中华肝胆外科杂志
中華肝膽外科雜誌
중화간담외과잡지
CHINESE JOURNAL OF HEPATOBILIARY SURGERY
2012年
5期
372-376
,共5页
牛坚%赵何伟%王文%于彬%刘斌%王学浩%张业伟
牛堅%趙何偉%王文%于彬%劉斌%王學浩%張業偉
우견%조하위%왕문%우빈%류빈%왕학호%장업위
肝癌细胞%人胰岛素样生长因子1类受体%野生型P53%慢病毒
肝癌細胞%人胰島素樣生長因子1類受體%野生型P53%慢病毒
간암세포%인이도소양생장인자1류수체%야생형P53%만병독
Liver cancer cell%Human insulin like growth factor receptor 1%Wild type P53%Lentivirus
目的 研究anti-AFP scFv(抗AFP单链抗体)介导的慢病毒(lentivirus)载体对表达AFP肝癌细胞特异性基因转移以及双靶点基因系统对肝癌细胞生长的抑制作用.方法 用脂质体Lipofectamine 2000将目的基因转移载体、包装结构及包膜结构质粒共转染包装细胞293T,大量收集病毒上清,过滤、浓缩.用携带AFP-WtP53-pPRIME-miR30-shRNA-IGF1R融合基因的慢病毒体外转染培养的肝癌细胞HEP3B.荧光显微镜下观察感染效果,用PCR、Western blotting鉴定WtP53、miR30-shRNA-IGF1R在细胞中的整合转录结果.CCK8观察重组慢病毒对肝癌细胞生长的影响,TUNEL检测细胞凋亡.结果 成功构建anti-AFP scFv介导的慢病毒；滴度为4.58×109 PFU/ml;PCR、Western blotting均显示阳性条带,证明anti-AFP scFv介导的慢病毒在靶细胞中整合且转录表达.重组慢病毒对肝癌细胞的生长有明显的抑制作用且有促进细胞凋亡的作用.结论 anti-AFPscFv介导的慢病毒可将双靶点基因系统高效靶向性转染、杀伤肝癌细胞.
目的 研究anti-AFP scFv(抗AFP單鏈抗體)介導的慢病毒(lentivirus)載體對錶達AFP肝癌細胞特異性基因轉移以及雙靶點基因繫統對肝癌細胞生長的抑製作用.方法 用脂質體Lipofectamine 2000將目的基因轉移載體、包裝結構及包膜結構質粒共轉染包裝細胞293T,大量收集病毒上清,過濾、濃縮.用攜帶AFP-WtP53-pPRIME-miR30-shRNA-IGF1R融閤基因的慢病毒體外轉染培養的肝癌細胞HEP3B.熒光顯微鏡下觀察感染效果,用PCR、Western blotting鑒定WtP53、miR30-shRNA-IGF1R在細胞中的整閤轉錄結果.CCK8觀察重組慢病毒對肝癌細胞生長的影響,TUNEL檢測細胞凋亡.結果 成功構建anti-AFP scFv介導的慢病毒；滴度為4.58×109 PFU/ml;PCR、Western blotting均顯示暘性條帶,證明anti-AFP scFv介導的慢病毒在靶細胞中整閤且轉錄錶達.重組慢病毒對肝癌細胞的生長有明顯的抑製作用且有促進細胞凋亡的作用.結論 anti-AFPscFv介導的慢病毒可將雙靶點基因繫統高效靶嚮性轉染、殺傷肝癌細胞.
목적 연구anti-AFP scFv(항AFP단련항체)개도적만병독(lentivirus)재체대표체AFP간암세포특이성기인전이이급쌍파점기인계통대간암세포생장적억제작용.방법 용지질체Lipofectamine 2000장목적기인전이재체、포장결구급포막결구질립공전염포장세포293T,대량수집병독상청,과려、농축.용휴대AFP-WtP53-pPRIME-miR30-shRNA-IGF1R융합기인적만병독체외전염배양적간암세포HEP3B.형광현미경하관찰감염효과,용PCR、Western blotting감정WtP53、miR30-shRNA-IGF1R재세포중적정합전록결과.CCK8관찰중조만병독대간암세포생장적영향,TUNEL검측세포조망.결과 성공구건anti-AFP scFv개도적만병독；적도위4.58×109 PFU/ml;PCR、Western blotting균현시양성조대,증명anti-AFP scFv개도적만병독재파세포중정합차전록표체.중조만병독대간암세포적생장유명현적억제작용차유촉진세포조망적작용.결론 anti-AFPscFv개도적만병독가장쌍파점기인계통고효파향성전염、살상간암세포.
Objective To investigate the targeting infection of single chain antibody againstAFP (scFv anti-AFP) directed lentivirus and the inhibitory effects of a dual-growth inhibition systemon hepatocarcinoma cells.Methods Plasmids WtP53-pPRIME-miR30-shRNA-IGF1R,pMD2G-Anti-AFP,and psPAX2 have previously been constructed to cotransfect to the packaging cell line 293Tusing Lipofectamine2000.The infection results were observed through fluorescence microscopy.PCRand Western blotting were used to demonstrate the successful transduction and transcription of theWtP53-pPRIME-miR30-shRNA-IGF1R gene.The effects of reconstructed lentivirus infected liver cellgrowth were assessed by the cell growth curve of CCK8 cells. Apoptosis was evaluated by theTUNEL assay.Results Recombined lentivirus was successfully constructed with the functional PFUtiters of recombined lentivirus at 4.58× 109PFU/ml.This positive result was confirmed by PCR andWestern blotting.Conclusions The targeted therapy mediated by anti-AFP scFv could significantlyinhibit the proliferation of HEP3B cells and promote the apoptosis.
