2型登革病毒对原代人肝窦内皮细胞凋亡、自噬以及相关基因表达的影响  

Effects of dengue type 2 virus on the apoptosis and autophagy of primary HHSECs and the expression of related genes

作　　者：戴雪婷 左丽[1] 赵军[1] 袁静[1] 裴华[1] 孔维莹 

机构地区：[1]贵州医科大学免疫学教研室,贵阳550025

出　　处：《中华微生物学和免疫学杂志》2016年第5期340-345,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金（31260224,81560263）;贵州省教育厅“十二五”重大科技专项（黔教合重大专项字[2012]008号）

摘　　要：目的研究2型登革病毒（DENV-2）感染原代人肝窦内皮细胞（HHSECs）后，HHSECs的凋亡、自噬，细胞间黏附分子-1（ICAM-1）及Beclin-1 mRNA水平的表达，分析DENV-2致病过程中可能的作用机制。方法用DENV-2（MOI＝1）作用于HHSECs，Real-time PCR动态检测DENV-2 NS1部分序列；CCK-8法动态检测DENV-2对HHSECs的毒性作用。real-time PCR动态检测被感染HHSECs的ICAM-1、Beclin-1 mRNA的表达水平。流式细胞术检测被感染HHSECs的细胞凋亡率，以及LC3B和ICAM-1的表达。结果被DENV-2作用后的HHSECs有NS1部分基因序列的表达。DENV-2感染细胞后，CCK-8法检测细胞的抑制率分别为（10.90±1.24）%（12 h）、（16.40±0.42）%（24 h）、（17.00±0.46）%（36 h）、（29.60±0.26）%（48 h）。Real-time PCR检测HHSECs中Beclin-1及ICAM-1 mRNA的表达，以未感染组为对照，2^－△△Ct值于24 h达峰值，分别为46.77±2.55和40.97±4.91。流式细胞术动态观察被DENV-2感染的HHSECs的凋亡，于12 h较明显，凋亡率为13.17%；36 h时LC3B的表达率（35.50%）达到峰值；ICAM-1的表达率分别为9.17%（12 h）、14.7%（24 h）、12.7%（36 h）、11.6%（48 h）。结论HHSECs对DENV-2易感。DENV-2可激活HHSECs，诱导ICAM-1上调；可促进自噬的发生，于早期诱导HHSECs细胞凋亡。Objective To investigate the effects of dengue type 2 virus （DENV-2） on the apoptosis and autophagy of primary human hepatic sinusoidal endothelial cells （HHSECs） and the expression of ICAM-1 and Beclin-1 at mRNA level and to analyze the possible pathogenic mechanism of DENV-2. Methods Immunohistochemistry （IHC） and flow cytometry analysis （FCM） were performed to identify HHSECs by detecting factor Ⅷ and CD31. The DENV-2 strain was identified by using PCR and Hind Ⅲ. The 50% tissue culture infective dose （TCIDs0） of DENV-2 was calculated after infecting C6/36 cells with DENV-2. Dynamic changes of DENV-2 NS1 were measured by real-time PCR after infecting HHSECs with DENV-2. CCK-8 was used to dynamically detect the cytotoxicity of DENV-2 to HHSECs. The transcriptional levers of Beclin-1 and ICAM-1 in DENV-2-infected HHSECs were detected by real-time PCR. FCM was performed to analyze the apoptosis of HHSECs and the expression of LC3B and ICAM-1. Results The cells in the experimental group were stained brown by DAB and the positive expression rate of CD31 reached 87.1%. The TICD50 of DENV-2 to C6/36 cells was 10^-6.845/0. 1 ml. Compared with the uninfected cells, partial sequences of NS1 gene were expressed in DENV-2-infected HHSECs. DENV-2 suppressed the cell activities of HHSECs. The suppression rates of DENV-2 to HHSECs at 12 h, 24 h, 36 h and 48 h were respectively （ 10.90±1.24）%, （ 16.40±0.42）% , （ 17.00±0.46）% and （29.60±0.26）% （P〈0.05）. The transcriptional levels of Beelin-1 and ICAM-1 in HHSECs were significantly increased at the time point of 24 h after DENV-2 infection, the 2^-△△Ct values of which were 46.77±2.55 and 40.97±4.91, respectively. The expression of LC3B and ICAM-1 in DENV-2-infected HHSECs were increased, the peaks of which were reached at 24 h （14.7%） and 36 h （35.5%）, respectively. The apoptosis of DENV-2-infected HHSECs was remarkably enhanced at 12 h with an apoptosis rate of 13.17%. Conclusion HHSECs was susceptible to DENV-2.

关 键 词：登革病毒 原代人肝窦内皮细胞 凋亡 自噬 BECLIN-1 ICAM-1 

分 类 号：R373[医药卫生—病原生物学]