The Influence Of Cx43cDNA Transfection On Bystander Effect In Glioma Chemotherapy

Objective:To further investigate the bystander effect of glioma chemotherapy and evaluate the relationship between the gap junctional intercellular communication(GJIC) and the bystander effect of glioma chemotherapy. Methods: ①Transformation,amplification,purification and identification of plasmids:the plasmids were transformed into JM109 bacterium through calcium chlorid method,filtrated with Ampenicillin on Luria-Bertani medium,then purified with Wizard PureFection Plasmid DNA Purification System,and identified with endonuclease with EcoRI and HindIII.②Gene transfection: at first,C6 glioma cells were cultured in medium with different doses G418 to find the lowest dose of all C6 cells killed,eg filtrating dose. Then C6 cells were transfected by plasmid DNA through LipofectAMINE2000.The transfected cells were obtained by using the filtrating dose of G418.The Cx43mRNA expression of the transfected C6 cells was detected by RT-PCR,The GJIC functions were investigated with scrape loading dye transfer test (SLDT).The inhibiting growth rate after transfected C6 cells was investigated by MTT method.③Mixed culture to Investigate the bystander effect In chemotherapy:the transfected cells were divided into VM-26-treated and VM-26-untreated groups.The two groups were co-cultured with different ratio mixed.The survival rates of each mixed group were detected with MTT,and apoptotic rate with flow cytometry.④Study the relationship between bystander effect and gap junctions by adding GJIC Inhibitor, 18-α-glycyrrhetinic acid(AGA),to culture medium:The mixed co-culture were repeated after adding AGA to the culture medium for detecting the influence of AGA on bystander effect. Result:①The high-purified,transfectable plasmids were successful obtained. ○2 According to the different transfected plasmids,C6-Cx43 and C6-Non,relevant to different plasmids ,were havested.The level of Cx43mRNA was obviously elevated in C6-Cx43.And the dye transfer ability of C6-Cx43 was stronger than that of C6-Non. ○3 No bystander effect or weak bystander effect could be detected in glioma chemotherapy probably due to the poor GJIC of C6 cells.While bystander effect In vitro was obviously detected due to the amplified cytotoxicity of VM-26 after GJIC of C6-Cx43 was upregulated by Cx43-cDNA transfected.○4the amplification of VM-26 cytotoxicity,bystander effect,on C6-Cx43 was obviously inhibited by AGA. Conclusion:○1 Weak or no bystander effect can be detected in chemotherapy on C6; ○2 Cx43cDNA transfection can elevate the level of Cx-43mRNA in cells,and upregulate GJIC of C6 cells. ○3 The bystander effect of glioma chemotherapy can be obviously detected after the GJIC being upregulated. ○4 The GJIC is one of the substantial fundment of the bystander effect of glioma chemotherapy. ○5 We first reporte the induced bystander effect in C6 cells chemotherapy,and suggest a new pattern of integrated treatment for tumor,that is improving GJIC function of tumor cells is the precondition for subsequent chemotherapy after tumor surgical excision.