临床儿科杂志
臨床兒科雜誌
림상인과잡지
2015年
1期
64-68
,共5页
人巨细胞病毒%干扰RNA%即刻早期基因%基因表达
人巨細胞病毒%榦擾RNA%即刻早期基因%基因錶達
인거세포병독%간우RNA%즉각조기기인%기인표체
human cytomegalovirus%siRNA%immediate-early gene%gene expression
目的：探讨RNA干扰技术在体外抑制人巨细胞病毒(HCMV)基因表达时siRNA转染和病毒感染的先后顺序对抑制效果的影响。方法设计并化学合成靶向即刻早期(IE)基因的siRNA。应用阳离子脂质体法将siRNA导入人胚肺成纤维(HELF)细胞，并分为先转染siRNA后感染HCMV、先感染HCMV后转染siRNA以及同时转染siRNA和感染HCMV 3组，同时设置正常对照组、阴性对照组、阳性对照组和转染试剂对照组。采用荧光定量PCR和琼脂糖凝胶电泳方法分别检测分析各组转染的siRNA对IE基因和阳性对照基因GAPDH的抑制效果。结果阳性对照组GAPDH基因mRNA表达量较阴性对照组和转染试剂对照组有明显下降，siRNA抑制率为70.4%，差异有统计学意义(P<0.05)。先转染siRNA后感染HCMV组、先感染HCMV后转染siRNA组、同时转染siRNA和感染HCMV组以及阳性、阴性、转染试剂对照组的IE基因mRNA表达量比较，差异有统计学意义(F=146.93，P=0.000)。其中先转染siRNA后感染HCMV组以及同时转染siRNA和感染HCMV组对IE基因mRNA表达的抑制效果均好于先感染HCMV后转染siRNA组，差异有统计学意义(P<0.05)。结论在体外，siRNA能有效抑制靶基因的表达，且先转染siRNA再感染病毒的抑制效果相对更好，提示siRNA对HCMV感染具有一定的预防作用。
目的：探討RNA榦擾技術在體外抑製人巨細胞病毒(HCMV)基因錶達時siRNA轉染和病毒感染的先後順序對抑製效果的影響。方法設計併化學閤成靶嚮即刻早期(IE)基因的siRNA。應用暘離子脂質體法將siRNA導入人胚肺成纖維(HELF)細胞，併分為先轉染siRNA後感染HCMV、先感染HCMV後轉染siRNA以及同時轉染siRNA和感染HCMV 3組，同時設置正常對照組、陰性對照組、暘性對照組和轉染試劑對照組。採用熒光定量PCR和瓊脂糖凝膠電泳方法分彆檢測分析各組轉染的siRNA對IE基因和暘性對照基因GAPDH的抑製效果。結果暘性對照組GAPDH基因mRNA錶達量較陰性對照組和轉染試劑對照組有明顯下降，siRNA抑製率為70.4%，差異有統計學意義(P<0.05)。先轉染siRNA後感染HCMV組、先感染HCMV後轉染siRNA組、同時轉染siRNA和感染HCMV組以及暘性、陰性、轉染試劑對照組的IE基因mRNA錶達量比較，差異有統計學意義(F=146.93，P=0.000)。其中先轉染siRNA後感染HCMV組以及同時轉染siRNA和感染HCMV組對IE基因mRNA錶達的抑製效果均好于先感染HCMV後轉染siRNA組，差異有統計學意義(P<0.05)。結論在體外，siRNA能有效抑製靶基因的錶達，且先轉染siRNA再感染病毒的抑製效果相對更好，提示siRNA對HCMV感染具有一定的預防作用。
목적：탐토RNA간우기술재체외억제인거세포병독(HCMV)기인표체시siRNA전염화병독감염적선후순서대억제효과적영향。방법설계병화학합성파향즉각조기(IE)기인적siRNA。응용양리자지질체법장siRNA도입인배폐성섬유(HELF)세포，병분위선전염siRNA후감염HCMV、선감염HCMV후전염siRNA이급동시전염siRNA화감염HCMV 3조，동시설치정상대조조、음성대조조、양성대조조화전염시제대조조。채용형광정량PCR화경지당응효전영방법분별검측분석각조전염적siRNA대IE기인화양성대조기인GAPDH적억제효과。결과양성대조조GAPDH기인mRNA표체량교음성대조조화전염시제대조조유명현하강，siRNA억제솔위70.4%，차이유통계학의의(P<0.05)。선전염siRNA후감염HCMV조、선감염HCMV후전염siRNA조、동시전염siRNA화감염HCMV조이급양성、음성、전염시제대조조적IE기인mRNA표체량비교，차이유통계학의의(F=146.93，P=0.000)。기중선전염siRNA후감염HCMV조이급동시전염siRNA화감염HCMV조대IE기인mRNA표체적억제효과균호우선감염HCMV후전염siRNA조，차이유통계학의의(P<0.05)。결론재체외，siRNA능유효억제파기인적표체，차선전염siRNA재감염병독적억제효과상대경호，제시siRNA대HCMV감염구유일정적예방작용。
Objective To investigate the effect on in vitro inhibition of the human cytomegalovirus (HCMV) gene replication by RNA interference using different orders of small interfering RNA (siRNA) transfection and HCMV infection. Methods The siRNAs were designed and synthesized according to the sequence of the immediate early (IE) genes of HCMV. The siRNAs were transfected into human embryonic lung fibroblastic (HELF) cells by cationic liposome through different orders including before or after or at the same time as HCMV infection. Simultaneously, the control groups were set including normal control group, negative control group, positive control group and transfection reagent group. The inhibitory effects of siRNAs on HCMV-IE and a positive control gene of GAPDH were examined by lfuorescent quantitative PCR and agarose gel electrophoresis. Results The expression level of GAPDH mRNA in positive control group was signiifcantly lower than that in negative control group and transfection reagent group (P<0.05), and the inhibition rate of siRNA was 70.4%. Among different experimental groups of siRNAs transfection before, after or at the same time as HCMV infection and three control groups of negative control group, positive control group and transfection reagent group, the expression level of IE mRNA was signiifcantly different (F=146.93, P=0.000). In the groups of siRNAs transfection before HCMV infection or at the same time as HCMV infection, the expression levels of IE mRNA were signiifcantly lower than those in the group of siRNAs transfection after HCMV infection (P<0.05). Conclusions In vitro, siRNAs can effectively inhibit the expression of target gene and it is better to do siRNA transfection ifrst then followed by HCMV infection. It is suggested that siRNA might have a role in prevention from HCMV infection.
