医学分子生物学杂志
醫學分子生物學雜誌
의학분자생물학잡지
FOREIGN MEDICAL SCIENCES
2015年
4期
213-218
,共6页
杜贤进%张杰%董长垣%周晓光%王肖%周峰
杜賢進%張傑%董長垣%週曉光%王肖%週峰
두현진%장걸%동장원%주효광%왕초%주봉
溶瘤病毒%蓝舌病毒%肾肿瘤
溶瘤病毒%藍舌病毒%腎腫瘤
용류병독%람설병독%신종류
oncolytic virus%bluetongue virus%renal tumor
目的：研究蓝舌病毒湖北株（ BTV?HbC3）对原代培养二倍体人肾小管上皮细胞（ RTECs）、人肾癌细胞（A498）和小鼠肾癌细胞（Renca）的感染特性，并探讨其靶向性杀伤肾癌细胞作用及其机制。方法原代培养二倍体人肾小管上皮细胞（ RTECs）并应用透射电镜方法鉴定，以1MOI BTV?HbC3感染RTECs、 A498和Renca细胞48 h后，观察光镜下细胞病变效应和扫描电镜下超微结构变化； MTT检测分别在0?001、0?01、0?1、1和10 MOI的病毒感染RTECs、 A498和Renca3种细胞48 h后的存活率；用流式细胞仪Anexin V／FITC＋PI分析1MOI BTV?HbC3感染3种细胞48 h后凋亡情况，并检测A498和Renca细胞感染后的DNA ladder条带；流式细胞仪PI单染法分析感染前后细胞周期的变化；荧光显微镜观察感染后A498和Renca细胞核和内质网的变化。结果成功培养出二倍体的原代人肾小管上皮细胞， BTV?HbC3感染细胞A498和Renca细胞48 h后均出现显著的细胞病变效应，电镜下胞浆中均出现典型的病毒颗粒；而RTECs对病毒不敏感，电镜下形态无变化，胞浆内未见病毒颗粒；10 MOI的BTV?HbC3感染RTECs、 A498和Renca细胞48 h后存活率分别为：（99?25±3?27）％、（25?69±3?50）％和（22?63±5?85）％， A498和Renca细胞与RTECs比较有显著差异（P＜0?05）；流式细胞仪检测1 MOI的BTV?HbC3感染48 h后RTECs、A498和Renca早期凋亡率分别为（2?0±0?9）％、（52?0±3?4）％、（40?0±2?4）％（P＜0?01）， A498和Renca细胞呈现典型的凋亡条带。细胞周期检测显示感染后A498和Renca细胞出现了S期阻滞，荧光显微镜观察到A498和Renca细胞核染色质固缩，边聚，内质网出现大量空泡，未感染A498及感染A498细胞内质网荧光值分别为56?396±7?658、33?497±5?836（ P＜0?01）；而感染后Renca内质网也出现空泡，未感染A498及感染A498细胞内质网荧光值分别为70?146±4?754、42?291±6?893（P＜0?01）。结论 BTV?HbC3对原代培养二倍体肾小管上皮细胞不敏感，能通过凋亡途径分别诱导 A498和 Renca死亡，证实了BTV?HbC3具有选择性抗肾癌的作用。
目的：研究藍舌病毒湖北株（ BTV?HbC3）對原代培養二倍體人腎小管上皮細胞（ RTECs）、人腎癌細胞（A498）和小鼠腎癌細胞（Renca）的感染特性，併探討其靶嚮性殺傷腎癌細胞作用及其機製。方法原代培養二倍體人腎小管上皮細胞（ RTECs）併應用透射電鏡方法鑒定，以1MOI BTV?HbC3感染RTECs、 A498和Renca細胞48 h後，觀察光鏡下細胞病變效應和掃描電鏡下超微結構變化； MTT檢測分彆在0?001、0?01、0?1、1和10 MOI的病毒感染RTECs、 A498和Renca3種細胞48 h後的存活率；用流式細胞儀Anexin V／FITC＋PI分析1MOI BTV?HbC3感染3種細胞48 h後凋亡情況，併檢測A498和Renca細胞感染後的DNA ladder條帶；流式細胞儀PI單染法分析感染前後細胞週期的變化；熒光顯微鏡觀察感染後A498和Renca細胞覈和內質網的變化。結果成功培養齣二倍體的原代人腎小管上皮細胞， BTV?HbC3感染細胞A498和Renca細胞48 h後均齣現顯著的細胞病變效應，電鏡下胞漿中均齣現典型的病毒顆粒；而RTECs對病毒不敏感，電鏡下形態無變化，胞漿內未見病毒顆粒；10 MOI的BTV?HbC3感染RTECs、 A498和Renca細胞48 h後存活率分彆為：（99?25±3?27）％、（25?69±3?50）％和（22?63±5?85）％， A498和Renca細胞與RTECs比較有顯著差異（P＜0?05）；流式細胞儀檢測1 MOI的BTV?HbC3感染48 h後RTECs、A498和Renca早期凋亡率分彆為（2?0±0?9）％、（52?0±3?4）％、（40?0±2?4）％（P＜0?01）， A498和Renca細胞呈現典型的凋亡條帶。細胞週期檢測顯示感染後A498和Renca細胞齣現瞭S期阻滯，熒光顯微鏡觀察到A498和Renca細胞覈染色質固縮，邊聚，內質網齣現大量空泡，未感染A498及感染A498細胞內質網熒光值分彆為56?396±7?658、33?497±5?836（ P＜0?01）；而感染後Renca內質網也齣現空泡，未感染A498及感染A498細胞內質網熒光值分彆為70?146±4?754、42?291±6?893（P＜0?01）。結論 BTV?HbC3對原代培養二倍體腎小管上皮細胞不敏感，能通過凋亡途徑分彆誘導 A498和 Renca死亡，證實瞭BTV?HbC3具有選擇性抗腎癌的作用。
목적：연구람설병독호북주（ BTV?HbC3）대원대배양이배체인신소관상피세포（ RTECs）、인신암세포（A498）화소서신암세포（Renca）적감염특성，병탐토기파향성살상신암세포작용급기궤제。방법원대배양이배체인신소관상피세포（ RTECs）병응용투사전경방법감정，이1MOI BTV?HbC3감염RTECs、 A498화Renca세포48 h후，관찰광경하세포병변효응화소묘전경하초미결구변화； MTT검측분별재0?001、0?01、0?1、1화10 MOI적병독감염RTECs、 A498화Renca3충세포48 h후적존활솔；용류식세포의Anexin V／FITC＋PI분석1MOI BTV?HbC3감염3충세포48 h후조망정황，병검측A498화Renca세포감염후적DNA ladder조대；류식세포의PI단염법분석감염전후세포주기적변화；형광현미경관찰감염후A498화Renca세포핵화내질망적변화。