中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2012年
5期
419-424
,共6页
王官清%田雅兰%李晓霞%陈婷婷%吴宁俊%卢珍玲%井上直樹
王官清%田雅蘭%李曉霞%陳婷婷%吳寧俊%盧珍玲%井上直樹
왕관청%전아란%리효하%진정정%오저준%로진령%정상직수
白黎芦醇%抗病毒%水痘-带状疱疹病毒%即刻早期蛋白62
白黎蘆醇%抗病毒%水痘-帶狀皰疹病毒%即刻早期蛋白62
백려호순%항병독%수두-대상포진병독%즉각조기단백62
Resveratrol%Antiviral%Varicella-zoster virus%Immediate early protein 62
目的 应用构建的水痘-带状疱疹病毒(VZV)报告细胞系MV9G进一步研究白黎芦醇体外抑制VZV的作用机制.方法 将无细胞VZV直接感染MV9G细胞(CFVs直接感染)或将带细胞VZV与MV9G细胞共培养(CAVs共培养)以激发MV9G细胞表达报告基因萤火虫荧光素酶.在CFVs直接感染前或CAVs共培养不同时间点加入白黎芦醇,通过比较药物对CFVs或CAVs激发荧光素酶的抑制强度分析白黎芦醇直接灭活病毒、抑制病毒黏附和穿透、抑制病毒在细胞内复制及其时间点和可逆性:通过比较药物作用前后VZV即刻早期蛋白62(IE62)mRNA拷贝数和IE62表达强度变化分析白黎芦醇对IE62转录和表达的抑制作用.结果 白黎芦醇＞30.0 μg/ml时MV9G细胞三磷酸腺苷(ATPs)含量随药物浓度升高而逐渐降低,ATPs降低50％时白黎芦醇浓度(CD.)约60.3μg/ml.CFVs与白黎芦醇(25.0μg/ml)预混37℃水浴孵育2h后直接感染MV9G细胞,CFVs激发荧光素酶下降50％.MV9G细胞在含白黎芦醇培养基中37℃孵育2h后直接感染CFVs,CFVs激发荧光素酶随药物浓度升高而逐渐降低,但4℃孵育对无显著变化.在CAVs共培养中加入白黎芦醇后CAVs激发荧光素酶显著降低,药物抑制荧光素酶50％时浓度(IC.)约8.7 μg/ml.分别在CAVs共培养3、6、9、12、24、30和36 h时加入白黎芦醇,3～24h加药各组CAVs激发荧光素酶均显著高于对照组,但1h、3D h和36 h加药组与对照组间差异无统计学意义.CAVs共培养时撤除白黎芦醇后CAVs激发荧光素酶显著高于撤药前,尤以24h和72 h撤药组明显.VZV IE62 mRNA拷贝数和IE62抗体阳性细胞数随药物作用时间延长而逐渐降低.结论 白黎芦醇细胞毒性较强,MV9G细胞可耐受最高浓度为30.0μg/ml.白黎芦醇部分灭活CFVs、抑制CFVs穿透MV9G细胞但对CFVs黏附MV9G细胞无影响,以浓度依赖方式可逆性抑制CAVs细胞内复制.白黎芦醇可能通过抑制1E62基因的转录和表达而抑制VZV感染的早期阶段.
目的 應用構建的水痘-帶狀皰疹病毒(VZV)報告細胞繫MV9G進一步研究白黎蘆醇體外抑製VZV的作用機製.方法 將無細胞VZV直接感染MV9G細胞(CFVs直接感染)或將帶細胞VZV與MV9G細胞共培養(CAVs共培養)以激髮MV9G細胞錶達報告基因螢火蟲熒光素酶.在CFVs直接感染前或CAVs共培養不同時間點加入白黎蘆醇,通過比較藥物對CFVs或CAVs激髮熒光素酶的抑製彊度分析白黎蘆醇直接滅活病毒、抑製病毒黏附和穿透、抑製病毒在細胞內複製及其時間點和可逆性:通過比較藥物作用前後VZV即刻早期蛋白62(IE62)mRNA拷貝數和IE62錶達彊度變化分析白黎蘆醇對IE62轉錄和錶達的抑製作用.結果 白黎蘆醇＞30.0 μg/ml時MV9G細胞三燐痠腺苷(ATPs)含量隨藥物濃度升高而逐漸降低,ATPs降低50％時白黎蘆醇濃度(CD.)約60.3μg/ml.CFVs與白黎蘆醇(25.0μg/ml)預混37℃水浴孵育2h後直接感染MV9G細胞,CFVs激髮熒光素酶下降50％.MV9G細胞在含白黎蘆醇培養基中37℃孵育2h後直接感染CFVs,CFVs激髮熒光素酶隨藥物濃度升高而逐漸降低,但4℃孵育對無顯著變化.在CAVs共培養中加入白黎蘆醇後CAVs激髮熒光素酶顯著降低,藥物抑製熒光素酶50％時濃度(IC.)約8.7 μg/ml.分彆在CAVs共培養3、6、9、12、24、30和36 h時加入白黎蘆醇,3～24h加藥各組CAVs激髮熒光素酶均顯著高于對照組,但1h、3D h和36 h加藥組與對照組間差異無統計學意義.CAVs共培養時撤除白黎蘆醇後CAVs激髮熒光素酶顯著高于撤藥前,尤以24h和72 h撤藥組明顯.VZV IE62 mRNA拷貝數和IE62抗體暘性細胞數隨藥物作用時間延長而逐漸降低.結論 白黎蘆醇細胞毒性較彊,MV9G細胞可耐受最高濃度為30.0μg/ml.白黎蘆醇部分滅活CFVs、抑製CFVs穿透MV9G細胞但對CFVs黏附MV9G細胞無影響,以濃度依賴方式可逆性抑製CAVs細胞內複製.白黎蘆醇可能通過抑製1E62基因的轉錄和錶達而抑製VZV感染的早期階段.
