杨杨,左丽
YANG Yang,ZOU Li

• 1:贵州医科大学
• 2:河南南阳高等医学专科学校第一附属医院
• 3:贵州医科大学基础医学院免疫学教研室

摘要(Abstract)：

目的:研究登革2型病毒NGC株(DEN2)感染人血管内皮细胞(HUVEC)后对HUVEC自噬的影响。方法:采用免疫组织化学法检测Ⅷ因子表达,鉴定HUVEC,50%组织细胞感染量(TCID50)测定DEN2 NGC株对白纹伊蚊C6/36细胞的毒力,实时荧光定量PCR检测感染DEN2的HUVEC中病毒mRNA表达水平的变化,Western-blot检测DEN2感染对HUVEC自噬标记性蛋白LC3B表达的影响,激光共聚焦显微镜观察DEN2感染HUVEC自噬现象。结果:免疫组织化学法结果显示,细胞表达Ⅷ因子,符合HUVEC特点;接种DEN2后5～7 d C6/36细胞出现肿胀、融合、产生空泡等典型的细胞病变,TCID50为10-5. 6;随着感染DEN2量增加,HUVEC中DEN2 mRNA相对表达量呈上升趋势,在加入1×104TCID50病毒量时DEN2 mRNA相对表达量最高(P <0. 05);用巴伐洛霉素A1(Baf)抑制后,与未用Baf相比,相同TCID50 DEN2感染HUVEV后DEN2 mRNA表达减少(P <0. 05)。Western-blot结果显示,与空白对照组比较,1×102、1×103、1×104TCID50 DEN2感染组LC3BⅡ/Ⅰ比值升高,LC3BⅡ、Ⅰ条带灰度值增加(P <0. 05);用Baf抑制后,与未用Baf相比,1×102、1×103、1×104TCID50DEN2感染组LC3BⅡ/Ⅰ比值上高,LC3BⅡ、Ⅰ条带灰度增加(P <0. 05)。激光共聚焦扫描显微镜检测结果显示,在正常HUVEC胞浆内有LC3B(绿色荧光)及LAMP1(红色荧光),荧光融合后绿色、红色荧光重叠;用Baf抑制后,HUVEC胞浆内绿色红色荧光强度增加,荧光融合后绿色、红色荧光重叠部分减少;与正常HUVEC相比,1×104TCID50 DEN2感染组HUVEC胞浆内可观察到绿色、红色及橘黄色荧光(DEN2 NS1),绿色、红色荧光强度增加,在荧光融合后绿色、红色与橘黄色荧光重叠。结论:HUVEC在DEN2感染后24 h,随着感染量增加,病毒载量增加,加Baf抑制后DEN2增殖被抑制; DEN2感染HUVEC后,在胞浆内DEN2 NS1与LC3B、溶酶体标记蛋白LAMP1共定位; DEN2可诱导HUVEC自噬增强,Baf可抑制DEN2感染HUVEC自噬的发生。
Objective: To research the effect of autophagy on human vascular endothelial cells(HUVEC) infected with Dengue virus 2 NGC strain(DEN2),so as to provide a scientific basis for the study of the pathogenesis of injury of HUVEC infected with DEN2. Methods: Median tissue culture infectious dose(TCID50) was used to determine the virus virulence of DEN2; Changes of DEN2 mRNA levels of HUVEC infected with DEN2 was detected by real-time florescence quantitative PCR; The expression of autophagy marker protein LC3 B of HUVEC infected with DEN2 was detected by Westernblot; HUVEC autophagy infected with DEN2 was observed by laser scanning confocol microscopy. Results: 1. Adherent cells were spindle type,growth well and clear outline,which was consistent with HUVEC growth characteristics; The expression of HUVEC factor VШ was detected by immunohistochemistry,and after DAB staining,HUVEC was stained as brown compared to the control group. 2.DEN2 mRNA was detected by Real-time florescence quantitative PCR. In groups infected with DEN2 of 1 × 102,1 × 103,1 × 104 TCID50,DEN2 mRNA expression increased and showed a rising trend,with statistical significance(P < 0. 05); In groups infected with DEN2 of 1 × 102,1 × 103,1 × 104 TCID50 inhibited by Bafilomycin A1(Baf),DEN2 mRNA expression increased and showed a rising trend,with statistical significance(P < 0. 05); Compared with groups uninhibited by Baf,DEN2 mRNA expression decreased in groups inhibited by Baf infected with the same TCID50 DEN2,with statistical significance(P < 0. 05). 3. LC3 B II/I ratio of HUVEC was detected by Western-blot for 24 h.Compared with the control group,in groups infected with DEN2 of 1 × 102,1 × 103 TCID50,gray of LC3 B II,I bands increased and LC3 B II/I ratio of HUVEC turned up with a growing trend. In the group infected with DEN2 of 1 × 104 TCID50 gray of LC3 B II,I bands increased and LC3 B II/I ratio of HUVEC turned up,with statistical significance(P < 0. 05); Compared with control group,in groups infected with DEN2 of 1 × 102,1 × 103,1 × 104 TCID50 inhibited by Baf,gray of LC3 B II,I bands increased and LC3 B II/I ratio of HUVEC turned up; Compared with groups uninhibited by Baf,in groups inhibited by Baf infected with 1 × 102,1 × 103,1 × 104 TCID50 DEN2 gray of LC3 B II,I bands increased and LC3 B II/I ratio of HUVEC turned up. 4. HUVEC autophagy was observed by Laser confocal microscope for 24 h,autophagy marker protein LC3 B(Green),lysosomal marker protein LAMP1(red) and DEN2 NS1(orange). In the normal HUVEC,green and red fluorescence could be observed in HUVEC cytoplasm and after fluorescence was synthetized,green and red fluorescence were overlapped. Compared with the control group,in the group inhibited by Baf,the green and red fluorescence intensity increased and after fluorescence was synthetized,green and red fluorescence were partly overlapped. In 1 × 104 TCID50 DEN2 infected group,green,red and orange fluorescence were observed in cytoplasm and after fluorescence was synthetized,green,red and orange fluorescence were overlapped. Conclusions: 1. After infected with DEN2 for 24 h,as concentration of DEN2 increases,viral load in HUVEC increases; DEN2 replication is inhibited after autophagy was inhibited by Baf. 2.DEN2 can enhance HUVEC autophagy; Baf can inhibit HUVEC autophagy induced by DEN2. 3. After infected with DEN2,in HUVEC cytoplasm,DEN2 NS1,autophagy marker protein LC3 B and lysosomal marker protein LAMP1 were co-localized.

关键词(KeyWords)： 登革2型病毒;感染;人血管内皮细胞;自噬;LC3B;溶酶体标记性蛋白;巴伐洛霉素A1
Dengue virus 2;human vascular endothelial cells;autophagy;LC3B;lysosomal marker protein;bafilomycin A1

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金资助项目(81560263,81860289)

作者(Author): 杨杨,左丽
YANG Yang,ZOU Li

DOI: 10.19367/j.cnki.1000-2707.2018.10.013

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