骨形成蛋白-7论文：腺相关病毒介导bmp-7和sox9体外共同转染人退变椎间盘生物学效应的观察

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1、骨形成蛋白-7论文：腺相关病毒介导BMP-7和SOX9体外共同转染人退变椎间盘生物学效应的观察【中文摘要】构建骨形成蛋白-7(BMP7)腺相关病毒载体质粒pAAV-hBMP7-IRES-ZsGreen和SOX9腺相关病毒载体质粒pAAV-hSOX9-IRES-tdTomato并进行腺相关病毒包装,体外共同转染人椎间盘细胞评价其生物学效应。方法：用分子克隆技术从质粒pUC57-hBMP-7获得BMP-7cDNA并克隆至AAV包装质粒pAAV-IRES-ZsGreen上,构建成pAAV-hBMP7-IRES-ZsGreen的AAV重组质粒,经酶切及测序鉴定

2、后,由深圳百恩维公司对PAAV-hBMP7-IRES-ZsGreen进行包装；同法获得SOX9过表达腺相关病毒pAAV-hSOX9-IRES-tdTomato的包装；AAV-BMP-7与AAV-SOX9联合转染人退变的椎间盘纤维环细胞,Westernblotting检测BMP-7与SOX9蛋白表达以及髓核细胞II型胶原表达量的变化。结果：经酶切鉴定及基因测序证实AAV包装质粒pAAV-hBMP7-IRES-ZsGreen与pAAV-hSOX9-IRES-tdTomato构建完成,荧光免疫组化显示BMP-7与SOX9蛋白表达,百恩维公司包装出具有生物学功

3、能的AAV-BMP-7,AAV-SOX9构建成功。Westernblotting显示BMP-7与SOX9在纤维环细胞中表达,AAV-BMP-7与AAV-SOX9双基因联合转染的纤维环细胞II型胶原表达量明显较AAV-BMP-7与AAV-SOX9基因转染的细胞II型胶原表达量多。结论：构建AAV-BMP-7和AAV-SOX9可在体外表达,BMP-7在体外能够协同SOX9促进退变纤维环细胞II型胶原表达增加,为BMP-7与SOX9体内实验与基因逆转椎间盘的早期退变提供了理论依据。【英文摘要】:Toconstructtheadeno-associatedvi

4、rus(pAAV-hBMP7-IRES-ZsGreen)vectorwithhumanbonemorphogeneticprotein7(BMP7)andadeno-associatedvirus(pAAV-hSOX9-IRES-tdTomato)vectorwithSOX9,andpackageintovirusparticles,andassessitsbiologicalactivityandfunctionbytransductingintohumandisccellsinvitro.Methods:TheBMP-7cDNAsequenceam

5、plifiedfromplasmidpUC57-hBMP-7wasclonedintopackplasmidpAAV-IRES-ZsGreen.PlasmidpAAV-hBMP7-IRES-ZsGreenwasconstructedbypAAV-IRES-ZsGreen.TherecombinantplasmidpAAV-hBMP7-IRES-ZsGreenwasidentifiedbyPCR,restrictionenzymesanalysisandsequencinganalysis,andthenpackageintovirusparticles

6、.ThepackageofSOX9adeno-associatedvirus(pAAV-hSOX9-IRES-tdTomato)wasobtainedinthesameway.TherecombinantexpressionplasmidAAV-BMP-7andAAV-SOX9wasco-transfectedintohumanannulusfibrosuscellsofdegeneratedlumbarintervertebraldiscs.Westernblotwasusedtodetecttheexpressionchangesofcollage

7、ntypeⅡ.Results:TherecombinantpAAV-hBMP7-IRES-ZsGreenandpAAV-hSOX9-IRES-tdTomatowerecompletelyconstructedandconfirmedbyrestrictionenzymesanalysisandsequencinganalysis.TheproteinhBMP-7andhSOX9weredetectedsuccesfullybyimmuofluorescence.AAV-BMP-7andAAV-SOX9wereconstructedsuccessfull

8、y.TheexpressionofAAV-hBMP-7andAAV-hSOX9weremani