中华放射学杂志
中華放射學雜誌
중화방사학잡지
Chinese Journal of Radiology
2009年
1期
88-93
,共6页
赵纳%崔建岭%郭志远%郭志萍%孙英彩%刘计存
趙納%崔建嶺%郭誌遠%郭誌萍%孫英綵%劉計存
조납%최건령%곽지원%곽지평%손영채%류계존
基因表达调控%受体%肿瘤坏死因子%基因疗法%诊断显像
基因錶達調控%受體%腫瘤壞死因子%基因療法%診斷顯像
기인표체조공%수체%종류배사인자%기인요법%진단현상
Gene expression regulation%Receptors,yulnor necrosis factor%Gene therapy%Diagnostic imaging
目的 应用作者构建的同时携带人肿瘤坏死因子相关的诱导凋亡配体(hTRAIL)基因和萤火虫荧光素酶(lue)基因的腺病毒双表达载体(Ad-hTRAIL-lue),以luc为报告基因,体外监测hTRAIL基因的表达及其作用.方法 携带增强型绿色荧光蛋白(EGFP)基因的腺病毒载体(Ad-EGFP)以不同感染复数(MOI)感染肺癌A549细胞,48 h后检测腺病毒载体的感染效率.Ad-hTRAIL-luc分别以不同MOI感染A549细胞,用流式细胞仪48 h后分别检测hTRAIL的表达率及A549细胞的凋亡率;用液闪计数仪检测荧光素酶发光的每分钟计数(cpm).携带luc基因的腺病毒载体(Ad-luc)以不同MOI感染A549细胞,48 h后枪测荧光素酶发光的cpm值.Ad-hTRAIL-luc转染A549细胞后,对各组细胞的hTRAIL表达率及细胞凋亡率结果(自然对数转换后)、cpm值结果进行完全随机设计资料的方差分析,并对hTRAIL表达率及细胞凋亡率的2组数据进行直线相关分析.对Ad-EGFP感染后阳性率及Ad-luc感染后cpm值进行完伞随机设计多样本比较的秩和检验.结果 Ad-hTRAIL-luc转染A549细胞后,各组hTRAIL基因的表达率经自然对数转换后分别为(2.37 ± 0.04)/、(3.16±0.03)/、(3.64±0.03)/、(3.96±0.02)/、(4.24±0.02)/、(4.34±0.02)/,各组间差异具有统计学意义(F=7364,P＜0.01),两两比较差异均有统计学意义(P＜0.01);将各组细胞凋亡率进行自然对数转换后分别为(1.52±0.04)/、(2.93±0.02)/、(3.39±0.02)/、(3.64±0.02)/、(3.86±0.02)/、(4.08±0.02)/、(4.20±0.02)/,各组间差异具有统计学意义(F=12456.7,P＜0.01),两两比较差异均有统计学意义(P＜0.01);各组细胞检测到的cpm值分别为465 561±26 801、1 038 576±29 417、937 655±23 197、786 432±20 028、524 288±16 338、401 566±15 961,各组间差异具有统计学意义(F=1315.94,P＜0.01),差异两两比较均有统计学意义(P＜0.01).hTRAIL基因的表达率和A549细胞的凋亡率之间存在相关关系(r=0.984,P＜0.01).结论 腺病毒双表达载体Ad-hTRAIL-luc可有效将luc及hTRAIL基因传递到肺癌A549细胞中,从分子或细胞水平研究生物体的变化和疾病的进展,监测基因表达及疾病疗效.
目的 應用作者構建的同時攜帶人腫瘤壞死因子相關的誘導凋亡配體(hTRAIL)基因和螢火蟲熒光素酶(lue)基因的腺病毒雙錶達載體(Ad-hTRAIL-lue),以luc為報告基因,體外鑑測hTRAIL基因的錶達及其作用.方法 攜帶增彊型綠色熒光蛋白(EGFP)基因的腺病毒載體(Ad-EGFP)以不同感染複數(MOI)感染肺癌A549細胞,48 h後檢測腺病毒載體的感染效率.Ad-hTRAIL-luc分彆以不同MOI感染A549細胞,用流式細胞儀48 h後分彆檢測hTRAIL的錶達率及A549細胞的凋亡率;用液閃計數儀檢測熒光素酶髮光的每分鐘計數(cpm).攜帶luc基因的腺病毒載體(Ad-luc)以不同MOI感染A549細胞,48 h後鎗測熒光素酶髮光的cpm值.Ad-hTRAIL-luc轉染A549細胞後,對各組細胞的hTRAIL錶達率及細胞凋亡率結果(自然對數轉換後)、cpm值結果進行完全隨機設計資料的方差分析,併對hTRAIL錶達率及細胞凋亡率的2組數據進行直線相關分析.對Ad-EGFP感染後暘性率及Ad-luc感染後cpm值進行完傘隨機設計多樣本比較的秩和檢驗.結果 Ad-hTRAIL-luc轉染A549細胞後,各組hTRAIL基因的錶達率經自然對數轉換後分彆為(2.37 ± 0.04)/、(3.16±0.03)/、(3.64±0.03)/、(3.96±0.02)/、(4.24±0.02)/、(4.34±0.02)/,各組間差異具有統計學意義(F=7364,P＜0.01),兩兩比較差異均有統計學意義(P＜0.01);將各組細胞凋亡率進行自然對數轉換後分彆為(1.52±0.04)/、(2.93±0.02)/、(3.39±0.02)/、(3.64±0.02)/、(3.86±0.02)/、(4.08±0.02)/、(4.20±0.02)/,各組間差異具有統計學意義(F=12456.7,P＜0.01),兩兩比較差異均有統計學意義(P＜0.01);各組細胞檢測到的cpm值分彆為465 561±26 801、1 038 576±29 417、937 655±23 197、786 432±20 028、524 288±16 338、401 566±15 961,各組間差異具有統計學意義(F=1315.94,P＜0.01),差異兩兩比較均有統計學意義(P＜0.01).hTRAIL基因的錶達率和A549細胞的凋亡率之間存在相關關繫(r=0.984,P＜0.01).結論 腺病毒雙錶達載體Ad-hTRAIL-luc可有效將luc及hTRAIL基因傳遞到肺癌A549細胞中,從分子或細胞水平研究生物體的變化和疾病的進展,鑑測基因錶達及疾病療效.
