PTHLH基因对水牛卵泡颗粒细胞活性的影响及其分子调控机制的初步研究  

A Preliminary Study on the Effect of PTHLH Gene on the Viability of Buffalo Follicular Granulosa Cells and Its Regulatory Mechanism

作　　者：陆杏蓉 邓廷贤 郑海英 杨春艳 韦科龙 钟华配 段安琴 马小娅 徐媛媛 尚江华 LU Xingrong;DENG Tingxian;ZHENG Haiying;YANG Chunyan;WEI Kelong;ZHONG Huapei;DUAN Anqin;MA Xiaoya;XU Yuanyuan;SHANG Jianghua(Guangxi Buffalo Research Institute,Key Laboratory of Buffalo Genetics,Breeding and Reproduction Technology,Ministry of Agriculture and Rural Affairs,Guangxi Key Laboratory of Buffalo Genetics,Reproduction and Breeding,Nanning 530001,China)

机构地区：[1]广西水牛研究所,农业农村部水牛遗传繁育技术重点实验室,广西水牛遗传繁育重点实验室,南宁530001

出　　处：《中国草食动物科学》2024年第6期1-10,共10页China Herbivore Science

基　　金：广西自然科学基金面上项目(2021GXNSFAA220004,2022GXNSFAA035522);国家现代农业产业技术体系广西奶水牛产业创新团队(nycytxgxcxtd-2021-21-01)。

摘　　要：试验旨在探讨PTHLH基因过表达对水牛卵泡颗粒细胞活性的影响,以及PTHLH基因对增殖、表观修饰和多能性相关基因表达模式的调控作用,为理解卵泡颗粒细胞和卵泡发育的分子调控机制提供新的视角。采用脂质体转染法将PTHLH基因过表达载体转染到水牛卵泡颗粒细胞中;利用MTT细胞活性检测试剂测定细胞活性;用STRING在线分析PTHLH及相关蛋白的互作网络;用qRT-PCR检测转染后水牛卵泡颗粒细胞中增殖、表观修饰和多能性相关基因的表达情况,分析PTHLH基因过表达对水牛卵泡颗粒细胞相关基因表达的影响。结果显示,PTHLH基因过表达极显著降低了水牛卵泡颗粒细胞的活性(P<0.0001);过表达组细胞中增殖相关基因(CCNB1、CCND1、CDKN1B、CDKN1A和CDK2)的表达极显著下调(P<0.0001);甲基化相关基因DNMT1和羟甲基化相关基因TET3的表达极显著下调(P<0.001或P<0.01),甲基化相关基因DNMT3B和IDH1的表达显著下调(P<0.05),而羟甲基化相关基因TET1和TET2的表达显著上调(P<0.05);乙酰化相关基因(HDAC1、HDAC2、HAT1、P300和CBP)的表达极显著下调(P<0.001或P<0.0001);与此同时,多能性相关基因OCT4和SOX2的表达极显著上调(P<0.01或P<0.001),而NANOG基因的表达极显著下调(P<0.01)。综上,PTHLH基因过表达抑制了水牛卵泡颗粒细胞的细胞活性,对增殖相关基因(CCNB1、CCND1、CDKN1B、CDKN1A和CDK2)、甲基化相关基因DNMT1与羟甲基化相关基因TET3、乙酰化相关基因(HDAC1、HDAC2、HAT1、P300和CBP)和多能性相关基因NANOG的表达具有负调控作用,而对羟甲基化相关基因TET1和TET2,以及多能性相关基因OCT4和SOX2的表达具有正调控作用。In order to investigate the effect of PTHLH gene overexpression on the activity of buffalo follicular granulosa cells,and the regulatory effect of PTHLH gene on gene expression patterns related to proliferation,epigenetic modification and pluripotency,this paper provided a new perspective for understanding the molecular regulation mechanism of follicular granulosa cells and follicle development.In this study,the PTHLH gene overexpression vector was transfected into buffalo follicular granulosa cells by lipid transfection,the cell viability was determined by MTT cell viability assay reagent,the interaction network of PTHLH and related proteins was analyzed online by STRING,qRT-PCR was used to detect the expression of genes related to proliferation,epigenetic modification and pluripotency in buffalo follicle granulosa cells after transfection,and the effect of PTHLH gene overexpression on the expression of genes related to buffalo follicle granulosa cells was analyzed.The results showed that overexpression of PTHLH gene significantly reduced the activity of buffalo follicular granulosa cells(P<0.0001).The expression of proliferation-related genes(CCNB1,CCND1,CDKN1B,CDKN1A and CDK2)in the overexpression group was significantly down-regulated(P<0.0001).The expressions of methylation-related genes DNMT1(P<0.001)and hydroxymethylation-related genes TET3(P<0.01)were significantly down-regulated,the expressions of methylation-related genes DNMT3B and IDH1 were significantly down-regulated(P<0.05),and the expressions of hydroxymethylation-related genes TET1 and TET2 were significantly up-regulated(P<0.05).The expression of acetylation-related genes(HDAC1,HDAC2,HAT1,P300 and CBP)was significantly down-regulated(P<0.001 or P<0.0001).At the same time,the expression of pluripotency-related genes OCT4(P<0.01)and SOX2(P<0.001)was significantly up-regulated,while the expression of pluripotency-related genes NANOG was significantly down-regulated(P<0.01).In conclusion,overexpression of PTHLH gene inhibited the proliferative activ

关 键 词：水牛 PTHLH基因 卵泡颗粒细胞 基因表达 

分 类 号：S823.3[农业科学—畜牧学]