基质金属蛋白酶抑制剂_

人TIMP2重组腺病毒载体的构建及其在血管平滑肌细胞中的表达

http://www.100md.com 《中国普通现代外科进展》 2005年第5期

基质金属蛋白酶抑制剂,,基质金属蛋白酶抑制剂·腺病毒，人·肌，平滑，血管,1材料与方法,2结果,3讨论,参考文献

     【摘要】目的：构建含人TIMP2(hTIMP2)基因的重组腺病毒载体并在大鼠主动脉平滑肌细胞中表达，为血管疾病基因治疗提供实验基础。方法：利用基因重组技术将腺病毒骨架质粒pAdEasy1以及线性化的重组穿梭质粒pTrackCMVhTIMP2共转化BJ5183受体菌，并在其中发生同源重组，利用重组前后抗性的改变筛选出重组子；重组腺病毒质粒AdhTIMP2经过293细胞的包装，扩增和纯化后，测定病毒滴度。腺病毒体外感染大鼠主动脉平滑肌细胞，一步法提取细胞总RNA,RTPCR检测hTIMP2的mRNA。并收集细胞上清，进行Western blot检测TIMP2蛋白。结果：得到了携带hTIMP2基因的重组腺病毒，纯化后滴度为4×1011efu/ml，在感染VSMC后24h即检测到hTIMP2的表达。结论：成功地构建了携带hTIMP2的重组腺病毒载体并体外转染VSMC，为下一步应用于血管疾病基因治疗提供了基础。

    【关键词】基质金属蛋白酶抑制剂·腺病毒，人·肌，平滑，血管

    The expression of human tissue inhibitor of metalloproteinase2(TIMP2)gene in rat aortic smooth muscle cells mediated by adenoviral vector

    ZHAO Xin,ZHANG Peirui,YU Xianghai,LIAO Mingfang,JING Zaiping

    1Department of Hepatobiliary Surgery,302 Hospital of PLA(Beijing 100039,China)

    2Department of Vascular Surgery,Changhai Hospital of the Second Military Medical University(Shanghai 200003,China)

    【ABSTRACT】Objective:To construct an adenoviral vector carrying human tissue inhibitor of metalloproteinase2(TIMP2)gene in order to mediate the expression of TIMP2 gene in vascular smooth muscle cells(SMC)in vitro.Methods:A recombinant adenovirus(AdhTIMP2)containging human TIMP2 cDNA fragment was generated by homologous recombination in BJ5183 bacteria.Recombinant plasmids were screened by alteration of antibiotics.The adenovirus vector was then packaged and amplified in 293 cells.The SMC of rat aortic were isolated and cultured in vitro and been infected with AdhTIMP2.The expression of TIMP2 was detected by the techniques of Western blot and RTPCR.Results:The recombinant adenoviral vector carrying human TIMP2 was constructed. The titer was 4×1011efu/ml after purification.The AdhTIMP2 could infect the cultured VSMC efficiently(MOI=100 ......

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