TRAIL基因修饰脐带间充质干细胞联合5-氟尿嘧啶对U-87MG、A549及HeLa细胞杀伤作用研究  

Killing effect of TRAIL gene modified umbilical cord mesenchymal stem cells combined with 5-fluorouracil on U-87MG,A549 and HeLa cells

作　　者：苗丽 李欣 米一 徐立强 张晨亮 陈瑶瑶 刘拥军 刘广洋 MIAO Li;LI Xin;MI Yi;XU Liqiang;ZHANG Chenliang;CHEN Yaoyao;LIU Yongjun;LIU Guangyang(Beijing Baylx Biotech Co.,Ltd.,Beijing 100176,China;Stem Cell Biology and Regenerative Medicine Institution,Yi-Chuang Institute of Bio-Industry,Beijing 100176,China)

机构地区：[1]北京贝来生物科技有限公司,北京100176 [2]北京市亦创生物技术产业研究院干细胞与再生医学研究所,北京100176

出　　处：《药物评价研究》2021年第10期2065-2073,共9页Drug Evaluation Research

基　　金：北京市科技计划课题(Z211100002521006)。

摘　　要：目的研究TRAIL基因修饰的脐带间充质干细胞(TRAIL-MSCs)联合化疗药5-氟尿嘧啶(5-FU)对人脑胶质瘤细胞U-87MG、人肺癌细胞A549和人宫颈癌细胞HeLa的杀伤作用。方法通过基因工程手段结合慢病毒转染体系构建高表达十二聚体TRAIL蛋白(dTRAIL)的TRAIL-MSCs,应用流式细胞技术检测TRAIL-MSCs的生物学特性;通过ELISA技术检测TRAIL-MSCs分泌的TRAIL量来评估转染效果;CCK-8法检测低浓度(0、1、2、4、8、16μg/mL)5-FU对U-87MG、A549、HeLa细胞的增殖抑制率,并计算其对各细胞的半数抑制浓度(IC_(50));CCK-8法检测5-FU(IC_(50))、UC-MSCs培养上清、TRAIL-MSCs培养上清、5-FU(IC_(50))+UC-MSCs培养上清和5-FU(1/4 IC_(50)、1/2 IC_(50)和IC_(50))+TRAIL-MSCs培养上清对U-87MG、A549、HeLa细胞的增殖抑制率,计算5-FU和TRAIL-MSCs培养上清的相互作用系数(CDI);Western blotting法检测5-FU对U-87MG、A549、HeLa细胞死亡受体DR4、DR5蛋白表达的影响;台盼蓝染色法观察5-FU、UC-MSCs、TRAIL-MSCs、5-FU+UC-MSCs和5-FU+TRAIL-MSCs对U-87MG、A549、HeLa细胞的杀伤作用。结果生物学检测结果表明TRAIL-MSCs在维持UC-MSCs生物学特性的同时能够高表达TRAIL蛋白。5-FU对U-87MG、A549、HeLa细胞均有增殖抑制作用,抑制作用由高到底依次为HeLa细胞(IC_(50)为9.15μg/mL)>A549细胞(IC_(50)为10.62μg/mL)>U-87MG细胞(IC_(50)为22.37μg/mL)。与对照组比较,除A549细胞UC-MSCs培养上清组外,各处理组细胞增殖抑制率均显著升高(P<0.01、0.001);与相应各5-FU IC_(50)及TRAIL-MSCs培养上清组比较,U-87MG、HeLa细胞5-FU(1/4 IC_(50)、1/2 IC_(50)和IC_(50))+TRAIL-MSCs培养上清组细胞增殖抑制率均显著升高(P<0.001),A549细胞5-FU(IC_(50))+TRAIL-MSCs培养上清组细胞增殖抑制率显著升高(P<0.01、0.001);5-FU(1/2 IC_(50))+TRAIL-MSCs培养上清组对U-87MG的细胞增殖抑制协同效应最显著(CDI<0.7且P<0.001);5-FU(1/4 IC_(50))+TRAIL-MSCs培养上清组对A549的细胞增殖抑制具有协同作�Objective Investigation on the therapeutic effects of genetically modified umbilical cord mesenchymal stem cells(TRAIL-MSCs)combined with chemotherapy drugs 5-fluorouracil(5-FU)in tumor cells U-87MG,A549 and HeLa.Methods The TRAIL-MSCs were constructed by genetic engineering combined with lentivirus transfection system.The biological characteristics of TRAIL-MSCs were detected by flow cytometry.The expression of TRAIL was detected by ELISA technique to evaluate the transfection effect.CCK-8 method was used to detect the proliferation inhibition rate of U-87MG,A549 and HeLa cells with low concentration(0,1,2,4,8,16μg/mL)5-Fu,and calculate the IC_(50) of 5-FU to each cell.CCK-8 assay was used to detect 5-FU(IC_(50)),UC-MSCs supernatant,TRAIL-MSCs supernatant,5-FU(IC_(50))+UC-MSCs supernatant and 5-FU(1/4 IC_(50),1/2 IC_(50) and IC_(50))+TRAIL-MSCs culture supernatant on the proliferation inhibition rate of U-87MG,A549,HeLa cells,the interaction coefficient(CDI)between 5-FU and TRAIL-MSCs culture supernatant was calculated.Western blotting was used to detect the effects of 5-FU on the expression of U-87MG,A549 and HeLa cell death receptors DR4 and DR5.Trypan blue staining was used to observe the killing effect of 5-FU,UC-MSCs,TRAIL-MSCs,5-Fu+UC-MSCs and 5-Fu+TRAIL-MSCs on U-87MG,A549 and HeLa cells.Results The biological detection results showed that TRAIL-MSCs could maintain the biological characteristics of UC-MSCs and at the same time express TRAIL protein.5-FU could inhibit the proliferation of U-87MG,A549 and HeLa cells,and the order of inhibition was HeLa cells(IC_(50)9.15μg/mL)>A549 cells(IC_(50)10.62μg/mL)>U-87mg cells(IC_(50)22.37μg/mL).Compared with control group,the inhibition rate of cell proliferation was significantly increased in all treatment groups except the UC-MSCs culture supernatant group of A549 cell(P<0.01,0.001).Compared with the corresponding 5-FU IC_(50) and TRAIL-MSCs supernatant groups,the proliferation inhibition rate of U-87MG,5-FU(1/4 IC_(50),1/2 IC_(50) and IC_(50))+TRAIL-MSCs sup

关 键 词：间充质干细胞 5-氟尿嘧啶 TRAIL基因修饰MSCs(TRAIL-MSCs) 肿瘤细胞系 细胞增殖 DR4 DR5 

分 类 号：R965[医药卫生—药理学]