慢病毒介异的孕烷X受体sh-RNA稳定干扰HepG2细胞株的构建-论文.pdf

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1、癌变一变建变第26卷第3期圈■圈EstablishmentofaHepG2linewithstablePXRsilencing0byl1ent-iv-irus-med-·iated。RNAinterferenceN兀JYong-dong]’2CHENLi-ming3WUMan-peng4，。CHENGHe，xUHan，GAOFen-fei，SHIGang-gang1'f1．DepartmentofPharmacology,ShantouUniversityMedical汕头515041；2．上海市肿瘤研究所，癌

2、基因College,Shantou515041,"2．StateKeyLaboratoryofOncogenes及相关基因国家重点实验室，上海200032；andRelatedGenes,ShanghaiCancerInstitufShanghai200032；3．汕头大学医学院第一附属医院肿瘤科，广东14∞3．DepartmentofOncolog~,FirstAfiliatedHospitalofShantou汕头515041；4．汕头市第二人民医院儿科，Universi(vMedicalCollege,S

3、hantou515041,"Departmentof广东汕头515011)Pediatrics,TheSecondPeople'sHospitalofShantou,Shantou515011，Guangdong,Oa．a)【摘要】目的：构建慢病毒介导的人孕烷x受体(hPXR)sh—RNA干扰载体及筛选肝癌细胞稳定干扰株。方法：针对hPXR的特异性序列，设计干扰序列，构建重组克隆。转染HEK293T细胞，筛选有效干扰片段。HEK293T包装干扰病毒并转染肝癌HepG2细胞株，200mmol／L嘌呤霉素筛选2—3

4、周，Westernblot检测PXR表达。hPXR激动剂利福平处理PXRsh—RNA稳定干扰肝癌HepG2细胞株和Scramble对照细胞，抽提RNA并鉴定hPXR靶基因细胞色素P4503A4(CYP3A4)的表达。结果：筛选出了PXR有效干扰片段，并获得了PXR信号通路有效敲低的HepG2慢病毒稳定干扰株。结论：成功构建了慢病毒介导的PXRsh—RNA稳定干扰HepG2细胞株。【关键词】人孕烷X受体；原发性肝细胞癌；慢病毒；RNA干扰中图分类号：R365文献标志码：A文章编号：1004—616X(2014)0

5、3—0209—04doi：10．3969~．issn．1004—616x．2014．03．011[ABSTRACT]0BJECTIVE：ToestablishaHepG2celllinewithstablePXRsilencingeffectbyalentiviralvectorcarryingashorthairpinRNAfsh-RNA)．M【ETHoDS：Threedouble-strandedsh-RNAtargetingthePXRgeneweredesigned．synthesizedandclon

6、edintothepsi—sHlsh—RNAvector．Theeffectiverecombinantsh—RNAexpressionplasmidagainstPXR．whichwasselectedofHEK293Tcellswiththreedouble—strandedsh—RNAplasmids，waspackagedintoHEK293TcellsandusedtoinfectHeDG2cellsafter200mmol／Lpuromycinscreeningfor2—3weeks．Theexpr

7、essionlevelofPXRwasdetectedbywesternblot．theCYP3A4mRNAlevelsbothinsh—RNAPXRHepG2stablecellsandscramblecontrolHepG2cellsweredetectedbyreal—timePCR．RESUIS：Arecombinantsh—RNAexpressionplasmidagainstPXRgenewitheffectiveinterferencewassuccessfnllyselectedandaHepG

8、2celllinestablytransfectedwiththesh—RNAPXRwasestablished．CONCLUSION：AHepG2celllinestablytransfectedwiththesh—RNAPXRwasestablished．／／cueYwoRDS】humanpregnaneXreceptor；hepatocellularcarcinoma；lenti