Preparation And Study Of Replication-defective Adenovirus Targeted To Hepatocarcinoma

Hepatocarcinoma is one of the malignancies that menace to human health severely. Now, the therapeutical measures for the treatment of hepatocarcinoma include surgery, chemotherapy and radiotherapy etc. But the effects are not very satisfactory. Gene therapy was considered that it has a bright application prospect. The chief problem of gene therapy is its lack of specificity. Using the tissue specific regulatory elements to promote the expression of therapeutic gene in corresponding tissue is generally applied. AFP is increased obviously in hepatocarcinoma patients' serum. AFP promoter is widely used for targeting gene therapy of liver cancer. Studies have confirmed that inactivation of p16 gene is occurred altofrequently in human primary hepatocarcinoma. So introducing the exogenous pl6 gene to hepatocarcinoma cells may recover the inhibition of cell proliferation, induce cell apoptosis and suppress the progression of hepatocarcinoma.AdEasy XL Adenoviral Vector System is used to produce the recombination replication-defective adenovirus. At first we inserted the exogenous pl6 gene into the shuttle vectors respectively behind the AFP core promoter AF0.3, CMV promoter and HREAF hybrid promoter. Then the shuttle vectors were linearized with Pme I and transformed into BJ5183-AD-1 competent cells. Homologous recombination was occurred with pAdEasy-1 vector. The recombinants were digested with Pac I to expose LITR and RITR, then transfected respectively into HEK293 cells, and produced four types of recombination replication-defective adenoviruses which carried exogenous p16 gene: Ad-CMV-pl6, Ad-AFP-pl6, Ad-HREAF-pl6 and AdTrack-CMV-p16.Human hepatocellular carcinoma HepG₂ cell line and Bel-7402 cell line were infected with recombination adenovirus Ad-AFP-p16, and the high-level expression of exogenous p16 gene was detected in hepatocellular carcinoma cell lines, and cell growth was inhibited remarkably. But human normal hepatic cell LO2 and non-hepatocarcinoma cells showed no obvious growth inhibition. pl6 gene specifically...