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

Objective:To construct an adenoviral vect or 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)i n vitro. Methods:A recombinant adenovirus(AdhTIMP-2)containging human TIM P-2 cDNA fragment was generated by homologous recombination in BJ5183 bacteria. Recombinant plasmids were screened by alteration of antibiotics.The adenovirus v ector was then packaged and amplified in 293 cells.The SMC of rat aortic were is olated 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 TI MP-2 was constructed. The titer was 4×1011efu/ml after purification.The AdhTIMP-2 could infect the cultured VSMC efficiently(MOI=100,infection ra te=94%).The expression of TIMP-2 gene in those infected cells was detected by R T-PCR.After the cells were infected with AdhTIMP-2 24hours,TIMP-2 protein cou ld be detected in the conditioned medium by Western blot. Conclusion:The recombinant adenoviral vector carrying human TIMP -2 is successfully constructed and AdhTIMP-2 can efficently mediate the expres sion of TIMP-2 gene in cultured VSMC,paving the way for further application in vascular disease gene therapy.