The Effects Of Polyethyleneimine-mediated Bmk CT Gene On C6 Glioma Cells In Vitro And In Vivo

Objective:A neurotoxin protein, Bmk CT,can specifically combine with glioma cells but not with normal cell binding. The binding sites on glioma cell are mmp-9 and GCC respectively.The previous experiments show that Bmk CT protein can significantly inhibit the growth of subcutaneous glioma in rats by intraperitoneal injection. In order to reduce the immunogenicity and costs, prevent protein degradation, we built the recombinant virus Ad-Bmk CT. We injected Ad-Bmk CT into the tumor bearing mice, which can inhibit the growth and metastasis of glioma. For the safety problem of injection of virus vector in vivo, the recombinant plasmid, p EGFP-N1-Bmk CT, was constructed. We hope to achieve the therapeutic effect through non viral vector transfection.Methods:2 plasmids, pEGFP-N1 and p EGFP-N1-BmK CT were combined with PEI to form the composites,PEI/pEGFP-N1-Bmk CT and PEI/pEGFP-N1, which were used to transfect C6 glioma cells. Then, MTS test was performed to investigate the proliferation of cells. At 48 hours after transfection, the expression of PCNA, cyclinD1, c-myc, VEGF, MMP-9, AQP-1, p-ERK, p-AKT, p-P65 proteins were detected by Western-bloting. We establishmented in situ glioma rat model, and examined the rate of tumor formation by MRI,and then randomly divided experimental animals into 3 groups, control group, pEGFP-N1-Bmk CT gene therapy group, pEGFP-N1 empty vector group. Thirteenth days after modeling, normal saline, PEI/pEGFP-N1-Bmk CT and PEI/pEGFP-N1 was injected into tumor.After 48 hours, GFP protein was detected by immuno histochemistry, p-ERK, PCNA, cyclinD1, VEGF protein was detected by immunofluorescence.Results:At 48 hours after transfection,the proliferation of C6 glioma cells in the pEGFP-N1-Bmk CT group was significantly inhibited, while the expression levels of PCNA,cyclinD1,c-myc,VEGF,MMP-9,AQP-1,p-ERK,p-AKT,p-P65 were lower than pEGFP-N1 group(P <0.05). Immunohistochemistry showed that GFP protein was expressed in the cytoplasm of gliomas. Imunofluorescence showed that p-ERK, PCNA, cyclinD1, VEGF in the pEGFP-N1-Bmk CT group were lower than pEGFP-N1 group(P <0.05).Conclusion: The pEGFP-N1-Bmk CT recombinant plasmid was transfected in vivo and in vitro by PEI can obtain a considerable transfection rate, and inhibit tumor cell proliferation, depress tumor edema related protein expression. This may be related to the inhibition of excessive activation of ERK, AKT,NF-κB pathway.