中华生物医学工程杂志
中華生物醫學工程雜誌
중화생물의학공정잡지
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
2012年
5期
380-383
,共4页
程木华%李建芳%张峰%曾凤伟%谢良骏
程木華%李建芳%張峰%曾鳳偉%謝良駿
정목화%리건방%장봉%증봉위%사량준
癌,肝细胞%甲胎蛋白类%RNA干扰%基因表达调控%慢病毒属
癌,肝細胞%甲胎蛋白類%RNA榦擾%基因錶達調控%慢病毒屬
암,간세포%갑태단백류%RNA간우%기인표체조공%만병독속
Carcinoma,hepatocellular%alpha-Fetoproteins%RNA interference%Gene expression regulation%Lentivirus
目的 构建携带甲胎蛋白(AFP)基因小干扰RNA(siRNA)的慢病毒载体并转染肝癌细胞,评价其对AFP基因的沉默效率.方法 设计和构建针对AFP基因的阳性siRNA及不针对任何已知基因的阴性siRNA,将其分别插入携带绿色荧光蛋白(GFP)基因的重组慢病毒载体,然后转染到表达AFP基因的人肝癌细胞HepG2并筛选阳性表达细胞株,分别为慢病毒转染阳性siRNA组和慢病毒转染阴性siRNA组.同时设立脂质体转染阳性siRNA组、脂质体转染阴性siRNA组以及加AFP siRNA而未用转染试剂的siRNA组、空白对照组.荧光显微镜观察评估转染效率,荧光定量PCR和免疫印迹法检测各组HepG2细胞APF mRNA及蛋白的相对表达量,比较不同转染方法对AFP基因表达的抑制率.结果 慢病毒能够有效地转染siRNA到HepG2细胞内.荧光定量PCR显示慢病毒转染siRNA对AFP mRNA表达的抑制率显著高于脂质体转染siRNA(92.1％比74.3％,P＜0.05).免疫印迹亦显示慢病毒转染siRNA对AFP蛋白表达的抑制率显著高于脂质体转染siRNA(88.2％比63.7％,P＜0.05).结论 慢病毒转染AFP siRNA能够更有效地抑制HepG2细胞内AFP基因表达.
目的 構建攜帶甲胎蛋白(AFP)基因小榦擾RNA(siRNA)的慢病毒載體併轉染肝癌細胞,評價其對AFP基因的沉默效率.方法 設計和構建針對AFP基因的暘性siRNA及不針對任何已知基因的陰性siRNA,將其分彆插入攜帶綠色熒光蛋白(GFP)基因的重組慢病毒載體,然後轉染到錶達AFP基因的人肝癌細胞HepG2併篩選暘性錶達細胞株,分彆為慢病毒轉染暘性siRNA組和慢病毒轉染陰性siRNA組.同時設立脂質體轉染暘性siRNA組、脂質體轉染陰性siRNA組以及加AFP siRNA而未用轉染試劑的siRNA組、空白對照組.熒光顯微鏡觀察評估轉染效率,熒光定量PCR和免疫印跡法檢測各組HepG2細胞APF mRNA及蛋白的相對錶達量,比較不同轉染方法對AFP基因錶達的抑製率.結果 慢病毒能夠有效地轉染siRNA到HepG2細胞內.熒光定量PCR顯示慢病毒轉染siRNA對AFP mRNA錶達的抑製率顯著高于脂質體轉染siRNA(92.1％比74.3％,P＜0.05).免疫印跡亦顯示慢病毒轉染siRNA對AFP蛋白錶達的抑製率顯著高于脂質體轉染siRNA(88.2％比63.7％,P＜0.05).結論 慢病毒轉染AFP siRNA能夠更有效地抑製HepG2細胞內AFP基因錶達.
목적 구건휴대갑태단백(AFP)기인소간우RNA(siRNA)적만병독재체병전염간암세포,평개기대AFP기인적침묵효솔.방법 설계화구건침대AFP기인적양성siRNA급불침대임하이지기인적음성siRNA,장기분별삽입휴대록색형광단백(GFP)기인적중조만병독재체,연후전염도표체AFP기인적인간암세포HepG2병사선양성표체세포주,분별위만병독전염양성siRNA조화만병독전염음성siRNA조.동시설립지질체전염양성siRNA조、지질체전염음성siRNA조이급가AFP siRNA이미용전염시제적siRNA조、공백대조조.형광현미경관찰평고전염효솔,형광정량PCR화면역인적법검측각조HepG2세포APF mRNA급단백적상대표체량,비교불동전염방법대AFP기인표체적억제솔.결과 만병독능구유효지전염siRNA도HepG2세포내.형광정량PCR현시만병독전염siRNA대AFP mRNA표체적억제솔현저고우지질체전염siRNA(92.1％비74.3％,P＜0.05).면역인적역현시만병독전염siRNA대AFP단백표체적억제솔현저고우지질체전염siRNA(88.2％비63.7％,P＜0.05).결론 만병독전염AFP siRNA능구경유효지억제HepG2세포내AFP기인표체.
Objective To construct lentiviral vectors carrying small interference RNA (siRNA) of alpha-fetoprotein(AFP) followed by transfection into hepatocellular carcinoma cells and to assess the efficiency of AFP gene silencing.Methods The AFP-specific positive siRNA and non-specific negative siRNA were designed and constructed prior to insertion to the recombinant lentiviral vectors carrying green fluorescent protein (GFP) gene.This was followed by transfection into human hepatocellular carcinoma cells HepG2 and subsequent screening of cell strains with GFP expression,and were assigned into lentivirustransfected positive siRNA group and lentivirus-transfected negative siRNA group respectively.Furthermore,liposome-transfected positive siRNA group,liposome-transfected negative siRNA group,siRNA group which treated with AFP siRNA but without transfection reagent,and blank control group were established simultaneously.The efficiency of transfection was assessed under fluorescent microscope,and the relative expression of AFP mRNA and protein in HepG2 cells was determined via fluorescent quantitative polymerase chain reaction and immunoblotting assay.In addition,the inhibition rate of AFP gene expression was compared among all groups.Results Lentiviral vectors were associated with effective transfection of siRNA into HepG2 cells.The siRNA transfected with lentivirus yielded considerably higher inhibition rate of AFP mRNA than that with liposomes,as evidenced by fluorescent quantitative polymerase chain reaction (92.1％vs 74.3％,P＜0.05).Immunoblotting assay showed that higher inhibition rate of AFP protein was in favor of siRNA transfected with lentivirus,but not with liposomes (88.2％ vs 63.7％,P＜0.05).Conclusion AFP siRNA transfected with lentivirus is associated with more effective inhibition of AFP expression in HepG2 cells.