목적 응용구건적수두-대상포진병독(VZV)보고세포계MV9G진일보연구백려호순체외억제VZV적작용궤제.방법 장무세포VZV직접감염MV9G세포(CFVs직접감염)혹장대세포VZV여MV9G세포공배양(CAVs공배양)이격발MV9G세포표체보고기인형화충형광소매.재CFVs직접감염전혹CAVs공배양불동시간점가입백려호순,통과비교약물대CFVs혹CAVs격발형광소매적억제강도분석백려호순직접멸활병독、억제병독점부화천투、억제병독재세포내복제급기시간점화가역성:통과비교약물작용전후VZV즉각조기단백62(IE62)mRNA고패수화IE62표체강도변화분석백려호순대IE62전록화표체적억제작용.결과 백려호순＞30.0 μg/ml시MV9G세포삼린산선감(ATPs)함량수약물농도승고이축점강저,ATPs강저50％시백려호순농도(CD.)약60.3μg/ml.CFVs여백려호순(25.0μg/ml)예혼37℃수욕부육2h후직접감염MV9G세포,CFVs격발형광소매하강50％.MV9G세포재함백려호순배양기중37℃부육2h후직접감염CFVs,CFVs격발형광소매수약물농도승고이축점강저,단4℃부육대무현저변화.재CAVs공배양중가입백려호순후CAVs격발형광소매현저강저,약물억제형광소매50％시농도(IC.)약8.7 μg/ml.분별재CAVs공배양3、6、9、12、24、30화36 h시가입백려호순,3～24h가약각조CAVs격발형광소매균현저고우대조조,단1h、3D h화36 h가약조여대조조간차이무통계학의의.CAVs공배양시철제백려호순후CAVs격발형광소매현저고우철약전,우이24h화72 h철약조명현.VZV IE62 mRNA고패수화IE62항체양성세포수수약물작용시간연장이축점강저.결론 백려호순세포독성교강,MV9G세포가내수최고농도위30.0μg/ml.백려호순부분멸활CFVs、억제CFVs천투MV9G세포단대CFVs점부MV9G세포무영향,이농도의뢰방식가역성억제CAVs세포내복제.백려호순가능통과억제1E62기인적전록화표체이억제VZV감염적조기계단.
Objective To further investigate inhibitory mechanism(s) of resveratrol on varicellazoster virus (VZV) in vitro with our previously generated reporter cell line MV9G.Methods Cell-free VZVs were directly inoculated onto MV9G cells (CFVs direct-infection) or cell-associated VZVs were co-cultured with MV9G cells (CAVs co-culture) to activate expression of reporter gene firefly luciferase in MV9G cells.Resveratrol was added before or after virus infection,roles of resveratrol on direct inactivation,on viral attachment to and penetration into MV9G cells,on intracellular viral replication and its IC50,inhibitorytime points and reversibility were assayed by comparing the luciferase activities reduction by resveratrol.Thereductions of VZV IE62 mRNA copies and IE62-antibody positive cells by resveratrol were further assayed.Results ATPs contents of MV9G cells in the presence of resveratrol over 30.0 μg/ml were concentrationdependently reduced,the CD50 of which was around 60.3 μg/ml.CFVs were premixed with 25.0 μg/ml resveratrol and incubated at 37℃ waterbath for two hours and then directly inoculated onto MV9G cells,luciferases activated by resveratrol-treated CFVs were reduced to around half of the untreated controls.MV9G cells were pre-incubated with resveratrol at 37℃ for 2 h and then directly infected with CFVs at 37℃ for another 2 h,the CFVs-activated luciferase was concentration-dependently reduced,but no big change was observed in those pre-incubated at 4℃.MV9G cells were co-cultured with CAVs in the presence of resvertrolfor 72 h,the CAVs-activated luciferases were markedly reduced in a concentration-dependent manner,the IC50 of which was around 8.7 μgml.Resveratrol was added in CAVs co-culture at 1,3,6,9,12,24,30,and 36 h post infection,the CAVs-activated luciferase in those resveratrol was added at 3,6,9,12,and 24 h post infection were significantly higher than those of controls.Resveratrol was withdrawn from CAVs coculture media,the CAVs-activated luciferases after withdrawal were significantly higher than those before,especially in those withdrswn at 24 and 72 h post infection.The IE62 mRNA levels shown by cDNA copiesdetected with SYBR Green RT-PCR and IE62 positive cells shown by monoclonal anti-IE62 antibody of thevirus-infected cells treated with resveratrol were significantly reduced with increase of incubation time withresveratrol.Conclusion Resveratrol was cytotoxic to MV9G cells,and the maximum resistant concentrationon MV9G cells was around 30.0 μg/ml,the CD50 of which was around 60.3 μg/ml.Non-cytotoxic resveratrol partly inactivated CFVs,inhibited viral penetration into rather than attachment to MV9G cells.Resveratrol inhibited CAVs' intmcellular replication strongly but reversibly in a concentration-dependent manner,the IC50 of which was around 8.7 μ/ml.The inhibition of resveratrol on VZV in vitro might be through suppression of IE62 gene transcription and expression in the early stage of infection.
