基于转录组测序的EV71感染RD细胞的基因表达变化研究  

A research on the changes in gene expression of RD cells infected by EV71 based on transcriptome sequencing

作　　者：程凯 曹丽 张鑫艳 郝晋芳 张晓延 CHENG Kai;CAO Li;ZHANG Xin-yan;HAO Jin-fang;ZHANG Xiao-yan(Department of Medical Laboratory,Fenyang College of Shanxi Medical University,Shanxi 032200,China)

机构地区：[1]山西医科大学汾阳学院医学检验系,032200

出　　处：《天津医药》2020年第8期689-694,共6页Tianjin Medical Journal

基　　金：国家自然科学基金青年基金项目(81301426);山西医科大学汾阳学院科技发展基金(2018B04);山西省高等学校大学生创新创业训练项目(2018813)。

摘　　要：目的基于转录组测序技术探讨肠道病毒71型(EV71)感染对人横纹肌肉瘤(RD)细胞自噬和免疫的影响及其作用机制。方法用不同剂量EV71感染RD细胞确定感染复数(MOI)。在MOI=1时选择不同时间点(0、0.5、1、1.5、3、6、9和12 h)共8组进行后续实验。倒置显微镜观察细胞形态和数量变化;蛋白免疫印迹(Western blot)检测蛋白表达情况;运用转录组测序技术分析细胞功能及基因表达的变化;高内涵细胞成像技术观察细胞内蛋白表达情况。结果倒置显微镜可见细胞在病毒感染3 h由梭形贴壁逐渐变为圆形悬浮,细胞数量相比0 h明显减少(P<0.05)。Western blot结果显示细胞在感染后6 h、9 h、12 h,病毒在RD细胞内已经表达病毒衣壳蛋白1(VP-1),感染后9 h自噬相关蛋白LC-3Ⅱ/LC-3Ⅰ与0 h组相比明显升高(P<0.01)。转录组测序结果显示EV71感染RD细胞后3 h细胞内基因表达变化达到峰值,上调基因1199个,下调基因126个。KEGG分析提示改变的信号通路分子大多与自噬和免疫相关。高内涵细胞成像显示细胞在感染后数量减少,细胞内自噬蛋白平均荧光强度增加(P<0.05)。结论EV71感染RD细胞通过自噬相关信号通路促进病毒在细胞内完成复制和组装。Objective To investigate the effect of EV71 infection on autophagy and immune response of human rhabdomyosarcoma(RD)cells and its mechanism based on transcriptome sequencing.Methods Multiplicity of infection(MOI)was determined with different doses of EV71 infected RD cells.When MOI=1,the different time points(0,0.5,1,1.5,3,6,9 and 12 h)were selected for follow-up experiments.The changes in cell morphology and cell number were observed by inverted microscope.Western blot assay was used for observing protein expression.Changes in cell function and gene expression were analyzed by transcriptome sequencing.The intracellular protein expression was observed by highconglutinative cell imaging.Results The inverted microscope showed that cells gradually changed from fusiform adherence to circular suspension at 3 h after virus infection,and the number of cells decreased significantly compared with the 0 h group(P<0.05).Western blot results showed that the cells had synthesised viral capsid protein1(VP-1)at 6 h after infection,and values of LC-3Ⅱ/LC-3Ⅰwere significantly increased at 9 h after infection compared with those in the 0 h group(P<0.01).Transcriptome sequencing results showed that gene expression changes in cells reached the peak at 3 h after infected,with 1199 up-regulated genes and 126 down-regulated genes.KEGG analysis suggested that most of the altered signaling pathway molecules were related to autophagy and immunity.High-conglutinative cell imaging showed that the number of cells decreased and the average fluorescence intensity of intracellular autophagy protein increased after infection(P<0.05).Conclusion EV71 infects RD cells to promote the replication and assembly of the virus in cells through the autophagy related signaling pathway.

关 键 词：肠道病毒A型 人 转录组 基因表达 自噬 横纹肌肉瘤细胞 免疫应答 

分 类 号：R373.2[医药卫生—病原生物学] R349.64[医药卫生—基础医学]