原头蚴成囊过程中内参基因的确定及水通道候选基因的转录表达  

Identification of optimal reference genes during development of hydatid cysts from protoscoleces and transcriptional expression of candidate aquaporins

作　　者：王芬 刘许诺 吴宏烨 李锴 范俊杰 叶彬 WANG Fen;LIU Xunuo;WU Hongye;LI Kai;FAN Junjie;YE Bin(Department of Pathogenic Biology,College of Basic Medical Sciences;Research Center for Molecule Medicine and Tumor,Chongqing Medical University,Chongqing,400016,China)

机构地区：[1]重庆医科大学基础医学院病原生物学教研室,重庆400016 [2]重庆医科大学基础医学院分子医学与肿瘤研究中心,重庆400016

出　　处：《第三军医大学学报》2018年第17期1533-1541,共9页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81672045,30972567,30371258)

摘　　要：目的筛选合适的内参基因以检测细粒棘球绦虫水通道蛋白(aquaporins of Echinococcus granulosus,EgAQPs)基因在原头蚴发育为棘球蚴过程中的转录表达水平。方法通过geNorm软件分析5个候选内参基因(ND1、ATP6、elp、actin和ActⅡ)在原头蚴成囊过程中的稳定性,并运用RT-q PCR方法检测体外培养原头蚴及原头蚴在小鼠体内囊化形成的继发棘球蚴中EgAQPs基因转录表达量。结果actin和ActⅡ、ATP6和ND1分别为检测体外培养原头蚴和原头蚴囊化形成的继发棘球蚴囊壁生发层细胞EgAQPs基因转录表达状态的理想内参基因。EgAQP3和EgAQP9在体外培养第3天的原头蚴中转录表达量最高,随着培养时间的增加,转录表达量呈持续下降的趋势,且不同培养时间转录表达量的差异有统计学意义(P<0.05);EgAQP4在体外培养第25天的原头蚴中转录表达量最高(P<0.05),在体外培养第3、15、35天的原头蚴中转录表达量较低,这3个时间点的差异无统计学意义(P>0.05)。EgAQP3、EgAQP4和EgAQP9在原头蚴中转录表达量最高(P<0.05),随着感染时间的延长,EgAQP3、EgAQP4和EgAQP9的转录表达水平在接种原头蚴感染小鼠后第3~7个月的继发棘球蚴囊壁生发层细胞中转录表达水平很低,差异无统计学意义(P>0.05)。EgAQP、EgAQP1、EgAQPFA-CHIP和EgAQPAnG在原头蚴和继发棘球蚴囊壁生发层细胞中的转录表达水平均很低,基本上检测不到。结论通过geNorm软件筛选了原头蚴发育为棘球蚴过程中稳定的内参基因;原头蚴囊化的继发棘球蚴囊壁生发层细胞中EgAQPs基因的转录表达水平很低,可能与棘球蚴液形成或积累有关。Objective To select suitable reference genes for detecting transcriptional expression profiles of aquaporins genes of Echinococcus granulosus（ EgAQPs） during the development of hydatid cysts from protoscoleces. Methods geNorm software was used to evaluate the stability of 5 candidate reference genes,including ND1,ATP6,elp,actin and Act Ⅱ, and then RT-q PCR was employed to detect the transcriptional expression level of EgAQP in in vitro protoscoleces and in mice during the development of hydatid cysts from protoscoleces. Results Actin and ActⅡ were optimal for detecting transcriptional level of EgAQP gene in in vitro protoscoleces by RT-q PCR. While,ATP6 and ND1 were optimal for the transcriptional level of the EgAQP3 genes in the germinal cells of cyst walls of hydatids developed from protoscoleces in vivo.The transcriptional levels of EgAQP3 and EgAQP9 reached the highest in protoscoleces after 3 days of culture,and then were in a trend of decreasing with the time elapse,with significant difference in different time points（ P〈 0. 05）. The transcriptional expression of EgAQP4 was the highest in protoscoleces after 25 days＇ culture（ P 〈0. 05）,and comparatively lower in the days 3,15 and 35,though without notably difference（ P 〉0. 05）. In in vivo study,the transcriptional levels of EgAQP3,EgAQP4 and EgAQP9 were the highest in protoscoleces（ P 〈0. 05）,and were quite low in 3 to 7 months after the protoscoleces inoculation in the abdominal cavities of mice,but there were no statistical differences（ P 〉0. 05）. The expression levels of EgAQP,EgAQP1,EgAQPFA-CHIP,and EgAQPAnG were very low and even hardly detected in both the protoscoleces and the germinal cells of cyst walls of hydatids developed from protoscoleces. Conclusion The optimal reference genes for transcriptional expression are identified by geNorm during the development of hydatid cysts from protoscoleces. The transcriptional levels of EgAQPs genes are very low in germinal cells of cyst walls of hydatids developed from

关 键 词：细粒棘球绦虫 原头蚴 棘球蚴 棘球蚴液 水通道蛋白 内参基因 转录表达 

分 类 号：R383.33[医药卫生—医学寄生虫学] R394-33[医药卫生—基础医学]