携带gad67基因真核表达载体的构建与转染骨髓基质细胞的的研究

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1、华中科技大学硕士学位论文染大鼠骨髓基质细胞，观察其转染效果及转染后GAD67基因在骨髓基质细胞中的表达。[方法]利用基因重组技术，用NotI、SpeI分别对pCMV6-XL5-GAD67质粒进行酶切获得GAD67基因片段，然后将其插入pDC316-CMV-EGFP质粒构建pDC316-CMV-EGFP-GAD67真核表达载体，经酶切、测序鉴定正确；通过转基因技术利用脂质体将其导入通过贴壁方法分离培养出来的骨髓基质细胞，荧光显微镜下观察、流式细胞仪检测其转染效率，免疫组化检测GAD67基因的表达。[结果]经酶切、测序鉴定证实成功

2、构建pDC316-CMV-EGFP-GAD67真核表达载体，其中插入有3.5kbp的人类GAD67cDNA，转染骨髓基质细胞后荧光显微镜下观察可见大量带绿色荧光的细胞，经流式细胞仪检测其转染效率为37.3%，免疫组化检测证实GAD67基因在转染后的BMSCs中呈阳性表达。[结论]成功构建pDC316-CMV-EGFP-GAD67真核表达载体，且脂质体可有效介导人类GAD67基因转染骨髓基质细胞，为进一步研究基因修饰的骨髓基质细胞癫痫灶内定向移植治疗癫痫奠定了实验基础。[关键词]谷氨酸脱羧酶67；真核表达载体；转染；骨髓基质细胞

3、3华中科技大学硕士学位论文ConstructionofeukaryoticexpressionvectorwithGAD67geneandresearchoftransfectingitintobonemarrowstromalcellsMajor:NeurologyMasterdegreecandidate:XieSanPingSupervisor:ProfessorChenJiXiangAbstractBackgroundEpilepsyisaverycommonneurologicaldisease,anditcanoc

4、curoverawideagerange.Atpresent,prevalenceestimatesforepilepsyfromsurveyinChinaabout0.5%,therearemorethan6millionpatientsoverthewholecountry.Nowthemaintherapytoepilepsyisdrugtreatmentandabout80percentseizurescanbecontrolledbytherationalandscientificuseofantiepileptic

5、s,andabout70percentofepilepticpatientscanachievecompletecontrol.Butstillabout20percentofpatientsremainseizureafterdrugtreatment,andmostofthemeventuallyturnintorefractoryepilepsy,thatcontainstworeasons,onethatmanypatientsareunabletostandthehightoxicandadverseeffectof

6、antiepileptics,theotheroneisthatsomepatientsstillgetseizuresbecausetheantiepilepticsarenotfunctioningproperlyintheirbodies.Notonlythepathologicalchangecannotbereversedbutalsothenaturalprocessofepilepticdevelopmentcannotbeprohibitedunderdrugtreatment,becauseantiepile

7、pticscanonlyinhibitseizures.MoreovertheseriousADR(AdverseDrugReaction),prolongeduseandexpensivepricesofantiepilepticsbringgreatdifficultiestothepatientsandtheirfamilies.Sothereisapressingneedtofindasafe,effective,andfeasiblemethodinthetreatmentofepilepsy.Fromalotofr

8、esearchworkswecanseethatseizuresareassociatedwiththedistributionofGABAneurons4华中科技大学硕士学位论文degenerationandabsencewithinepileptogenicfocus.D