In Vitro And In Vivo Inhibitory Effect On Growth Of Human Gliomas With P21～(WAF-1/CIP1) Gene Mediated By Targeted Non-viral Vector GE7 System

OBJECTIVES: To construct the novel targeted non-viral vector of epidermal growth factor receptor ligand GE7 system and to investigate in vitro and in vivo effects on growth of human gliomas with the cell cycle regulator p21MF~1/CIP1 gene mediated by GE7 system.METHODS: The epidermal growth factor receptor mediated EGF-R targeting non-viral vector EGF-R ligand GE7 system was constructed. The EGF-R expression of human glioma specimen and human glioma cell line U251MG, U87MG and rat glioma cell line C6 were examined by histochemical examination. Choosed the cells with higher EGF-R expression as the tageted cells for transfecting in vitro. The tageted cells were transfected in vitro with 3 -galactosidase gene as reporter gene and p21WF"1/CIP1 gene as therapeutic gene using this gene delivery system. By means of X-gal staining, MTS, apoptosis DNA ladder, FACS examination, the transferring rate of exogenous gene and the apoptosis of the tumor cells were examined. The EGF-R expression of transfeced U251 cells were examined by histochemical examination and RT-PCR. The glioma animal models were established by implanting U251 cells subcutaneously in nude mouse. The p2lWF~1/CIP1 expression of tumor tissues were examined by histochemical examination. The U251 glioma-bearing animals were injected with GE7/DNA/HA20 complexes subcutaneously and intravenously. Observed the average volume of each group and the tumor inhibitory rate in different groups. Two weeks later animals were killed for histopathological analysis, DNA ladder, WAF-1 histochemical analysis.RESULTS: The EGF-R expression rate of human glioma tissues and adjacent tissues was 70. 3% and 32. 4%. The EGF-R expression rate of human glioma was positively correlated with tumor grade. U251MG was choosed as the tagetedcell for transfecting in vitro with the higher EGF-R expression rate than U87MG and C6. The transferring of exogenous ?-gal gene were weak at 24 hours and reached top value of 70% at 72 hours , disappeared after 168 hours. After transfected in vitro with p21MH/clP1 gene ,the growth of U251 cells were inhibited evidently, and the apoptosis DNA ladder was observed. FACS examination showed Gl period inhibition and the number of cells in G2 period declined remarkably. The histochemical examination and RT-PCR showed the p2jtAF-i/cipi expression Of U251 cells increased remarkably after transfeced with GE7/p21/HA20. After implanted subcutaneously in nude mouse, the EGF-R expression rate of U251 cells remained high. The U251 glioma cells-bearing animals were injected with GE7/DNA/HA20 complexes subcutaneously and intravenously. The two ways have the same therapeutic effects on human gliomas. The tumor growth inhibitory rate of subcutaneous injection was 56%, and the other one was 60%. The WAF-1 expression and DNA ladder were observed two weeks after subcutaneous injection.CONCLUSION: The EGF-R mediated EGF-R targeting non-viral vector GE7 system has the ability to transfer exogenous gene to tumor cells in vitro and in vivo efficiently, and the expression of the p21WAF~1/CIP1 gene can induce the apoptosis of the glioma cell and inhibit its growth. The subcutaneous and introvenous injection of the GE7 system have the same therapeutic effects on human gliomas. The therapeutic effect of the GE7 system is targeting on the EGF-R expression of the glioma cells. This study will be useful in the establishment of the in situ animal model of human gliomas and provide a more effective way to improve the efficiency of human glioma gene therapy.