杆状病毒介导的内耳基因治疗的实验研究

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1、上海交通大学硕士学位论文杆状病毒介导的内耳基因治疗的实验研究姓名：王俊申请学位级别：硕士专业：耳鼻咽喉科学指导教师：王士礼20070401［结论］杆状病毒可以在体外高效，安全，快速的转染毛细胞和螺旋神经节细胞，有希望成为内耳基因治疗的新型载体。［关键词］杆状病毒，基因治疗，丁酸钠，螺旋神经节细胞，Coili器EXPERIMENTALSTUDYOFBACULOVIRUS-MEDIATEDINNEREARGENETHERAPYABSTRACT[Objective]Tostudythefeasibilityandthecharacteristicsofrecombinantbac

2、ulovirusasgenetransfervectorinnerear.[Methods]AfterthegenerationofrecombinantbaculovirusBac-GFPaccordingtoBac-to-Bacbaculovirusexpressionsystem,spiralganglionneurons(SGNs)wereinfectedbyBac-GFPwithdifferentmultiplicitiesofinfection(MOI)anddifferentconcentrationsofsodiumbutyrate.Thetransduce

3、dcellrateandmeanfluorescencestrength(MFS)weredetectedbyfluorescencemicroscopyandflowcytometry.ToxicityeffectsofrecombinantbaculovirusvectorsandsodiumbutyrateonSGNsweredeterminedbyspectroscopicmeasurementoftheMTT・UsingtheculturetechnologyoforganofCortiinvitro,establishedthemodeloftheculture

4、ofCortiandthecochlearexplantswereinfectedbyBac-GFP・[Results]BaculoviruswasabletoinfectprimarySGNcultures.Thedose-responsecharacteristicsofBac-CMV-GFPweredeterminedonSGNs,andtheexpressionlevelcouldbeup-regulatedbysodiumbutyrate.InfectionwithBac-CMV-GFPintheabsenceorpresenceofsodiumbutyrate(

5、<10mM)isconsideredtobenon-cytotoxictoprimarySGNs.WealsofoundthatSGNshadexpressedGFPat6hpost-infectionandthehighestnumbersofcellsexpressingGFPwereobservedatapproximately48hpost-infection.TheorganofCortioftheneonatalratgrewwell48hoursafterthetissuecultivation.Baculoviruswasabletoinfectprimar

6、ySpiralganglionneurons(SGNs)culturesandhaircells,andtheexpressionlevelcouldbeup-regulatedbysodiumbutyrate.Atthesametime,Bac-GFPandsodiumbutyratedidn9tchangetheirorganizedstructure.[Conclusions]Baculovirusisanovelandpromisingtoolforgenetransferintothecochlearbothforstudiesofthefunctionoffor

7、eigngenesandthedevelopmentofgenetherapystrategies.[KEYWORDS]baculovirus,genetherapy,sodiumbutyrate,spiralganglion,Corti9sorgan耳聋是影响人类生活质量最主要的问题之一。明显后天听力障碍者约占全世界总人口的10%,其中绝大多数是感音神经性聋［1］。我国新生聋儿每年以2万人的速度递增。听力障碍影响着各个年龄阶段的人们，居各类残疾之首，严重影响着人们生活、学习和社会交往，给家庭和社会带来了沉重的负担