Xia Dong, Yan Lu-Nan, Tong Yu, Wang Xin-Ping, Zhang Ming-Man, Zhao Lan-Ying
Department of General Surgery, West China Hospital, Sichuan University, Chengdu 610041, China.
Hepatobiliary Pancreat Dis Int. 2005 May;4(2):259-64.
Overexpression of human tissue inhibitors of metalloproteinase-1 (TIMP-1) may play an antitumor role in inhibiting hepatocellular carcinoma (HCC) growth and progression. The aim of the study was to construct a recombinant adenovirus vector carrying hTIMP-1 cDNA for liver gene therapy and observe its expression in vitro.
The full-length cDNA of hTIMP-1 was positively cloned into the adenoviral shuttle vector pAdTrack-CMV, and then cotransformed into competent BJ5183 cells with the adenoviral backbone plasmid pAdEasy-1. Thus, a recombinant adenovirus AdhTIMP-1 containing full-length cDNA of hTIMP-1 was generated by homologous recombination in E. coli. AdhTIMP-1 was then packaged and amplified in adenoviral packaging cells, or human embryonic kidney 293 cells. The viral titer was checked by green fluorescent protein (GFP), and the expression of hTIMP-1 in vitro was detected by the techniques of Western blot and RT-PCR.
The recombinant adenovirus vector carrying hTIMP-1 was constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. The transcription of TIMP-1 mRNA in 293 cells was checked by RT-PCR and TIMP-1 protein could be detected in the culture by Western blot analysis.
The successful construction of recombinant adenoviral vector carrying human TIMP-1 and the effective expression in vitro has laid a foundation for further study of its antitumor function and may pave the way for future application in liver gene therapy.
人基质金属蛋白酶-1（TIMP-1）的过表达可能在抑制肝细胞癌（HCC）生长和进展中发挥抗肿瘤作用。本研究旨在构建携带hTIMP-1 cDNA的重组腺病毒载体用于肝脏基因治疗，并观察其在体外的表达。
将hTIMP-1的全长cDNA正向克隆到腺病毒穿梭载体pAdTrack-CMV中，然后与腺病毒骨架质粒pAdEasy-1共转化到感受态BJ5183细胞中。通过在大肠杆菌中的同源重组产生了含有hTIMP-1全长cDNA的重组腺病毒AdhTIMP-1。然后在腺病毒包装细胞或人胚肾293细胞中对AdhTIMP-1进行包装和扩增。通过绿色荧光蛋白（GFP）检测病毒滴度，并通过蛋白质印迹和RT-PCR技术检测hTIMP-1在体外的表达。
通过限制性内切酶分析和DNA序列分析构建并确认了携带hTIMP-1的重组腺病毒载体。通过RT-PCR检测293细胞中TIMP-1 mRNA的转录，并通过蛋白质印迹分析在培养物中检测到TIMP-1蛋白。
携带人TIMP-1的重组腺病毒载体的成功构建及其在体外的有效表达为进一步研究其抗肿瘤功能奠定了基础，并可能为未来在肝脏基因治疗中的应用铺平道路。
