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时间:2019-01-04
《shrna干扰沉默mcl-1基因对淋巴瘤细胞系增殖的影响》由会员上传分享,免费在线阅读,更多相关内容在工程资料-天天文库。
1、shRNA干扰沉默Mcl-1基因对淋巴瘤细胞系增殖的影响岳光星1,张明智1,张旭东1,许伟朋2,陈新峰1,陈昱丞1,曹玲1,杨黎3,盛誉乔3(450052郑州,郑州大学第一附属医院:肿瘤科1,骨科2,河南省高等学校临床医学重点学科开放实验室3)[摘要]目的研究慢病毒介导的髓细胞白血病基因(Mcl-1)短发夹状RNA(shRNA)干扰对淋巴瘤细胞系SNK-6增殖和凋亡的影响。方法设计以Mcl-1为靶点的短发夹状RNA(shRNA)并构建携带此shRNA的慢病毒载体,转染淋巴瘤细胞系SNK-6,荧光定量PCR(qPCR)及Westernblot
2、方法检测病毒感染前后Mcl-1mRNA及蛋白表达变化,四甲基偶氮唑盐法(MTT法)和流式细胞仪分析细胞增殖和凋亡变化的情况。结果成功构建Mcl-1-shRNA慢病毒表达载体,并可有效感染淋巴瘤细胞株SNK-6,感染后SNK-6细胞Mcl-1基因的mRNA水平及蛋白水平表达明显下调,,MTT法检测显示慢病毒介导的Mcl-1基因shRNA干扰与对照病毒组相比可明显抑制SNK-6细胞增殖[(31.6±3.3)%vs(5.8±2.7)%,P<0.01],流式细胞仪测定感染后细胞凋亡明显高于对照病毒组[(28.9±2.1)%vs(5.3±1.5)%,
3、P<0.01]。结论慢病毒介导的Mcl-1基因shRNA干扰技术可特异性阻断SNK-6细胞Mcl-1基因的表达,抑制细胞的增殖并促进细胞的凋亡。[关键词]淋巴瘤;髓细胞白血病基因-1;短发夹RNA;凋亡;慢病毒载体[中图法分类号][文献标志码]ATheeffectsofshRNAinterferencetargetingMcl-1onproliferationoflymphomacelllineYueGuangxing1,ZhangMingzhi1,ZhangXudong1,XuWeipeng2,ChenXinfeng1,ChenYuche
4、ng1,CaoLing1,YangLi3,ShengYuqiao3(Departmentof1oncology,2orthopedics,3InstituteofClinicalMedicine,theFirstAffiliatedHospitalofZhengzhouUniversity,ZhengzhouHenan450052,China)[Abstract]ObjectiveToexploretheeffectsoflentivirus-mediatedshRNAinterference(RNAi)targetingtheMyeloi
5、dleukemia-1(Mcl-1)onproliferationandapoptosisoflymphomacelllineSNK-6.MethodsShorthairpinRNA(shRNA)targetingMcl-1wasdesignedandthelentiviralvectorcarryingtheshRNAwasconducted.AfterinfecttionoflentiviralvectorinSNK-6cells,RealtimePCRandwesternblotwereusedtoanalyzetheexpressi
6、onofMcl-1mRNAandprotein.MTTassayandflowcytometrywereemployedtodetectthestatusofproliferationandapoptosis.ResultsTheshRNAtargetingMcl-1wassuccessfullyinsertedintothelentiviralvector,whichcaneffectivelyinfectthelymphomacelllineSNK-6.AfterinfectingtheSNK-6cells,theexpressiono
7、fMcl-1mRNAandproteinwasobviouslydecreasedandMTTassaysuggestedthattheproliferationofSNK-6cellswasinhabitedbyshRNAinterferencetargetingMcl-1againstcontrol-shRNAgroup(31.6%±3.3%vs5.8%±2.7%,P<0.001).FlowcytometryshowedthattheapoptosisrateofMcl-1-shRNA-infectedcellswassignifica
8、ntlyhigherthanthecontrolgroup(28.9%±2.1%vs5.3%±1.5%,P<0.001).ConclusionLentivirus-mediate
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