水泡口炎病毒M蛋白对小鼠Lewis肺癌LL/2c细胞增殖与凋亡的影响  

Effects of Vesicular Stomatitis Virus Matrix Protein on Proliferation and Apoptosis of Mouse LL/2c Cells

作　　者：赵菊梅[1] 刘涛[2] 徐健蓉[1] 王国庆[1] 杜小波[1] 文艳君[1] 

机构地区：[1]四川大学华西医院肿瘤中心生物治疗国家重点实验室 [2]延安大学医学院附属医院耳鼻咽喉科,陕西延安716000

出　　处：《癌症》2007年第3期230-235,共6页Chinese Journal of Cancer

基　　金："973"课题(No.2004CB518807);国家自然科学基金(No.30200339)~~

摘　　要：背景与目的:水泡口炎病毒(vesicular stomatitis virus,VSV)具有明显的抗肿瘤作用,在人的A549肿瘤模型和小鼠的LL/2c模型中已取得了明显的治疗效果。但复制完全型病毒在临床应用中存在着一定的安全隐患,且研究报道VSV抗肿瘤作用与其基质蛋白(matrix protein,M蛋白)有关。本研究旨在探讨VSV-M蛋白对小鼠的LL/2c肿瘤细胞增殖和凋亡的影响。方法:采用分子克隆技术构建VSV-M蛋白重组真核表达质粒pcDNA3.1-M,经酶切、PCR、测序鉴定得到阳性重组子,体外转染LL/2c细胞,RT-PCR、Western blot检测M蛋白的表达。采用噻唑蓝(MTT)法检测了M蛋白质粒对LL/2c肿瘤细胞增殖的影响。DNALadder、Hoechst33528染色检测LL/2c肿瘤细胞凋亡。结果:构建了VSV-M蛋白真核表达质粒pcDNA3.1-M。重组质粒体外转染LL/2c细胞后,检测到M蛋白的表达;倒置显微镜下观察到细胞形态学的改变,且48h后细胞生长抑制明显,抑制率达41.3%(P<0.05),而空载体转染组只有6.5%的抑制率(P>0.05)。琼脂糖凝胶电泳可见DNA ladder形成,Hoechst33528染色检测到LL/2c细胞凋亡,细胞核改变。结论:VSV-M蛋白可抑制小鼠LL/2c细胞增殖,并诱导细胞凋亡。BACKGROUND ＆ OBJECTIVE： Vesicular stomatitis virus （VSV）, an oncolytic virus, is an attractive candidate for tumor therapy. Although previous studies showed obvious antitumor effects of VSV on human A549 and mouse LL/2c tumor models, the clinical application of a live virus is confronted with the problem of bio-safety. The matrix （M） protein of VSV is related to the antitumor effect of VSV. This study was to investigate the effects of VSV-M protein on the proliferation and apoptosis of mouse LL/2c tumor cells. METHODS. A eukaryotic expression plasmid pcDNA3.1-M encoding VSV-M protein was constructed by molecular cloning technique, and analyzed by enzyme digestion, polymerase chain reaction （PCR）, and DNA sequencing, then transfected into LL/2c cells. The expression of VSV-M protein in LL/2c cells was detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot. The effect of VSV-M protein on proliferation of LL/2c cells was assessed by MTT assay; its effect on cell apoptosis was assessed by DNA ladder and Hoechst 33528 staining. RESULTS. A plasmid pcDNA3.1-M encoding VSV-M protein was constructed successfully and identified. After transfection, the expression of VSV-M protein was detected in LL/2c cells, morphologic changes of LL/2c cells was observed under microscope, inhibition rate of cell survival was 41.3% （P〈0.05）, DNA ladder was detected, apoptotic nuclei was observed. CONCLUSION. VSV-M transfection could inhibit proliferation and induce apoptosis of LL/2c cells.

关 键 词：水泡口炎病毒 M蛋白 细胞增殖 凋亡 肺肿瘤 

分 类 号：R734.2[医药卫生—肿瘤]