摘要
目的:观察登革2型病毒NGC株(Dengue virus-2 NGC strain,DENV-2 NGC)诱导的HUVEC凋亡及其Fas/FasL表达的变化。方法:利用BALB/c乳鼠脑内接种以及C6/36细胞增殖病毒,RT-PCR鉴定病毒,细胞半数感染量(TCID50)测定法确定病毒滴度。直接免疫荧光法观察DENV-2吸附HUVEC;AO/EB染色法检测DENV-2感染后HUVEC细胞凋亡形态;流式细胞术动态测定不同滴度DENV-2诱导HUVEC凋亡率的变化,检测不同剂量病毒感染48小时的细胞感染情况及HUVEC膜表面Fas/FasL的表达水平。组间比较采用独立样本t检验。结果:DENV-2能够吸附于HUVEC并诱导HUVEC凋亡,凋亡率达(18.44±1.29)%,与未接种病毒组比较(5.78±1.43)%,差异有显著性(P<0.05),但凋亡率并未随着时间延长和病毒剂量增加,呈现出明显的上升或下降的趋势。DENV-2对HUVEC的感染率并未与细胞凋亡率呈现明显的相关性。与对照组相比,接种病毒组的HUVEC膜表面Fas的表达水平均有明显的增加,但FasL表达水平仅在接种病毒的1 000TCID50组显著增加。结论:DENV-2 NGC能够感染HUVECs并诱导其凋亡,可能是DHF/DSS发病机制中血管内皮屏障损伤导致血浆外渗的主要原因之一,Fas/FasL激活的凋亡死亡受体途径可能在DENV-2 NGC诱导的HUVECs凋亡中发挥了作用。
Objective：To investigate the apoptosis of HUVECs induced by DENV-2 and the changes expression of Fas/FasL on the cell surface. Methods： The combination of HUVECs and DENV-2 was detected by Direct Immunofluorescence. Apoptotic morphol- ogy of HUVEC was observed by AO/EB staining. Dynamic alteration in the rate of apoptosis in HUVEC subjected to DENV-2, the rate of infection in HUVECs with various doses of DENV-2 for 48 h and the expression levels of Fas/FasL on membrane of HUVECs were detected by means of flow cytometry. Between-group comparisons were performed using the independent -Samples Student＇ s t test with SPSS16.0 software for Windows. Results： The DENV-2 could bind to HUVECs in vitro and induce HUVEC apoptosis with an early ap- optosis rate of （ 18.44 ± 1.29 ） % , which was obviously higher than the rate （5.78 ± 1.43 ） % of control group （ P 〈 0.05 ）. However, there was no significant upward or downward trend as the time prolonged and viral concentrations increased. There was also no signifi- cant relationship between apoptotic and DENV-2 infection in HUVECs. The expression of Fas on HUVECs increased in all virus-infec- ted groups compared with the conrol group. But the expression of FasL on HUVECs increased only in the 1 000TCID50 group. Conclu- sion： DENV-2-mediated HUVECs apoptosis may play a role in the disruption of vessel endothelium and contribute to the pathogenesis of vascular leakage in DHF/DSS, and the activation of death receptor pathway mediated by Fas/FasL may play an important role in the apoptosis of HUVECs induced by DENV-2.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2013年第12期1244-1248,共5页
Chinese Journal of Immunology
基金
国家自然科学基金项目(No.31260224)资助
