端粒逆转录酶基因(TERT)修饰对蒙古羊输卵管上皮细胞体外增殖能力的影响  

Effect of Telomerase Reverse Transcriptase Gene(TERT)Modification on Proliferation of Mongolian Sheep(Ovis aries)Oviduct Epithelial Cells In vitro

作　　者：刘洋[1,2] 海勒思 刘永斌 刘春霞 曹俊伟[1,2] 张焱如 LIU Yang;HAI Le-Si;LIU Yong-Bin;LIU Chun-Xia;CAO Jun-Wei;ZHANG Yan-Ru(College of Life Sciences,Inner Mongolia Agricultural University,Hohhot 010018,China;Inner Mongolia Autonomous Region Key Laboratory of Biomanufacturing,Hohhot 010018,China;Inner Mongolia Academy of Agriculture and Animal Husbandry Sciences,Hohhot 010031,China)

机构地区：[1]内蒙古农业大学生命科学学院,呼和浩特010018 [2]内蒙古自治区生物制造重点实验室,呼和浩特010018 [3]内蒙古自治区农牧业科学院,呼和浩特010031

出　　处：《农业生物技术学报》2022年第4期686-694,共9页Journal of Agricultural Biotechnology

基　　金：内蒙古自治区科技重大专项(2020ZD0003)。

摘　　要：输卵管上皮细胞(oviduct epithelial cells,OECs)能够提供理想的生理生化环境、支持胚胎发育,但其体外增殖能力较弱,无法满足实验研究与应用需求。本研究将外源性端粒逆转录酶基因(telomerase reverse transcriptase,TERT)转入蒙古羊(Ovis aries)输卵管上皮细胞(sheep OCEs,SOECs)并观察TERT基因修饰的蒙古羊输卵管上皮细胞(TERT-SOECs)的体外增殖能力。首先经酶消化法建立了SOECs系,将重组质粒pCI-neo-TERT以脂质体法转染至原代SOECs;经G418抗性筛选,得到稳定表达TERT基因的SOECs,qPCR检测了TERT基因在细胞中的表达;免疫荧光法检测上皮细胞特异性标志物角蛋白18(cytokeratin18,CK18)并对TERT-SOECs进行生长曲线拟合和活性测定。结果显示,成功将TERT基因导入了SOECs,PCR检测表明细胞表达了276 bp的TERT基因片段,TERT基因表达量极显著高于未转染和转染pCI-neo空载体的SOECs(P<0.001);转染后的细胞仍保留了SOECs的生物学特性,P5 TERT-SOECs倍增时间为46.61 h,而P5 SEOCs倍增时间为65.03 h,TERT-SOECs体外增殖速度明显高于SOECs。综上,本研究成功建立了有较强体外增殖能力的TERT-SOECs系,为种质资源细胞库提供了种子细胞。Oviduct epithelial cells(OECs)can provide an ideal physiological and biochemical environment to support embryonic development,but their proliferation ability in vitro is weak,which can not meet the needs of experimental research and application.In this study,exogenous telomerase reverse transcriptase gene(TERT)was transferred into Mongolian sheep(Ovis aries)oviduct epithelial cells(sheep OCEs,SOECs),and the proliferation ability of Mongolian sheep oviduct epithelial cells modified by telomerase gene was observed in vitro.Firstly,the SOECs system was established by enzyme digestion,and the recombinant plasmid pCI-neo-TERT was transfected into the primary SOECs by liposome method.The results showed that the expression of TERT gene was detected by qPCR;The growth curve and activity of cytokeratin18(CK18),a specific marker of epithelial cells,were detected by immunofluorescence.The results showed that TERT gene was successfully introduced into SOECs,and PCR showed that 276 bp TERT gene fragment was expressed in the cells,and the expression of TERT gene was significantly higher than that of the uninfected and transfected pCI-neo empty vector(P<0.001).The transfected cells still retained the biological characteristics of SOECs.The doubling time of P5 TERT-SOECs was 46.61 h,while that of P5 SOECs was 65.03 h.It was obvious that the proliferation rate of TERT-SOECs was significantly higher than that of SOECs.In conclusion,the study successfully established the TERT-SOECs system with strong in vitro proliferation ability,which laid an important foundation for the seed cell supply of germplasm cell bank.

关 键 词：端粒逆转录酶(TERT) 蒙古羊输卵管上皮细胞(SOECs) 体外增殖 脂质体法 

分 类 号：S826.82[农业科学—畜牧学]