Experimental Study On The Efficacy Of βG Gene And βG Prodrug System In The Human Bladder Cancer Cell

Bladder carcinoma is the most common malignancy in the urinary, accounting for 20% of the malignancy of all the systems. The managements remain to be surgery and chemotherapy. High frequent recurrence and bad efficiency in the late stage call for new therapies for it. The suicide gene therapy is one of the most promising gene therapies for the tumor. f3-Glucuronidase is a kind of acid hydrolase in lysosome, involved in the processes of physiology, pathyology and metabolism of the drugs. It can hydrolysis glucuronide glycoside bond, releasing the glucuronic acid and ligand. It was well established that the prodrug of 13G can selectively kill the tumor cells. By combining the j3G, prodrugs and the gene therapy, a new oriented molecular chemotherapy may be developed. The bladder tumor cells with high expression of ~G take the prodrug and will be killed selectively. The aim of this study is to look for a new and effective therapy for the bladder carcinoma. The experiment was done in the following ways: 1 .The retrovirus eukaryotic expression vector with the f3G gene was constructed by the same EcoR I site of the PGEM-4 and pcDNA3.1(+). 2.By the lipofectAMlNE, a retrovirus eukaryotic expression vector pcDNA3.1 (+) containing the whole length sequence of the f3G cDNA was transfected into the human bladder tumour cell line T24. After the G41 8 screening, a clone that get the stable transfection of the BG was obtained. It was confirmed that the ~G gene had been stably integrated into the genomic DNA of the 124 cell and got a highly expression by the means of RNA blotting, in situ hybridization, Immunoflurescence, Jmmunohistochemistry, Western Blot. The stably transfected cell was named T24/ f3G. The biological characteristics of the fG transfected cell were studied by the methods of microscopy and electron microscop. 3.The series studies of the in vitro effects of the prodrug Epi-bus on T24/ f3G showed that the stably transfected cells are more sensitive to the Epi-bus by 1000 fold than the parent, with the IC50 0.1 7g/L. The Epi-bus with a concentration of 2.92g/l sustaining for 4-5 days had little affection on the growth of the transfected cells, but will kill all the cells with high j3G expression. The concentration of 0.292g11 for 6-7 days will kill the transfected cells, showing the obvious uicide effects The T24/G cells were mixed with parental cells not transfected with various proportion and 70%-90% of the mixed cells were killed when the 124/ f3G cell accounting for 1 0%-20%. It showed the potent stand-by effect. The study showed that the 13G gene and its prodrug could be introduced into the suicide gene system and the j3G gene transfection mediated by the LipofectAMlNE- retrovirus eukariotic expression vector combining the 13G prodrug managment will be the promising strategy for bladder therapy.