RNAi沉默MCM7基因对人肝癌细胞SMMC-7721裸鼠移植瘤影响研究  

Effects of RNA interference of MCM7 on subcutaneous tumor of human hepatocellular carcinoma cell line SMMC-7721 in nude mice

作　　者：孙雯[1,2] 曹骥[1] 卢晓旭[1,2] 朱伶群[1,2] 杨春[1] 欧超[1] 骆成飘[1] 李瑗[1] 苏建家[1] 

机构地区：[1]广西壮族自治区肿瘤防治研究所实验研究部,广西南宁530021 [2]广西医科大学研究生学院,广西南宁530021

出　　处：《中华肿瘤防治杂志》2015年第1期7-12,共6页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(30960428);广西壮族自治区卫生厅自筹经费科研(Z2012352)

摘　　要：目的探讨微小染色体维持蛋白7(miniehromosome maintenanceprotein7,MCM7)基因RNAi(RNA interference)的重组慢病毒载体,对人肝癌细胞SMMC-7721 MCM7基因表达和裸鼠移植瘤生长的影响。方法构建重组逆转录慢病毒载体MCM7-shRNA,以MCM7基因沉默重组慢病毒颗粒(LV-shRNA-MCM7)感染SMMC-7721细胞,作为实验组;以对照慢病毒颗粒(LV-shRNA-NC)感染SMMC-7721细胞,作为阴性对照组;空白对照组常规培养,不做任何处理。通过嘌呤霉素筛选出稳定转染细胞株。3组细胞分别接种至裸鼠皮下,建立人肝癌裸鼠移植瘤模型。观察裸鼠成瘤情况、移植瘤生长情况并绘制肿瘤生长曲线;4周后测定肿瘤体积和质量,并用RT-PCR、实时荧光定量PCR、蛋白质印迹法及免疫组织化学法检测移植瘤中MCM7的表达情况。结果 MCM7-shRNA慢病毒载体构建成功。裸鼠接种癌细胞后第6天均有肿瘤形成,与空白对照组和阴性对照组相比,实验组的肿瘤生长速度明显减慢,实验组、阴性对照组和空白对照组的瘤体平均体积分别为(27.72±7.80)、(81.86±10.91)和(79.75±16.61)mm3,差异有统计学意义,F=61.949,P<0.05;实验组、阴性对照组和空白对照组的瘤体平均质量分别为(0.19±0.06)、(0.501±0.14)和(0.509±0.18)g,差异有统计学意义,F=18.41,P<0.05。实验组MCM7mRNA的相对表达量为0.253±0.198,阴性对照组1.213±0.548,空白对照组1.201±0.744,实验组相比阴性对照组和空白对照组明显下降,差异有统计学意义,F=4.091,P<0.05;实验组MCM7蛋白相对表达量为0.207±0.015,阴性对照组1.116±0.062,空白对照组1.088±0.040,实验组相比阴性对照组和空白对照组明显下降,差异有统计学意义,F=292.778,P<0.05。MCM7蛋白在阴性对照组和空白对照组中阳性表达率均为100%(10/10),在实验组中为30%(3/10),实验组明显低于阴性对照组和空白对照组,差异有统计学意义,χ2=18.261,P<0.001。结论慢病毒沉默SMMC-7721细胞MCM7基因表达能OBJECTIVE To study the effects of the recombinant lentiviral vector for RNAi（RNA interference）of MCM7 gene on the expression of MCMTgene and the growth of subcutaneous tumor of human hepatocellular carcinoma cell line SMMC-7721 in nude mice. METHODS The recombinant retroviral vector MCMT-shRNA was constructed. Human hepatocellular carcinoma SMMC-7721 cells were seeded in 6 well plates and divided into three groups：the experimental group, the normal control group and the negative control group. For the experimental group, Human hepatocellular carcinoma SMMC-7721 cells were transfected with the recombinant lentivirirus vector （LV-shRNA MCMT）, while the negative control with an control lentiviral vector（LV shRNA NC） and the normal control with no treatment. All the transfected cells were then selected through puromycin. BALB/（7 nude mice were randomly divided into three groups, 10 mice in each group. Nude mice were inoculated subcutaneously with cells of each group to establish the subcutaneous tumor model of hepatocellular carcinoma. The developments of rumor in nude mice were observed, and the tumor growth curve was drawn. Tumor volume and weight were estimated four weeks after cell inoculation. The expressions of MCM7 were analyzed by transcription polymerase chain reaction （RT-PCR）, quantitative real time polymerase chain reaction （qPCR） ,Western blot and Immune hismchemical method. RESULTS The recombinant retroviral vector MCMT-shRNA was constructed successfully. At least 6 days after inoculation of cells, the tumor formation was observed. Compared with the normal control group and the negative control group, experimental group of tumor growth slowed significantly （F=61. 949,P〈0.05）. The mean volume of experimental group, negative control group and normal control group were （27.72±7.80）,（81.86±10. 91） and （79.75±16.61） mm^3; the mean weight were（0. 19±0.06）,（0.501±0. 14） and （0. 509± 0. 18） g respectively. The experimental group was different from nega

关 键 词：肝肿瘤 RNA干扰 基因 MCM7 动物模型 基因治疗 印迹法 蛋白质 裸鼠 

分 类 号：R735.7[医药卫生—肿瘤]