Experimental Studies On CYP2B1/IFO Suicide Gene Therapy Of Human Glioma Cell

GLIOBLASTOMA is a most common malignant tumor with a very high morbidity and mortality rate worldwide, it is accd the curative effect of surgery resection, radiatetherapy, chemotherapys , conservative medical treatments also improved, but, the prognosis of glioma patient, especially ount for 40% in the encephalic carcinoma^[1-2]. In the recent years, the diagnosis rate of early human glioma has increased an malignant glioma patient is still awfully not better^[3-6]. Thus efficient nonsurgical methods are urgent^[7-10]. Along with research progression of oncomolecularbiology , molecular genetics and the advance study on neurospongioma pathogenesy, as well as development of DNA recombinant , gene transfection technology , neurospongioma gene therapy is become possible^[11-13]. Although these experiments are in the early stages of development but show some promise^{[14-15,16-18][30-31]}, In this study, safe, neotype suicide gene system was used in the therapy of neurospongioma, and get better effect. nontoxic and low plasmid was used for CYP2B1/IFO gene system, the therapeutic effect and its mechanism were studied in glioma models in vitro一, The construction of eukaryotic a vector containing CYP2B1 gene and the detection of its expressions in cell lines derived from human tumorsObjective To construct an expression vector harboring CYP2B1 gene, and detect its expressions in three tumor cell lines. Method PCR amplification was performed using primer based on murine gene sequence from gene bank. pc3/2b1 as template. PCR product was directly inserted effective eukaryotic expressing plasmid pcDNA3.0. an expression vector "containing CYP2B1 gene driven by the CMV promoter, pcDNA3.0/CYP2B1 was constructed by using recombinant DNA techniques. The recombinant was analyzed and identified by restriction enzyme, PCR and sequencing. The expression vector carrying CYP2B1 gene was transfected into three tumor cell lines by liposome-mediated method. The expressions of CYP2B1 gene in all cell lines were detected by RT-PCR method. Result The target fragment was cloned into the corresponding vector. The expressions of CYP2B1 gene in different tumor cell lines were positive. Conclusion The expression vector containing CYP2B1 gene under the control of an CMV promoter, is an novel effective expression vector for tumor gene therapy.二 The constructions of two eukaryotic vectors harboring CYP2B1 gene under the control of two different promoters and the analysis of their different expressionsAIM: To construct two expression vectors carrying CYP2B1 gene under a cytomegalovirus (CMV) promoter and a hybrid α-fetoprotein (AFP) tissue-specific promoter, to investigate its expression in different eukaryotic cells lines.Method: hybrid promoter [HRE] was cloned into pcDNA3.0 vector, and an recombinant vector controlled by the hybrid[HRE] CMV promoter, p[HRE]CMV was constructed. Inserting CYP2B1 gene into pcDNA3.0 and p[HRE]CMV vectors separately, two CYP2B1 gene expression vectors driven by two different ¹promoters, pcDNA3.0/CYP2B1 and p[HRE]CMV/CYP2B1, were constructed by using recombinant DNA techniques. The recombinants were analyzed and identified by restriction enzyme, PCR and -sequencing. pcDNA3.0/CYP2B1 and p[HRE]CMV/CYP2B1 were transfected into human glioma U 251M G cell lines by liposome-mediated method. The expressions of CYP2B1 gene in four different cell lines were detected by RT-PCR method.Result: All target fragments were separately cloned into corresponding vectors. We can detect the expressions of CYP2B1 gene under the control of CMV promoter in all cell lines, and the tissue-specific expression of CYP2B1 gene under the control of [HRE]CMV promoter in AFP positive and negative human glioma U 251M G cell lines were positive. Conclusion: Two expression vectors harboring CYP2B1 gene are novel effective vectors for human human glioma U 251M G cell lines gene therapy, and p[HRE]CMV/CYP2B1 is an target-expressing vector in AFP positive, especially in AFP negative human glioma U 251M G cell lines.三, Experimental studies on glioma gene therapy with CYP2B1/IFO suicide gene systemObjective To investigate the killing effect of CYP2B1/IFO suicide gene system on hepatoma cells. Methods Inserting CYP2B1 gene into pcDNA3.0, an eukaryotic expression vector containing CYP2B1 gene, pcDNA3.0/CYP2B1, was constructed by using recombinant DNA techniques. Then it was transfected into a hepatoma cell line, human glioma U 251M G cell lines, by liposome-mediated method. The human glioma U 251M G cell lines cell line with stable CYP2B1 gene expression, human glioma U 251M G cell lines/CYP2B1, was established with G418 selection. The cell growth curve were determined with trypan blue stain. The sensitivity of human glioma U 251M G cell lines/CYP2B1 to IFO and bystander effects were assayed with MTT and FCM methods. The enzymatic activity of the product of CYP2B1 gene was determined with HPLC method. Results The cytotoxic effects of IFO on human glioma U 251M G cell lines/CYP2B1 cells were obvious (IC₅₀=4-5μM) and all human glioma U 251M G cell lines/CYP2B1 cells were killed after 4d with the dosage of 100μM IFO. As for the bystander effect, in mixed cultures containing increasing percentages of human glioma U 251M G cell lines/CYP2B1 cells, total population killing was demonstrated when human glioma U 251M G cell lines/CYP2B1 cells accounted for as few as 5% of all human glioma U 251M G cell lines cells after 8d with the dosage of 100μM IFO. By high-pressure liquid chromatography, it is positive that the product of CYP2B1 gene could convert IFO into phosphoramide mustard.Conclusion CYP2B1/IFO suicide gene system has an advantage over traditional suicide gene systems in hepatoma gene therapy. The results suggest that the high bystander effect of this system will result in significant anti-tumor responses in glioma gene therapy, especially in vivo.Through above reseach,we can make a below conclusion1. The express system of cyp2b1/IFO gene wase constructed successfully.2. CYP2B1 under controlling p[HRE] CMV prometer has high specificity in human glioma U 251M G cell linesr.3. The finding suggest that this system of CYP2B1/IFO will bring about significant anti-tumor responses in human glioma U 251M G cell linesr gene therapy,.The construction of PcAF/CYP2B1 and Pc[HRE]CMV/cyp2b1 established the ground for the next study of treatment of human glioma U 251M G cell lines in vivo.