MCMV诱导鼠T细胞淋巴瘤EL4细胞凋亡及其作用机制

Apoptosis of Murine T-Cell Lymphoma EL4 Cells Induced by Murine Cytomegalovirus and Its Mechanism

目的:观察鼠T细胞淋巴瘤细胞株EL4细胞能否被小鼠巨细胞病毒(Murine cytomegalovirus,MCMV)感染及其感染前后对EL4细胞形态的变化及凋亡的影响。方法:分别以感染复数(multiplicity of infection,MOI)为1、10、100的MCMV smith株感染EL4细胞,于感染后1、3和5 d收集细胞;设立正常对照组、MOI=1感染组、MOI=10感染组、MOI=100感染组;另外设有更昔洛韦(ganciclovir,GCV)组、MCMV+GCV组。应用瑞氏-姬姆萨染色后光学显微镜下观察细胞形态,台盼蓝抗染法计数细胞存活率,实时荧光定量PCR(Real-time quantitative PCR,RT-qPCR)检测感染细胞内MCMV DNA基因拷贝数,流式细胞技术检测细胞凋亡,RT-qPCR检测感染细胞P53、P21、cFlip、Caspase8的mRNA表达水平,Western blot检测Caspase8、P53、BAX、BCL-2、Cleaved-Caspase3的蛋白表达水平。结果:瑞氏-姬姆萨染色后光学显微镜下可见被MCMV感染的EL4细胞体积增大,细胞核不规则并见折叠扭曲。与正常对照组相比,随着感染复数的增加以及感染时间的延长,各感染组EL4细胞存活率降低,凋亡率增加(P<0.05),并均可上调凋亡蛋白P53、BAX/BCL-2、Cleaved-caspase3和Caspase8表达;且细胞存活率下降、凋亡率升高及各凋亡蛋白上调均以MOI=10感染EL4细胞后d 5最为明显。与MCMV感染组(MOI=60)比较,MCMV+GGV组(MOI=60,GCV 25μg/ml)EL4细胞内MCMV DNA含量降低(P<0.05),EL4细胞凋亡率及凋亡蛋白P53、Caspase8、BAX/BCL-2表达降低(P<0.05);与正常对照组比较,GCV组EL4细胞凋亡率及凋亡蛋白P53、Caspase8、BAX/BCL-2的表达无明显差异。结论:鼠T细胞淋巴瘤细胞株EL4细胞可被MCMV感染,呈现出明显凋亡现象。EL4细胞在MCMV感染后凋亡蛋白P53、BAX/BCL-2、Cleaved-caspase3、Caspase8表达上调。更昔洛韦抑制感染的EL4细胞内MCMV

Objective： To detect whether the murine T-cell lymphoma cell line EL4 could be infected by murine cytomegalovirus（ MCMV）,and to observe the morphological changes and apoptosis of El4 cells before and after infection. Methods： EL4 cells were infected with MCMV smith strain with 1,10 and 100 multiplicity of infection（ MOI） respectively. The morphology of the cells was observed by light microscopy and Wright＇s-Giemsa staining. The survival rate was calculated by trypan blue staining. RT-PCR-based assay was used to detect the copy number of MCMV-DNA in the infected ELA cells. Flow cytometry was used to detect apoptosis. RT-qPCR was used to detect the mRNA expression levels of P53,P21,cFlip and Caspase 8. The protein expression levels of Caspase8,P53,BAX,BCL-2 and Cleaved-Caspase3 proteins were detected by Western blot. Results： After Wright-Giemsa staining,it was found that the infected EL4 cells displayed larger volume,irregular nuclei and the folded twist under light microscopy.Compared with the normal control group,the survival rate of EL4 cells decreased,and the apoptosis rate statistically significantly（ P〈0. 05） increased with the increasing MOI and the infected time in each group. While,the level of apoptosis protein P53,BAX/BCL-2,Cleaved-caspase3 and Caspase8 were up-regulated. And the survival rate,apoptosis rate and the apoptosis protein level of infected EL4 cells with MOI = 10 were the most obvious at the 5^（th） day.Compared with MCMV infection group（ MOI = 60）,the content of MCMV DNA in EL4 cells was decreased in MCMV＋ GGV group [MOI = 60,GCV 25（ g/ml） ],and the cell apoptosis rate and apoptosis protein expression of P53,Caspase8,BAX/BCL-2 were decreased（ P〈0. 05）. Conclusion： Murine T-cell lymphoma cell line EL4 can be infected by MCMV and displayes an obvious apoptosis phenomenon. MCMV may up-regulate the expression levels of apoptosis protein P53,BAX-BCL-2,Cleaved-caspase3 and Caspase8 in EL4 cells. The drug ganciclovir reduces the copy mumber of MCMV DNA in infected EL4 cells and inhibited the killing effect of MCMV on EL4 cells.