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目的探讨LSm1-7复合体在登革病毒(DENV)RNA复制中的作用。方法 DENV感染HepG2细胞后,在不同时间点(2、24、36、48h)收集细胞和提取总mRNA,实时定量PCR(RT-qPCR)分析LSm1表达水平;将LSm1的siRNA和非特异对照siRNA两次转染HepG2细胞后感染DENV-2,24h后收集细胞,提取总mRNA,RT-qPCR分析LSm1表达水平与病毒RNA表达水平;利用LSm1抗体与dsRNA抗体对DENV感染细胞进行免疫荧光染色,激光共聚焦分析LSm1与dsRNA共定位情况。结果与2h比较,随着时间的推移,DENV-2 RNA和LSm1 RNA水平逐渐升高;与对照组siNC比较,siLSm1组LSm1 RNA水平明显降低,沉默效率达78.2%;与对照组siNC比较,siLSm1组DENV RNA含量降低35.6%;LSm1-7复合体在DENV感染HepG2细胞后募集到核周围,与DENV dsRNA共定位。结论 LSm1-7复合体可能作为DENV复制复合物的组成部分,正调控DENV RNA的复制。
Objective To investigate the role of LSm1-7 complex in the replication of dengue virus (DENV) RNA. Methods After HepG2 cells were infected with DENV, the cells were collected at different time points (2, 24, 36, 48 hours) and total mRNA was extracted. LSm1 expression was analyzed by real-time quantitative PCR (RT- qPCR) The transfected HepG2 cells were infected with DENV-2 and transfected for 24 hours. The total mRNA was extracted and the expression of LSm1 and the expression of viral RNA were analyzed by RT-qPCR. The infected cells were immunofluorescently stained with LSm1 antibody and dsRNA antibody. Focus analysis of co-localization of LSm1 and dsRNA. Compared with control group, the level of LSm1 RNA in siLSm1 group was significantly decreased and the silencing efficiency was 78.2%. Compared with the control group, siLSm1 Group DENV RNA content decreased 35.6%; LSm1-7 complex in HepG2 cells infected with DENV after recruitment to the nucleus co-localization with DENV dsRNA. Conclusion The LSm1-7 complex may be part of the DENV replication complex and positively regulates DENV RNA replication.