Construction Of The Targeting Recombinant Adenoviral Vector Carrying MDR1-Antisense RNA And Its Effect In Reversing The MDR Hepatocellular Cells

Hepatocellular carcinoma (HCC) is one of the most commonmalignant tumors in some areas of the world with an estimated 500,000 to1,000,000 new cases per year. It has an annual incidence of up to 500 casesper 100,000 populations in certain regions of Asia and sub-Saharan Africa. The prognosis of HCC patients is generally very poor. Most studieshave shown a five-year survival rate of less than 5% in symptomaticpatients. HCC has been found to be quite resistant to radio- orchemotherapy. Investigations of the natural history and clinical course ofHCC revealed a long-term survival of patients only with smallasymptomatic HCC that could be treated surgically or nonsurgically. Forpatients with advanced symptomatic HCC, novel therapeutic strategiessuch as gene therapy are urgently needed. Based on rapid scientific 9重庆医科大学博士学位论文advances, molecular diagnosis, gene therapy and molecular prevention arebecoming increasingly part of our patient management and will eventuallycomplement or in part replace the existing diagnostic, therapeutic andpreventive strategies. In recent years, several strategies of gene therapy and moleculartherapy against malignant tumor have been developed. To suppress aharmful gene, anti-sense gene and anti-sense oligonucleotide, hammerheadribozymes as well as the novel developed RNA interference (RNAi) areshowing promising strategies. The development of multiple drug resistance (MDR) in tumor cells isa significant problem in cancer clinic therapy. Various tumor cells wereobserved to develop multidrug resistance when the cells were treated withchemotherapy drugs. It has been recognized as a major hindrance tosuccessful chemotherapy. In human, one of the underlying mechanisms ofMDR is cellular over-production of P-glycoprotein (P-gp170), a 170-kDamember of the ATP-binding cassette transmembrane transportersuperfamily which serves as a drug efflux pump. By rapid elimination ofdrugs that enter cells and thus decreasing their intracellular accumulationand efficacy, tumor resistance can occur despite high levels of drugs in thesurrounding tissue or blood. P-gp170 is encoded by a small group ofclosely related genes termed mdr, only mdr1 is known to confer drug 10重庆医科大学博士学位论文resistance. Various attempts have made to block mdr1 and its product P-gp170over expression. Theoritically, P-gp170 can be blocked at 3 levels: DNA,RNA and protein. Chemical compounds such as verapamil, a calciumchannel blocker and cyclosporine-A, an immunosuppressive peptide aretwo most studied agents to reverse the drug resistance. However, they arenot effective and specific to P-gp170 expressing tumor cells. Sinceamplification of the mdr1 gene is not pre-requisite to P-gp170-relatedresistance in human tumor cells, the potential loci of inhibition can benarrowed to mRNA or protein. One means to intercept a target mRNA is touse an antisense RNA. Antisense technology offers the potential to block the expression ofspecific genes within cells. It involves the suppression of constitutive geneexpression, using plasmid vectors or virus vector, or the suppression oftransient gene expression using oligonucleotides. Antisense molecules havebeen used predominantly in animal cell culture systems to inhibitoncogenes as well as other harmful genes. In fact, a number of researchershave demonstrated antisense effects on mdr1 gene in cell culture byantisense oligonucleotides. However, the use of antisense oligonucleotidesfaces the problems of degration, since short oligonucleotides are easilydegraded by DNase present in medium or in blood when they are used 11重庆医科大学博士学位论文clinically in future, high concentration may produce toxic effect to the cells.In our study, 200bp mdr1 gene was cloned into an adenoviral vector inantisense orientati...