결과성공배양출이배체적원대인신소관상피세포， BTV?HbC3감염세포A498화Renca세포48 h후균출현현저적세포병변효응，전경하포장중균출현전형적병독과립；이RTECs대병독불민감，전경하형태무변화，포장내미견병독과립；10 MOI적BTV?HbC3감염RTECs、 A498화Renca세포48 h후존활솔분별위：（99?25±3?27）％、（25?69±3?50）％화（22?63±5?85）％， A498화Renca세포여RTECs비교유현저차이（P＜0?05）；류식세포의검측1 MOI적BTV?HbC3감염48 h후RTECs、A498화Renca조기조망솔분별위（2?0±0?9）％、（52?0±3?4）％、（40?0±2?4）％（P＜0?01）， A498화Renca세포정현전형적조망조대。세포주기검측현시감염후A498화Renca세포출현료S기조체，형광현미경관찰도A498화Renca세포핵염색질고축，변취，내질망출현대량공포，미감염A498급감염A498세포내질망형광치분별위56?396±7?658、33?497±5?836（ P＜0?01）；이감염후Renca내질망야출현공포，미감염A498급감염A498세포내질망형광치분별위70?146±4?754、42?291±6?893（P＜0?01）。결론 BTV?HbC3대원대배양이배체신소관상피세포불민감，능통과조망도경분별유도 A498화 Renca사망，증실료BTV?HbC3구유선택성항신암적작용。
Objective To study the different characteristic of BTV?HbC3 infected primary hu?man renal tubular epithelial cells ( RTECs) , mouse renal carcinoma cells ( Renca) and human re?nal carcinoma cells ( A498 ) and the mechanism of its action?Method Primary human RTEc were established and identified by Transmission electron microscopy?RTECs, A498 and Renca cells were infected with BTV?HbC3 at a multiplicity of infection ( MOI) of 1?The cytopathic effect ( CPE) and the ultramicrostructure were observed by phase contrast microscope and transmission electron micros?copy respectively?The survival rate of three cells which infected with BTV at 0?001 , 0?01 , 0?1 , 1 and 10 MOI was assessed by MTT?The early apoptosis of the three cells which were induced by BTV at 1 MOI were detected with Annexin V? FITC/PI assay?The DNA fragments of the post infection A498 and Renca cell were also measured?FCM analyzed each group's cell?cycle changes?The chan?ges of nuclear and endoplasmic reticulum of the two post infection cancer cell were detected by fluo?rescence microscopy?Results Primary RTEc were established and identified successfully?CPE was observed at the postinfection A498 and Renca cell?The electron microscopy examination showed characteristics of necrocytosis and lots of BTV?HbC3 appeared intra?cellular cytoplasm?But the char?acteristic of the primary RTECs was not changed by phase contrast microscope and Transmission e?lectron microscopy examination?The survival rate of three cells which infected with BTV at 10 MOI were (99?25 ± 3?27) %、 (25?69 ± 3?50) % and (22?63 ± 5?85) % respectively?The early ap?optosis of the RTECs, A498 and Renca cells which were induced by BTV at 1MOI were detected with flow cytometer were (2?0 ± 0?9) %、 (52?0 ± 3?4) %、 (40?0 ± 2?4) % (P<0?01), re?spectively?Apoptotic DNA fragments were detected in A498 cells and Renca cells?Comparing with control cells, cell numbers of postinfection in S stages increased?There were apoptotic body forma?tion, chromatin condensation Vacuoles were derived predominantly from the endoplasmic reticulum in postinfection A498 and Renca cells, the florescence of the normal control cells and infected cell were 56?396 ± 7?658、 33?497 ± 5?836 ( P<0?01 ) in A498 cells and 70?146 ± 4?754、 42?291 ± 6?893 (P<0?01) respectively in Renca cells?Conclusion BTV?HBC3 could not infect the pri?mary human RTECs, but it could induce A498 and Renca cells death by apoptotic pathways.