목적 응용작자구건적동시휴대인종류배사인자상관적유도조망배체(hTRAIL)기인화형화충형광소매(lue)기인적선병독쌍표체재체(Ad-hTRAIL-lue),이luc위보고기인,체외감측hTRAIL기인적표체급기작용.방법 휴대증강형록색형광단백(EGFP)기인적선병독재체(Ad-EGFP)이불동감염복수(MOI)감염폐암A549세포,48 h후검측선병독재체적감염효솔.Ad-hTRAIL-luc분별이불동MOI감염A549세포,용류식세포의48 h후분별검측hTRAIL적표체솔급A549세포적조망솔;용액섬계수의검측형광소매발광적매분종계수(cpm).휴대luc기인적선병독재체(Ad-luc)이불동MOI감염A549세포,48 h후창측형광소매발광적cpm치.Ad-hTRAIL-luc전염A549세포후,대각조세포적hTRAIL표체솔급세포조망솔결과(자연대수전환후)、cpm치결과진행완전수궤설계자료적방차분석,병대hTRAIL표체솔급세포조망솔적2조수거진행직선상관분석.대Ad-EGFP감염후양성솔급Ad-luc감염후cpm치진행완산수궤설계다양본비교적질화검험.결과 Ad-hTRAIL-luc전염A549세포후,각조hTRAIL기인적표체솔경자연대수전환후분별위(2.37 ± 0.04)/、(3.16±0.03)/、(3.64±0.03)/、(3.96±0.02)/、(4.24±0.02)/、(4.34±0.02)/,각조간차이구유통계학의의(F=7364,P＜0.01),량량비교차이균유통계학의의(P＜0.01);장각조세포조망솔진행자연대수전환후분별위(1.52±0.04)/、(2.93±0.02)/、(3.39±0.02)/、(3.64±0.02)/、(3.86±0.02)/、(4.08±0.02)/、(4.20±0.02)/,각조간차이구유통계학의의(F=12456.7,P＜0.01),량량비교차이균유통계학의의(P＜0.01);각조세포검측도적cpm치분별위465 561±26 801、1 038 576±29 417、937 655±23 197、786 432±20 028、524 288±16 338、401 566±15 961,각조간차이구유통계학의의(F=1315.94,P＜0.01),차이량량비교균유통계학의의(P＜0.01).hTRAIL기인적표체솔화A549세포적조망솔지간존재상관관계(r=0.984,P＜0.01).결론 선병독쌍표체재체Ad-hTRAIL-luc가유효장luc급hTRAIL기인전체도폐암A549세포중,종분자혹세포수평연구생물체적변화화질병적진전,감측기인표체급질병료효.
Objective To detect the expression and effect of human tumor necrosis facctor related apoptosis-inducing ligand(hTRAIL)in vitro by using a novel double expressing adenoviral vector encoding hTRAIL and firefly lueiferase (luc) gene (Ad-hTRAIL-luc),in which luc wag used, as reporter gene.Methods A549 cells were transduced with the adenoviral vector encoding enhanced green fluorescent protein (EGFP) gene(Ad-EGFP)at variable multiplicity of infection(MOI).Adenoviral transducfion efficiency wag determined 48 h later.A549 cells were transduced with Ad-hTRAIL-luc at variable MOI.and the following tests were performed 48h later,respectively:the expressive ratio of hTRAIL and the apeptotic ratio of A549 cells were meagnred by flow eytometer;counts per minute(cpm)of luminescence were measurde by scintiUation counters. A549 cells were transduced with Ad-luc at variable MOI, and cpm of luminescence was measured by scintillation counters 48 h later. After A549 cells were transduced with AdhTRAIL-luc,the expressive ratio of hTRAIL,the apoptotic ratio of A549 cells and cpm of luminescence were analyzed by one-way ANOVA.The positive ratio of EGFP and cpm of luminescence (Ad-luc) were analyzed bv nonparametric ANOVA.Results After A549 cells were transfected with Ad-hTRAIL-luc,the expressive ratio of hTRAIL on the cell membrane of the groups were(2.37±0.04)/,(3.16±0.03)/,(3.64±0.03)/,(3.96±0.02)/,(4.24±0.02)/,(4.34±0.02)/ respectively,which showed significant difference between each other (P＜0.01);and the apoptotic ratio of A549 cells were (1.52±0.04)/,(2.93±0.02)/,(3.39±0.02)/,(3.64±0.02)/,(3.86±0.02)/,(4.08±0.02)/,(4.20±0.02)/,respectirely,and it showed significant difference between each other (P＜0.01);cpm of luminescence were 465 561±26 801,1 038 576±29 417,937 655±23 197,786 432±20 028,524 288±16 338,401 566±15 961,respectively,and it also showed significant difference between each other(P＜0.01).There was a positive relationship between the expressive ratio of hTRAIL and cell apoptotic ratio of A549 cells (r=0.984,P＜0.01).Conclusion The double expressing adenoviral vector Ad-hTRAIL-luc can transfer luc and hTRAIL gene to A549 cells efficiently,and the activity of luc may reflect the effect of hTRAIL as well as the expression of hTRAIL.
