亡受体凋亡通路相关基因Bcl-2、Bax、Caspase-3、Fas及FasL的表达变化。对照组为转染pcDNA 3.1(+)空载体和siRNA阴性对照(NC)片段的卵泡颗粒细胞。结果显示,Crabp2过表达载体转染猪卵泡颗粒细胞可使细胞Crabp2的表达极显著升高(P<0.01),同时显著降低细胞凋亡率(P<0.05),而细胞凋亡通路中抑凋亡基因Bcl-2的表达显著升高(P<0.05),促凋亡基因Bax、Caspase-3、Fas极显著降低(P<0.01),FasL显著降低(P<0.05)。与此相反,Crabp2 siRNA片段转染猪卵泡颗粒细胞可使细胞Crabp2的表达极显著降低(P<0.01),同时显著增加细胞凋亡率(P<0.05),而Bcl-2的表达显著降低(P<0.05),Bax、Caspase-3和FasL极显著升高(P<0.01),Fas显著升高(P<0.05)。结果表明,Crabp2可能通过抑制Fas/FasL介导的死亡受体凋亡通路和线粒体凋亡通路,进而抑制猪卵泡颗粒细胞凋亡,从而参与调控猪卵泡的发育。
In order to reveal the regulation effect and mechanism of cytoplasmic retinoic acid binding protein 2(Crabp2) on porcine follicular development,Crabp2 overexpression vector was constructed and Crabp2 si RNA was designed to investigate their effects on apoptosis in porcine follicular granulosa cells.Total RNA was extracted from pig ovary from slaughterhouses and reverse transcribed into c DNA in this study.The specific primer of Crabp2 gene was designed.The Crabp2 gene was cloned by PCR,and Crabp2 overexpression plasmid was constructed successfully.Meanwhile,Crabp2 si RNA with the highest interference efficiency was designed and screened.After the Crabp2 overexpression plasmid and si RNA was respectively transfected into the porcine follicular granulosa cells for 24 h,the expression level of Crabp2 in the granulosa cells was detected by real-time quantitative PCR(q RT-PCR) and Western-blot,respectively.Flow cytometry was used to detect cell apoptosis rate and q RT-PCR was used to analyse the expression levels of related genes(Bcl-2,Bax,Caspase-3,Fas and Fas L) in the mitochondrial apoptosis pathway and death receptor apoptosis pathway in the granulosa cells.The control group was follicular granulosa cells transfected with pc DNA3.1(+) empty vector and si RNA negative control(NC)fragment,respectively.The results showed that compared with the control group,the expression of Crabp2 in the Crabp2 overexpression group was extremely significantly increased(P<0.01).Meanwhile,Crabp2 overexpression significantly reduced the apoptosis rate of porcine follicular granulosa cells(P<0.05) and the m RNA expression level of anti-apoptotic gene Bcl-2 in the apoptosis pathway was significantly increased(P<0.05),while the m RNA expression levels of pro-apoptotic genes Bax,Caspase-3 and Fas were extremely significantly decreased(P<0.01),and Fas L was significantly reduced(P<0.05).On the contrary,after Crabp2 si RNA was transfected into follicular granulosa cells,the expression of Crabp2 was extremely significantly decreased(P<0.01).C
作者
张虹亮
包俐
吴夏梦
李沐馨
张灿
王英
雷磊
王水莲
ZHANG Hong-liang;BAO Li;WU Xia-meng;LI Mu-xin;ZHANG Can;WANG Ying;LEI Lei;WANG Shui-lian(College cf Veterinary Medicine,Hunan,Agricultural University ,Changsha 410128,China;Agriculture and Rural Management Service Station of Canggang Town,Changde 415914,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2022年第1期111-119,共9页
Chinese Veterinary Science
基金
国家自然科学基金青年基金项目(31802151)
湖南省自然科学基金青年基金项目(2019JJ50257)
中国博士后科学基金面上项目(2018M642981)
湖南省教育厅优秀青年项目(18B099)。
