| Adenoviral vectors have recently played an important role in gene transfer technology. There are many advantages in using Ad vector. Ad vector can accommodate up to 7.5 kb of foreign DNA and replicate efficiently to high titers. Ad has a broad host range of replicative and non-replicative cells and it remains epichromosomal in all know cells except eggs and therefore dose not interfere with other host genes. These features make it the best-suited vector system for gene therapy applications.Extracellular matrix (ECM) is a key regulator for cell adhesion, migration, proliferation and angiogensis. Matrix metalloproteinases (MMPs) are one family of structurally related enzyme (zinc-dependent endopeptidases) that have the cooprative ability to degrade nearly all extracellular matrix molecules. An important mechanism for the regulation of the activity of MMPs is via their binding to a family of homologous protein referred to as tissue inhibitors of metalloproteinases(TIMPs). The effects of MMPs and TIMPs in the course of cancer formation and tumor metastasis has been reported. The anti-angiogenic therapy aimed at MMPs, including the application of MMPs inhibitor and gene therapy of adenoviral vectors, can decrease the tumor's ability of invasion and metastasis, and it is probably a promising treatment of cancer.In this paper, we constructed an adenoviral vector carrying TIMP1, pAdl/TIMPl, according to the simplified system for generating homologous recombinant adenoviruses in bacteria, and transfected it into QBI-293A cells in which the recombinant adenovirus are packaged and amplified. The process could be conveniently followed with green fluorescent protein(GFP), encoded by a gene incorporated into the viral backbone. The correct construction and expression of TIMP1 in 293 A were detected by PCR and Western blot. The titer of the virus was 1 X 10l2--10l3efu/ml after purification, paving the way for further application in cancer gene therapy.To observe the effects of the TIMP1 on the adhesion and motility of human livercarcinoma cell line BEL-7402 in vitro, the cells were infected with Adl/TIMPl virus to overexpress TIMP1. Infected cells were collected by trypsinization after 48h and reseeded at the same density into 96 well dishes coated with fibronectin,collagenl and the blank. The culture dishes were dramatically shaked to remove nonadherent cells after 2 hrs. The number of left adherent cells indicate the ability of cell adhesion to the culture matrix. The results showed that FN and Coll could increase the tumor cell's ability of adhesion. And the number of adherent cell infected with TIMP1 on FN and blank increased 22% and 28% compared with that of control cells. While the effect of Coll was smaller, the increase of adherent cells infected with TIMP1 was 6%.Motility assay was carried out in two assay systems: agarose globelet method and scraping method. Both experiments showed that cells infected with TIMP1 migrated much slower than the control cells. The migration rate reduced 52% and 62% respectively. It is indicated that TIMP1 inhibited the migration of BEL-7402 cells.There were always different opinions about the effect of TIMP 1 on cell growth in different cells. To understand its effect on BEL-7402, MTT and FACS method were carried out to examine the effect of high expression of TIMP 1 on BEL-7402 cell cycle. MTT studies demonstrated that TIMP 1 could slightly inhibit the cell growth but not significantly. FACS has got the similar results. Sum up, TIMP1 did not effect BEL-7402 cell growth.Some other recombinant adenoviruses were constructed with the simplified system for generating homologous recombinant adenoviruses in bacteria, they were pAdl-STAB(WT), pAdl-STAT3(CYF), pAdl-TCF4, PAdl-dnTCF4 and pAdl-B catenin. |
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