hIGF1基因转染促进退变椎间盘Ⅱ型胶原的表达

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1、hIGF-1基因转染促进退变椎间盘Ⅱ型胶原的表达作者：黄宗强，刘尚礼，郑召民【摘要】［目的］探讨人胰岛素样生长因子（humaninsulin-likegrowthfactor-1,hIGF-1)基因在退变椎间盘中的表达及对椎间盘中Ⅱ型胶原表达的影响。［方法］参考文献〔1〕制备出腰椎间盘退变（intervertebraldiskdegeneration,IVDD)动物模型24只，随机分为携带hIGF-1基因重组腺病毒（Ad/CMV-hIGF-1)、hIGF-1生长因子及PBS组。L4、5、L5、6椎间盘中分别注射25μl第2代Ad/CMV-hIGF-1(8

2、×108PFU)、hIGF-1生长因子（100μg/L)、PBS。注射后1、2、4、8周，Westernblot检测hIGF-1蛋白表达；半定量RT-PCR检测Ⅱ型胶原mRNA的表达。［结果］hIGF-1蛋白带出现在7540.00u。Ad/CMV-hIGF-1组hIGF-1蛋白表达持续达4周以上，hIGF-1组表达持续约2周；PBS注射组无hIGF-1蛋白表达。Ⅱ型胶原电泳条带出现在200～300bp；在注射后1～4周，Ad/CMV-hIGF-1组内Ⅱ型胶原mRNA相对表达量进行性增加，8周轻度下降，4个时期比较（F=12.18，P＜0.001)；注射后

3、1～8周，hIGF-1注射组、PBS注射组Ⅱ型胶原mRNA相对表达量进行性下降（F=27.38、21.56，P＜0.001)。相同时间点3组比较，Ⅱ14型胶原mRNA相对表达量Ad/CMV-hIGF-1组最高，PBS组最低（F=27.31～39.85，P＜0.001）。［结论］hIGF-1基因在退变椎间盘中的表达能够促进合成Ⅱ型胶原。【关键词】椎间盘；退变；表达；hIGF-1Abstract［Objective］TostudytheroleofhIGF-1geneoncollagentypeⅡexpressionofdegenerativeinterve

4、rtebraldisk.［Method］Twenty-fourmaleNew-Zealandrabbitsintervertebraldiskdegenerontion(IVDD)modelsweredoneaccordingtoreferenceandrandomlydividedintoAd-CMV-hIGF-1,hIGF-1growthfactorandPBSgroup.TwentyfivemicrolitrethesecondgenerationAd/CMV-hIGF-1(T=80×109PFU/L),hIGF-1growthfactor(100

5、μg/L)andPBSwererespectivelyinjectedintoL4、5,L5、6intervertebraldiskunderfluoroscopicguidance.One,two,fourandeightweekspost-operation,rabbitsweresacrificed,intervertebraldisksampleswereharvested.Totalproteinsofequalmassintervertebraldiskswereextracted,isolatedinSDS-Polyacrylamidege

6、lelectrophoresis(SDS-PAGE)andtransferredtopolyvinylidenedifluoride(PVDF)Millipore.ThehIGF-1growthfactorexpressionwereindentifiedwithWesternblot.CollagentypeⅡgenefragmentswereamplifiedwithRT-PCR,andrelativeexpressionwasdonewithGAPDHasinterncontrol.［Result］ThehIGF-114interestprotei

7、nexistedat7.6Kilo-Dalton.Atoneweekafterinjection,itsexpressionquantitieswerealmostequalbetweenAd/CMV-hIGF-1andhIGF-1growthfactorgroup.Attwoweekafterinjection,itobviouslydeclinedinhIGF-1growthfactorgroup.Atfourweekafterinjection,itstillexpressedinAd/CMV-hIGF-1group.Ateightweekafte

8、rinjection,itdidnotexpressinthereegroups