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时间:2020-04-27
《RNA干扰抑制CD133表达对CD133+肝癌干细胞增殖和化疗敏感性的影响-论文.pdf》由会员上传分享,免费在线阅读,更多相关内容在行业资料-天天文库。
1、2012;32(12)南方医科大学学报(JSouthMedUniv)·174l·RNA干扰抑制CD133表达对CD133+肝癌干细胞增殖和化疗敏感性的影响兰曦,王勇,曹妹,邹冬玲,李芳,李少林重庆医科大学放射医学教研室,重庆400016摘要:目的利用慢病毒介导的RNA干扰技术下调CD133HepG2肝癌干细胞中CD133的表达,探讨其对肝癌干细胞增殖及化疗敏感性的影响。方法利用流式分选技术筛选CD133HepG2细胞并检测分选前后CD133表达量,体外成球及体内成瘤实验鉴定其“干性”;随后以CD133mRNA编码序列为干扰靶点,采用慢病毒介导的shRNA转
2、染CD133细胞。应用RT-PCR和WesternBlot检测CD133mRNA及蛋白表达,克隆形成实验检测转染前后细胞增殖情况,CCK.8法和流式细胞仪检测在化疗药物顺铂作用下转染前后细胞的生长抑制率及凋亡情况。结果分选前后CD133表达率分别为(3.36~1.80)%,(88.74~3.19)%:CD133细胞相对于CD133细胞有更强的体外成球和体内成瘤能力;转染CD133shRNA后的细胞CD133mRNA表达明显受到抑制(P3、P<0.01),且其在顺铂作用下的凋亡率也显著增~JIl(P4、133livercancerstemcellsandincreasestheircisplatinchemosensitivityLANXi,WANGYong,CAOShu,ZOUDongling,LIFang,LIShaolinDepartmentofRadiology,ChongqingMedicalUniversity,Chongqing400016,ChinaAbstract:ObjectiveTostudytheeffectsofCD133suppressionbylentivirus—mediatedRNAinterference(RNAi)o5、ntheproliferationandchemosensitivitvofCD133cancerstemcellsfCSCs1sortedfromHepG2cellline.MethodsCD133andCD133。cellsweresortedfromHepG2celllinebyflowcytometry,andtheexpressionofCD133beforeandaftercellsortingweredetected.ThestemcellpropertyofsortedCD133cellswerevalidatedbysphere-form6、ingassayinvitroandxenograftexperimentsvivo.Lentivirus—mediatedshorthaipinRNA(shRNA1targetingCD133weretransfectedintoCD133cells,andCD133mRNAandproteinexpressionsofthetransfectedcellsweredetectedbyRT—PCRandWesternblotting,respectively.Beforeandafterthetransfection,theproliferafiveab7、i1ityofCD133cellswasevaluatedbycolonyformafionassaVandthecellgrowthinhibitionrateandapoptosisfollowingciplatinexposureweredetectedusingCCK一8assayandflowcytometry.ResultsThesortedCD133ceilsshowedahighpurltyof(88.74±3.19)%,ascomparedwiththepuftyof(3.36~1.8O)%beforecellsorting.CD133c8、ellsshowedahightumorsphereformati
3、P<0.01),且其在顺铂作用下的凋亡率也显著增~JIl(P4、133livercancerstemcellsandincreasestheircisplatinchemosensitivityLANXi,WANGYong,CAOShu,ZOUDongling,LIFang,LIShaolinDepartmentofRadiology,ChongqingMedicalUniversity,Chongqing400016,ChinaAbstract:ObjectiveTostudytheeffectsofCD133suppressionbylentivirus—mediatedRNAinterference(RNAi)o5、ntheproliferationandchemosensitivitvofCD133cancerstemcellsfCSCs1sortedfromHepG2cellline.MethodsCD133andCD133。cellsweresortedfromHepG2celllinebyflowcytometry,andtheexpressionofCD133beforeandaftercellsortingweredetected.ThestemcellpropertyofsortedCD133cellswerevalidatedbysphere-form6、ingassayinvitroandxenograftexperimentsvivo.Lentivirus—mediatedshorthaipinRNA(shRNA1targetingCD133weretransfectedintoCD133cells,andCD133mRNAandproteinexpressionsofthetransfectedcellsweredetectedbyRT—PCRandWesternblotting,respectively.Beforeandafterthetransfection,theproliferafiveab7、i1ityofCD133cellswasevaluatedbycolonyformafionassaVandthecellgrowthinhibitionrateandapoptosisfollowingciplatinexposureweredetectedusingCCK一8assayandflowcytometry.ResultsThesortedCD133ceilsshowedahighpurltyof(88.74±3.19)%,ascomparedwiththepuftyof(3.36~1.8O)%beforecellsorting.CD133c8、ellsshowedahightumorsphereformati
4、133livercancerstemcellsandincreasestheircisplatinchemosensitivityLANXi,WANGYong,CAOShu,ZOUDongling,LIFang,LIShaolinDepartmentofRadiology,ChongqingMedicalUniversity,Chongqing400016,ChinaAbstract:ObjectiveTostudytheeffectsofCD133suppressionbylentivirus—mediatedRNAinterference(RNAi)o
5、ntheproliferationandchemosensitivitvofCD133cancerstemcellsfCSCs1sortedfromHepG2cellline.MethodsCD133andCD133。cellsweresortedfromHepG2celllinebyflowcytometry,andtheexpressionofCD133beforeandaftercellsortingweredetected.ThestemcellpropertyofsortedCD133cellswerevalidatedbysphere-form
6、ingassayinvitroandxenograftexperimentsvivo.Lentivirus—mediatedshorthaipinRNA(shRNA1targetingCD133weretransfectedintoCD133cells,andCD133mRNAandproteinexpressionsofthetransfectedcellsweredetectedbyRT—PCRandWesternblotting,respectively.Beforeandafterthetransfection,theproliferafiveab
7、i1ityofCD133cellswasevaluatedbycolonyformafionassaVandthecellgrowthinhibitionrateandapoptosisfollowingciplatinexposureweredetectedusingCCK一8assayandflowcytometry.ResultsThesortedCD133ceilsshowedahighpurltyof(88.74±3.19)%,ascomparedwiththepuftyof(3.36~1.8O)%beforecellsorting.CD133c
8、ellsshowedahightumorsphereformati
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