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1、中国微侵袭神经外科杂志(CMINSJ),2008,13(7)·321··实验研究·Bex2对人脑胶质瘤干细胞增殖、生长和凋亡影响的体外实验研究12122于如同,刘明峰,石琼,陈冲,饶海承(1.徐州医学院附属医院神经外科;2.徐州医学院研究生部,江苏徐州221002)摘要:目的观察Bex2真核表达载体对脑胶质瘤干细胞增殖、生长和凋亡的影响,为胶质瘤治疗提供理论基础。方法采用免疫磁珠系统分离、Nestin免疫组化鉴定人脑胶质瘤干细胞;将RT-PCR法获得的Bex2与质粒pcDNA3.1(+)连接,脂质体法转染胶质瘤干
2、细胞,于转染18h、36h、48h、72h、96h后,采用WesternBlot检测Bex2表达,MTT法及Hoechst染色试剂盒检测生长抑制率和凋亡情况。结果分离、培养出人脑胶质瘤干细胞,并成功构建Bex2的真核表达载体。转染后18hBex2即有表达,48h达到高峰,后渐下降;转染36h、48h、72h、96h时,细胞生长抑制率和凋亡率明显高于空载体组和空白对照组(P<0.05);转染72h时,细胞生长抑制率和凋亡率最高(P<0.05)。结论Bex2能够抑制脑胶质瘤干细胞的增殖和生长,诱导胶质瘤干细胞凋亡。关
3、键词:Bex2基因;真核表达载体;胶质瘤干细胞;细胞凋亡中图分类号:R730.264,Q25文献标志码:A文章编号:1009-122X(2008)07-0321-04EffectsofBex2geneonmultiplication,growthandapoptosisofhumanbraingliomastemcellsinvitroYURutong1,LIUMingfeng2,SHIQiong1,etal.1.DepartmentofNeurosurgery,AffiliatedHospitalofXuzho
4、uMedicalCollege,Xuzhou221002,China;2.GraduatedFaculty,XuzhouMedicalCollege,Xuzhou221004,ChinaAbstract:ObjectiveTostudyeffectsofBex2onmultiplication,growthandapoptosisinhumangliomastemcellsandproviderationaleforgenetherapyofglioblastoma.MethodsHumanbrainglioma
5、stemcellswereisolatedandindentifiedbyusingthemagneticactivatedcellssorting(MACS)andnestinimmunohistochemistry.ThecDNAofBex2wasamplifiedbyRT-PCR,andthenBex2ligatedtoplasmidpcDNA3.1(+).Therecombinantwastransfectedintohumangliomastemcells.On18,36,48,72and96hafte
6、rtransfection,theexpressionofBex2wasobservedbyWesternblotting.CellinhibitionratiowasdetectedbyMTT.GrowthinhibitionrateandapoptosisprofileofthecellswereassayedbyHoechst33258staining.ResultsHumanbraingliomastemcellswereisolatedandcultured,andrecombinantofBex2-p
7、cDNA3.1(+)constructedsuccessfully.Aftertransfection,Bex2begantoexpressat18h,reacheditspeakat48handthendecreasedgradually.MTTassaymanifestedthatgrowthinhibitionrateofBex2groupat72hwashigherthanthoseatothertimepoints(P<0.05).Moreover,growthinhibitionratesofBex2
8、groupat36,48,72,96hwereobviouslyhigherthanthoseofthevectorandcontrolgroups(P<0.05).TheresultsofHoechst33258assaywerecoincidentwiththosebyMTT.ConclusionBex2genecaninhibitproliferationandgr
