| Hepatocellular carcinoma (HCC) is one of most common cancer of China and its mortality rate ranks second among malignant diseases. Despite aggressive approaches made in the therapy of HCC in the past decades, there is no suitable surgical treatment for the patients with terminal stage of HCC and the postoperative patients with multiple recurrence or metastasis of HCC . Now nosurgical treatments such as chemotherapy, radiotherapy, immunotherapy, Chinese traditional medicine, microwave coagulation therapy, ablation of radiofreguency et al are used to treat them., but the long term outcome is uncertain. To improve the prognosis of HCC, new treatment with new strategy is needed. With the development of oncology, melocular biology and virology, gene therapy for tumor become a real therapeutic method from a dream. Gene therapy with a distinct antitumor strategy offer us anew clue for treatment of HCC.Adenoviruses are capable of infecting dividing and nondividing cells. In order to efficiently replicate, the expression of adenoviral El gene complex is vital. El gene complex consists of two regions. El A and E1B. El A binds to and inhibits the action of the cellular tumor suppressor pRB. This serves to create a cellular environment favorable for the synthesis of multiple copies of the adenoviral genome. E1B-55 kD expression is required to inhibit function of the cellular tumor suppressor p53. If it were not for adenoviral E1B expression, cellular levels of p53 would rise in response to adenoviral infection with resulting apoptosis or cell cycle arrest attenuating viral replication. Incidence of mutation of pRB and p53 are very frequent in rumor, so adenovirus with mutation of El A or E1B can selectively replicate in these tumor cell; but the replication of these adenovirus are limited in the cell with nomal path of p53 and pRB.6Adenovirus mutant HI01 invented by Shanghai Sanwei Pharmaceutic corporation. It contains a deletion of the E1B-55 kD gene, and this mutant virus exhibits significant cytopathic effects in the p53 mutant cancer cells, but only limited cytotoxicity in normal human cell with normal p53 function. In china , now HI 01 has entered the clinical trial.,but most of them were limited to the superficial tumor. Deep-seated tumors such as HCC, gastric cancer , cancer of colon and esophageal carcinoma are not touched. The mutation of p53 have been reported in 30-50% of HCC. Mutant p53 correlates with tumor dedifferentiation,metastatic potential, and other poor prognostic indicators such as capsular invasion and portal vein involvment. This experiment studied the antitumor effect of H10Ion the HCC cell lines in vitro and in vivo, and its relation with expression and mutation of p53 and p21 . Adriarnycin was also used to evaluated the synergy of HI01 and Adriarnycin for the inhibiting of turner growth of HCC cell lines. Our present study includes as fellows:Part 1: The experimental study of antitumor effect of H101 on HCC cell lines in vitro. Objective: To study the antitumor effect of HI 01 on the HCC cell lines in vitro, and itsrelationship between p53,p21WAF1 and antitumor effect of H101. Method: Three human HCC cell lines including two p53-wild type cell lines (HepG2 andBEL7404 ) and a p53-null cell line (Hep3B) were used . A immortalized nomal liver cell line (L02) which status of p53 was unknown was also used. The p53 status of L02 was assayed by polymerase chain reaction ( PCR) and single strand conformation polymorphism (SSCP). The viral titer of H101 were determined by End-Point Dilution Assay. Four cell lines were infected with HlOlwith different multiple of infection (MOI). 8 days after infection, the percentage of survival cells was determined by colorimetric assay. HepG2, Hep3B and L02 were infected with 5 MOI of HI01. After 24 hours or 36 hours- infection of H101, specimens were taken to assay the change of expression of p53 and p21 WAF1 by immuhistochemistry staining. Result: 1. There is no muatations or deletions of p53 of L02.2. At 100, 10,1,0.1 a...
|
PDF Full Text Request