医学分子生物学杂志
醫學分子生物學雜誌
의학분자생물학잡지
FOREIGN MEDICAL SCIENCES
2010年
1期
39-44
,共6页
肝干细胞%白蛋白启动子%荧光素酶报告基因%稳定细胞株
肝榦細胞%白蛋白啟動子%熒光素酶報告基因%穩定細胞株
간간세포%백단백계동자%형광소매보고기인%은정세포주
liver stem cell%ALB promoter%luciferase reporter gene%stable cell line
目的 构建稳定表达ALB启动子及荧光素酶报告基因的肝干细胞株.方法 PCR扩增获得ALB启动子,并与pBGLuc连接获得携带ALB启动子及荧光素酶报告基因的pBGLuc-ALB质粒,脂质体转染质粒到不同细胞,ALB-GLuc活性检测功能.构建逆转录病毒,感染HP14.5肝干细胞株获得携带ALB启动子及荧光素酶报告基因的稳定细胞株,经Dex、HGF体外诱导后第3、6、9、12天ALB-GLuc检测荧光素酶活性,免疫荧光检测ALB的表达.结果 PCR、酶切及测序结果显示ALB启动子正确插入至荧光素酶GLuc基因上游,HEK293、HP14.5、LC14d及Hepa1-6细胞中ALB-GLuc活性与免疫荧光结果一致.HP14.5 ALB-Gluc稳定细胞株在高浓度的稻瘟菌素中存活,免疫荧光结果显示Dex、HGF诱导后细胞中ALB的表达逐渐增强,并与ALB-Gluc活性升高一致.结论 成功构建了稳定表达ALB启动子及荧光素酶报告基因的肝干细胞株,为研究肝干细胞的体外成熟分化提供了重要的细胞手段.
目的 構建穩定錶達ALB啟動子及熒光素酶報告基因的肝榦細胞株.方法 PCR擴增穫得ALB啟動子,併與pBGLuc連接穫得攜帶ALB啟動子及熒光素酶報告基因的pBGLuc-ALB質粒,脂質體轉染質粒到不同細胞,ALB-GLuc活性檢測功能.構建逆轉錄病毒,感染HP14.5肝榦細胞株穫得攜帶ALB啟動子及熒光素酶報告基因的穩定細胞株,經Dex、HGF體外誘導後第3、6、9、12天ALB-GLuc檢測熒光素酶活性,免疫熒光檢測ALB的錶達.結果 PCR、酶切及測序結果顯示ALB啟動子正確插入至熒光素酶GLuc基因上遊,HEK293、HP14.5、LC14d及Hepa1-6細胞中ALB-GLuc活性與免疫熒光結果一緻.HP14.5 ALB-Gluc穩定細胞株在高濃度的稻瘟菌素中存活,免疫熒光結果顯示Dex、HGF誘導後細胞中ALB的錶達逐漸增彊,併與ALB-Gluc活性升高一緻.結論 成功構建瞭穩定錶達ALB啟動子及熒光素酶報告基因的肝榦細胞株,為研究肝榦細胞的體外成熟分化提供瞭重要的細胞手段.
목적 구건은정표체ALB계동자급형광소매보고기인적간간세포주.방법 PCR확증획득ALB계동자,병여pBGLuc련접획득휴대ALB계동자급형광소매보고기인적pBGLuc-ALB질립,지질체전염질립도불동세포,ALB-GLuc활성검측공능.구건역전록병독,감염HP14.5간간세포주획득휴대ALB계동자급형광소매보고기인적은정세포주,경Dex、HGF체외유도후제3、6、9、12천ALB-GLuc검측형광소매활성,면역형광검측ALB적표체.결과 PCR、매절급측서결과현시ALB계동자정학삽입지형광소매GLuc기인상유,HEK293、HP14.5、LC14d급Hepa1-6세포중ALB-GLuc활성여면역형광결과일치.HP14.5 ALB-Gluc은정세포주재고농도적도온균소중존활,면역형광결과현시Dex、HGF유도후세포중ALB적표체축점증강,병여ALB-Gluc활성승고일치.결론 성공구건료은정표체ALB계동자급형광소매보고기인적간간세포주,위연구간간세포적체외성숙분화제공료중요적세포수단.
Objective To construct a liver stem cell line with stable expression of functional ALB promoter and luciferase reporter gene.Methods ALB promoter was amplified by PCR and then ligated into luciferase reporter gene pBGLuc to get pBGLuc-ALB plasmid.The resultant plasmid was transfected into different cell lines to detect ALB-GLuc activity.Afterward,HP14.5 cells were infected with ALB-GLuc retrovirus to establish stable cell lines containing functional ALB promoter and luciferase reporter gene.Stable cell lines were cultured with Dex and HGF induction in vitro.ALB-GLuc activity and immunofluoresce were checked at 3,6,9,12 days after induction.Results ALB promoter was correctly cloned upstream GLuc gene.Relative ALB-GLuc activity in HEK293,HP14.5,LC14d and Hepa1-6 cells were consistent with immunofluorecence results.HP14.5 ALB-Gluc stable cell line selectively survived in medium containing high concentration of Blasticidin.The expression of ALB and ALB-Gluc activity gradually increased after Dex and HGF induction.Conclusion A liver stem cell line with stable expression of ALB promoter and luciferase reporter gene was successfully constructed,providing a tool for future study of hepatocyte differentiation.
