JEV和EV71感染SH-SY5Y细胞内吞途径的初步研究  

Endocytosis pathways of Japanese encephalitis virus and enterovirus 71 in SH-SY5Y cells

作　　者：谭雨豪 吴月明[1,2] 徐庆强[1] 朱耐伟 彭浩然[1] 朱勇喆[1] 

机构地区：[1]第二军医大学微生物学教研室,上海200433 [2]第二军医大学海军医学系学员5队,上海200433

出　　处：《中国热带医学》2016年第8期753-757,共5页China Tropical Medicine

基　　金：第二军医大学创新能力培养计划(No.FH2015145;FH2015146)

摘　　要：目的初步探讨乙型脑炎病毒(Japanese encephalitis virus,JEV)和肠道病毒71型(enterovirus 71,EV71)感染人神经细胞的内吞途径机制。方法通过蛋白酶K实验检测JEV和EV71感染人神经母细胞瘤SH-SY5Y细胞的入侵速率。采用MTT法检测针对不同内吞途径关键分子的小干扰RNA(small interfering RNA,si RNA)以及化学抑制剂对SH-SY5Y细胞的细胞毒性。用JEV和EV71感染si RNA或化学抑制剂预处理的SH-SY5Y细胞,荧光定量PCR(quantitative real-time PCR,q RT-PCR)检测不同处理对两种病毒m RNA和靶蛋白表达的影响。结果确定针对网格蛋白重链的si RNA(si CHC)最高工作浓度为50 nmol/L,针对小窝蛋白1的si RNA(si CAV1)的最高工作浓度为100 nmol/L。q RT-PCR结果显示,si CHC转染神经细胞能抑制EV71 m RNA的表达(t=17.153,P<0.01),但不能抑制JEV m RNA的表达(P>0.05);而si CAV1转染神经细胞能抑制JEV m RNA的表达(t=11.406,P<0.01),但不能抑制EV71 m RNA的表达(P>0.05)。针对网格蛋白途径的化学抑制剂氯丙嗪(Chlorpromazine)可抑制EV71 m RNA的表达(t=4.114,P<0.05),且呈剂量依赖性,而氯丙嗪并不影响JEV m RNA的表达(P>0.05);针对小窝途径的化学抑制剂非律平(Filipin)可抑制JEV m RNA的表达(t=4.064,P<0.05),且呈剂量依赖性,而非律平并不影响EV71 m RNA的表达(P>0.05),与si RNA结果相一致。结论初步推测JEV入侵人神经细胞是通过小窝蛋白1依赖性的内吞作用入胞,而EV71感染人神经细胞则是通过网格蛋白依赖型的内吞途径。Objective To investigate the endocytosis pathways of Japanese encephalitis virus（JEV）and enterovirus 71（EV71）in human neuroblastoma SH-SY5Y cells. Methods Proteinase K assay was used to determine the kinetics of JEV and EV71 internalization into SH-SY5Y cells. MTT assay was employed to detect the cytotoxicity of siRNAs or chemical inhibitors targeting key molecules in different endocytosis pathways on SH-SY5Y cells. SH-SY5Y cells pretreated with specific siRNAs were inoculated by JEV and EV71,and the expression of JEV and EV71 mRNA and target proteins were analyzed by quantitative real-time PCR（qRT-PCR）. Results The highest working concentrations of siRNA targeting clathrin heavy chain（siCHC）and caveolin 1（siCAV1）were 50 and 100 nmol/L,respectively. qRT-PCR showed that siCHC transfection significantly inhibited the expression of EV71 mRNA（t=17.153,P〈0.01）,but had no effect on JEV mRNA（P〉0.05）. And siCAV1 transfection dramatically suppressed the expression of JEV mRNA（t=11.406,P〈0.01）,but showed no effect on EV71 mRNA（P〉0.05）. Chlorpromazine,a chemical inhibitor targeting clathrin,significantly suppressed the expression of EV71 mRNA in a dose-dependent manner（t=4.114,P〈0.05）,but it did not inhibit JEV mRNA（P〉0.05）. Filipin,a chemical inhibitor against caveolin 1,suppressed the expression of JEV mRNA in a dose-dependent manner（t=4.064,P〈0.05）,but it did not suppressed EV71 mRNA（P〉0.05）,which were consistent with the results of siRNA. Conclusion JEV invades human neuronal cells through caveolin 1-dependent endocytosis pathway,and EV71 enters the cells through clathrin-dependent endocytosis pathway.

关 键 词：乙型脑炎病毒 肠道病毒71型 神经细胞 内吞途径 

分 类 号：R512.5[医药卫生—内科学]