vegf基因体外转染大鼠骨髓间充质干细胞的实验研究

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1、VEGF基因体外转染大鼠骨髓间充质干细胞的实验研究998中国美容医学2006年9月第15卷第9期ChineseJournalofAestheticMedicine.Sept.2006.Vo1.15.No.9?基础研究?VEGF基因体外转染大鼠骨髓问充质干细胞的实验研究郑岩,易成刚1,郭树忠,刘丹,黄博,夏炜,潘华,隋继强(1.第四军医大学西京医院全军整形外科研究所陕西西安710032;2.第四军医大学生化教研室)?论着?[摘要]目的:探讨脂质体介导血管内皮细胞生长因子(VEGF)基因转染大鼠骨髓间充质干细胞(MSCS)应用于基因治疗的可行性,安

2、全性.方法:体外分离,培养,鉴定MSCs,PcDNA3.1(一)/VEGF165质粒转染MSCs,转染后用免疫荧光和ELISA检测MSCs表达VEGF蛋白的情况,MTT检测MSCs对VEGF质粒转染的敏感性.结果:骨髓中分离得到MSCs,流式细胞检测显示MSCs不表达CD34和CD45,但表达CD90.透射电镜观察可见细胞浆中含大量粗面内质网和分泌颗粒.VEGF基因转染MSCs后第5天抗VEGF免疫荧光染色约90%的MSCs呈阳性,ELISA检测结果显示PcDNA3.I(一)/VEGF165质粒转染组细胞培养上清液中VEGF含量明显高于对照组,

3、并于转染后第5天达到峰值.MTT检测结果显示VEGF质粒转染对MSCs增殖无影响.结论:MSC可作为VEGF基因转染的靶细胞用于基因治疗.[关键词]大鼠骨髓间充质干细胞;血管内皮细胞生长因子;基因治疗[中图分类号]Q8I3.I[文献标识码]A[文章编号]1008—6455(2006)09—998—03StudyofVEGFgonetransductiononratmesenchymalstemcellsinvitroZHENGYan,YICheng-gang,GUOShu—zhong,LIUDan,HUANGBo,XIAWei,PANHua,S

4、UIJi—qiang(DepartmentofP1asticSurgery,XijingHospital,theFourthMilitaryMedicalUniversity,Xian710032,Shanxi,China)Abstract:0bjectiveToinvestigatethefeasibleandsecurityofVascularendothelialgrowthfactor(VEGF)transductiononratmesenchymalstemcells(MSCs)mediatedbyliposome.MethodsMS

5、CswereisolatedfromSprague—Dawley(SD)ratbonemarrowandculturedjnvitro.CulturedMSCswereanalyzedbyfluorescence—activatedcellsorting(FACS).PlasmidPcDNA3.1(一)NEGF165containingVEGFgenewastransducedintotheMSCsbyIiposome.MSCswithouttransductionwereusedascontrols.HumanVEGFexpressionin

6、vitrowasassessedbyjmmunofluorescencestainingandELISA.M-I-ImethodwasusedtodetecttheeffectoftransductiononMSCs.ResultsTheMSCsobtainedfrOmratsbonemarrowdidnotexpressCD34andCD45.however.almostallthecellsexpressedCD90.Approximately90%ofcellsinVEGF—transducedMSCsgroupwerestainedpo

7、sitivelyonday5aftergenetransduction.TherewasnoVEGFexpressioninnon—transducedMSCsgroup.TheIeveIofhumanVEGFjntheculturemedium0fVEGF—transducedMSCswassignificantlyjncreasedandreachedapeakatday5.andnoVEGFwasdetectedjntheculturemediumofnon—transducedMSCs.ItwasntfoundthatVEGFtrans

8、ductionhadasignificantlyinhibitoryeffectonMSC.ConclusionMSCcanbetargetcelIf