间充质干细胞端粒酶催化亚基RT-qPCR方法的建立  

Establishment of RT-qPCR method for telomerase catalytic subunits of mesenchymal stem cells

作　　者：陈晓菲 李慧婷 董莹莹 曹译丹 付欣悦 刘明月 张瑞瑞 王艳辉 王新乐 崔梦姗 张峒 庞琳 饶春明 CHEN Xiao-fei;LI Hui-ting;DONG Ying-ying;CAO Yi-dan;FU Xin-yue;LIU Ming-yue;ZHANG Rui-rui;WANG Yan-hui;WANG Xin-yue;CUI Meng-shan;ZHANG Tong;PANG Lin;RAO Chun-ming(JOINN pharmaceutical Quality Research and Testing(Beijing)Co.,Ltd.,Beijing 102605,China)

机构地区：[1]北京昭衍药物检定研究有限公司,北京102605

出　　处：《药物分析杂志》2025年第1期20-29,共10页Chinese Journal of Pharmaceutical Analysis

基　　金：北京市科技计划课题:基因修饰免疫细胞和基因治疗药物质量控制关键技术与服务平台建设(Z221100007922015)。

摘　　要：目的:建立一种简便、高效的端粒酶活性检测方法,用于评价间充质干细胞产品的成瘤性风险。方法:针对端粒酶催化亚基(TERT)的保守结构域设计特异性引物和探针,并对TERT基因的引物和探针进行优化筛选,同时设置内参基因的引物和探针,在一个反应体系内使用双色荧光探针进行多重定量PCR反应,建立RT-qPCR探针法。应用该法对人间充质干细胞中是否表达TERT基因来间接判断细胞中是否存在端粒酶活性。结果:该方法可稳定特异地检测到阳性对照细胞293T/17的TERT基因,Ct平均值为23.96,RSD为1.5%。内参基因GAPDH均可以正常检出,Ct平均值为14.13,RSD为1.3%。阴性对照细胞MRC-5内参基因GAPDH可以正常检出,Ct平均值为12.81,RSD为0.46%,TERT基因未检出,该细胞端粒酶活性为阴性。人间充质干细胞HMSC内参基因GAPDH可以正常检出,Ct平均值为13.01,RSD为3.8%,TERT基因未检出,人间充质干细胞HMSC的端粒酶活性为阴性。结论:本研究建立的RT-qPCR探针法重复性好,特异性高,能够准确检测端粒酶阳性细胞催化亚基mRNA的转录。可用于间充质干细胞的端粒酶活性分析,间接评估间充质干细胞成瘤性风险。Objective:To establish a simple and efficient method for detecting telomerase activity for evaluating tumorigenic risk of cell products.Methods:Specific primers and probes were designed for the conserved domain of telomerase catalytic subunit(TERT),and the primers and probes of TERT gene were optimized and screened,and the primers and probes of internal reference genes were set up.Multiple quantitative PCR reaction was performed using two-color fluorescent probes in a reaction system,and RT-qPCR probe method was established.The expression of TERT gene in human mesenchymal stem cells HMSC was used to determine whether telomerase activity existed in the cells indirectly.Results:TERT gene of 293T/17 positive control cells could be stably and specifically detected by this method,with a mean Ct value of 23.96 and a RSD of 1.5.The internal reference gene GAPDH could be detected successfully,the mean Ct value was 14.13,the RSD was 1.3%.The reference gene GAPDH in MRC-5 could be detected in negative control cells,the mean Ct value was 12.81,the RSD was 0.46%,the TERT gene was not detected,and the telomerase activity was negative.The reference gene GAPDH in HMSC could be detected,the Ct mean value was 13.01,and the RSD was 3.8%,whereas,the telomerase activity of HMSC was negative.Conclusion:The real-time fluorescent quantitative RT-qPCR probe method established in this study can accurately detect the expression of catalytic subunit mRNA in telomerase positive cells with good repeatability and high specificity.It can be used to analyze telomerase activity of stem cells and indirectly evaluate tumorigenic risk of cell products derived from stem cells.

关 键 词：荧光定量RT-PCR探针法 端粒酶催化亚基 端粒酶活性 干细胞 成瘤性 

分 类 号：R917[医药卫生—药物分析学]