摘要
目的:构建含人TI MP-2(hTI MP-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-hTI MP-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AdhTI MP-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hTI MP-2的mRNA。并收集细胞上清,进行Western blot检测TI MP-2蛋白。结果:得到了携带hTI MP-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hTI MP-2的表达。结论:成功地构建了携带hTI MP-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。
Objeetive：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（SMO）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 adenovlrus 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 tech- niques of Western blot and RT-PCR. Results ： The recombinant adenoviral vector carrying human TIMP-2 was constructed. The titer was 4 × 10^11 efu/ml after purification. The AdhTIMP-2 could infect the cultured VSMC efficiently（MOI = 100, infection rate = 94%）： The expresslon of TIMP-2 gene in those Infected cells was detected by RT-POR. After the cells were Infected with AdhTIMP-2 24hours, TIMP-2 protein could be detected In the conditioned medium by Western blot. Conelusion ：The recombinant adenoviral vector carrying human TIMP-2 Is sucoessfully constructed and AdhTIMP-2 can efflcently mediate the expression of TIMP-2 gene In cultured VSMO, ioavlng the way for further application in vascular disease gene therapy.
出处
《中国现代普通外科进展》
CAS
2005年第5期288-291,共4页
Chinese Journal of Current Advances in General Surgery
关键词
基质金属蛋白酶抑制剂·腺病毒
人·肌
平滑
血管
Tissue inhibitor of metal loprotelnase · Adenoviruses, human · Muscle, smooth, vascular
