蓝舌病毒选择性诱导肾癌细胞凋亡的研究  

Study on the Potential of Bluetongue Virus Targeting Inducing Renal Carcinoma Cellsapoptosis

作　　者：杜贤进[1] 张杰[2] 董长垣[3] 周晓光[2] 王肖[2] 周峰[2] 

机构地区：[1]武汉大学人民医院急诊科,武汉市430060 [2]武汉大学人民医院泌尿外科,武汉市430060 [3]武汉大学病毒学国家重点实验室,武汉市430060

出　　处：《医学分子生物学杂志》2015年第4期213-218,共6页Journal of Medical Molecular Biology

摘　　要：目的研究蓝舌病毒湖北株（BTV-HbC3）对原代培养二倍体人肾小管上皮细胞（RTECs）、人肾癌细胞（A498）和小鼠肾癌细胞（Renca）的感染特性，并探讨其靶向性杀伤肾癌细胞作用及其机制。方法原代培养二倍体人肾小管上皮细胞（RTECs）并应用透射电镜方法鉴定，以1MOI BTV—HbC3感染RTECs、A498和Renea细胞48h后，观察光镜下细胞病变效应和扫描电镜下超微结构变化；MTT-F检测分别在0．001、0．01、0．1、1和10MOI的病毒感染RTECs、A498和Renca3种细胞48h后的存活率；用流式细胞仪AnexinV／FITC±P1分析1MOI BTV-HbC3感染3种细胞48h后凋亡情况，并检测A498和Renca细胞感染后的DNA ladder条带；流式细胞仪PI单染法分析感染前后细胞周期的变化；荧光显微镜观察感染后A498和Renca细胞核和内质网的变化。结果成功培养出二倍体的原代人肾小管上皮细胞，BTV-HbC3感染细胞A498和Renca细胞48h后均出现显著的细胞病变效应，电镜下胞浆中均出现典型的病毒颗粒；而RTECs对病毒不敏感，电镜下形态无变化，胞浆内未见病毒颗粒；10MOI的BTV-HbC3感染RTECs、A498和Renea细胞48h后存活率分别为：（99．25±3．27）％、（25．69±3．50）％和（22．63±5．85）％，A498和Renca细胞与RTECs比较有显著差异（P〈0．05）；流式细胞仪检测1MOI的BTV．HbC，感染48h后RTECs、A498和Renea早期凋亡率分别为（2．04-0．9）％、（52．04-3．4）％、（40．04-2．4）％（P〈0．01），A498和Renca细胞呈现典型的凋亡条带。细胞周期检测显示感染后A498和Renca细胞出现了s期阻滞，荧光显微镜观察到A498和Renca细胞核染色质固缩，边聚，内质网出现大量空泡，未感染A498及感染A498细胞内质网荧光值分别为56．396±7．658、33．497±5．836（P〈0．01）；而感染后Renea内质网也出现空泡，未感染A498及感染A498细胞内质网荧光值分别为70．146±4．754、42．Objective To study the different characteristic of BTV-HbC3 infected primary hu- man renal tubular epithelial cells （RTECs） , mouse renal carcinoma cells （Renca） and human re- nal carcinoma cells （A498） and the mechanism of its action. Method Primary human RTEc were established and identified by Transmission electron microscopy. RTECs, A498 and Renca ceils were infected with BTV-HbC3 at a multiplicity of infection （MOI） of 1. The cytopathic effect （CPE） and the uhramicrostructure were observed by phase contrast microscope and transmission electron micros- copy respectively. The survival rate of three cells which infected with BTV at 0. 001, 0. 01 , 0, 1, 1 and 10 MOI was assessed by MTT. The early apoptosis of the three cells which were induced by BTV at 1MOI were detected with Annexin V- FITC/PI assay. The DNA fragments of the post infection A498 and Renca cell were also measured. FCM analyzed each groupg cell-cycle changes. The chan- ges of nuclear and endoplasmic reticulum of the two post infection cancer cell were detected by fluo- rescence microscopy. Results Primary RTEc were established and identified successfully. CPE was observed at the postinfection A498 and Renca cell. The electron microscopy examination showed characteristics of necrocytosis and lots of BTV-HbC3 appeared intra-cellular cytoplasm. But the char- acteristic of the primary RTECs was not changed by phase contrast microscope and Transmission e- lectron microscopy examination. The survival rate of three cells which infected with BTV at 10 MOI were （99.25 ± 3.27） %, （25.69 ± 3.50） % and （22.63 ± 5.85） % respectively. The early ap- optosis of the RTECs, A498 and Renca cells which were induced by BTV at 1MOI were detected with flow cytometer were （2.0±0.9） %, （52.0±3.4） %, （40.0±2.4） % （P〈0.01）, re- spectively. Apoptotic DNA fragments were detected in A498 cells and Renca cells. Comparing with control cells, cell numbers of postinfection in S stages increased. There were apoptotic body for

关 键 词：溶瘤病毒 蓝舌病毒 肾肿瘤 

分 类 号：R373[医药卫生—病原生物学]