摘要
目的:通过利用登革Ⅱ型病毒(DENV-2)感染原代人脐静脉内皮细胞(HUVECs)后,研究病毒诱导HUVECs产生自噬和对细胞活力的影响。方法:利用DENV-2 NGC株感染HUVECs,Real-time PCR检测HUVECs中DENV-2 NS1基因部分系列的表达,给予自噬诱导剂雷帕霉素(RAPA)和自噬抑制剂氯喹(CQ)预处理观察细胞自噬通量变化,激光共聚焦显微镜观察自噬体形成;CCK-8检测细胞活力;Western blot检测自噬通量标志性蛋白LC3、P62的蛋白表达量。结果:DENV-2感染HUVECs组病毒NS1基因在各时间点均有表达;被感染的HUVECs在24 h时细胞活力下降不明显,36、48 h细胞活力分别下降至未处理组82.46%和78.47%,差异具有显著统计学意义(P<0.05);HUVECs被DENV-2感染后,LC3-Ⅱ蛋白量表达明显增高,自噬底物P62表达明显降低,36、48 h时间点相较未处理组具有统计学意义(P<0.05),与RAPA处理组结果相似(P>0.05)。CQ处理组LC3-Ⅱ及P62蛋白量表达均明显升高,36、48 h时间点相较未处理组具有显著性差异(P<0.05)。HUVECs被DENV-2感染36 h,激光共聚焦显微镜观察到LC3-Ⅱ表达明显高于未处理组,自噬诱导剂RAPA处理HUVECs后,LC3-Ⅱ表达明显增加,自噬强度更加明显。DENV-2与CQ联合处理组相较DENV单独处理组,36 h细胞活力下降了13.61%,差异具有显著统计学意义(P<0.05)。结论:DENV-2感染可抑制HUVECs的生长;而DENV-2诱导HUVECs产生的自噬却有利于HUVECs的存活。
Objective： To investigate the autophagy induced by dengue virus type 2（ DENV-2） and the effect of DENV-2 on viability of primary human umbilical vein endothelial cells（ HUVECs）. Methods： HUVECs were infected with DENV-2 NGC,and the relative expression of NS1 in HUVECs was detected via Real-time PCR. HUVECs were treated with rapamycin（ RAPA） as autophagy inducers,or pretreated with chloroquine（ CQ） as autophagy inhibitor to detect the changes in autophagy flux. The formation of autophagosomes induced by DENV-2 in HUVECs was observed by Laser confocal microscopy. The viability of HUVECs was detected via CCK-8. The expressions of LC3 and P62 were detected by Western blot. Results： The NS1 gene was expressed in HUVECs infected with DENV-2 on all time points. HUVECs were cultured on the second day at the logarithmic growth stage. The vitality of HUVECs infected with DENV-2 were declined; compared with the untreated group,the vitality of HUVECs was not decreased significantly on 24 h; compared with the untreated group,the vitality of HUVECs were decreased significantly on 36 h and 48 h（ P〈0. 05）,which were decreased in 82. 46% and 78. 47%,related to the untreated group,respectively. The expression of LC3-Ⅱ was increased significantly in HUVECs infected with DENV-2. There was a significant difference in the expression of P62 as autophagy substrate,which was decreased significantly in HUVECs infected with DENV-2（ P〈0. 05）,similar to the result of the group treated with RAPA（ P〉0. 05）,compared with untreated group on 36 h and 48 h（ P〈0. 05）. There was a significant difference in the expressions of LC3-Ⅱ and P62,which were obviously higher in the group treated with CQ,compared with untreated group on 36 h and 48 h（ P〈0. 05）; Laser confocal microscopy show that the expression of LC3-Ⅱ was significantly higher in the infected group than that in the untreated group on 36 h,which was more obvious in the group treated with RAPA. The group treated with DENV-2 combined wit
作者
毛佳璇
左丽
孔维莹
张妮
王克
袁静
陈俊豪
来涛
罗玉
MAO Jia-Xuan;ZUO Li;KONG Wei-Ying;ZHANG Ni;WANG Ke;YUAN Jing;CHEN Jun-Hao;LAI Tao;LUO Yu(Department of Immunology,Guizhou Medical University,Guiyang 550025,Chin)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2018年第8期1131-1136,共6页
Chinese Journal of Immunology
基金
国家自然科学基金项目(81560263)
贵州省教育厅"125"重大科技专项[黔教合重大专项字(2012)008号]
贵州省优秀科技教育人才省长专项[黔省专合字2009(82)号]资助
