Study Of Transfection Of Connexin CX43 Bystander Effect Of Glioma Cells Chemotherapy In Vivo Or In Vitro

Objective:To construct pCMV-Cx43cDNA plasmid transfected into C6 cells, increased expression of connexin CX43, enhanced gap junction function. Discussion Transfection of connexin CX43 bystander effect of gliomas chemotherapy in vivo or in vitro.Method:1. Connexin CX43 by constructing eukaryotic expression plasmid pCMV-Cx43cDNA, and transfected into C6 cells, C6 cells overexpressing connexin CX43, RT-PCR and weston-blot detection of CX43 mRNA and protein levels. Scratch loading dye transfer experiments applied to assay of gap junction protein CX43 gap junction after the function.2. TRASWELL cells in vitro co-culture model, transfection of pCMV-Cx43cDNA, empty vector cells were divided into chemotherapy treatment with Temozolomide Temozolomide untreated cells, and then two groups of cells at a certain percentage of cells were seeded TRASWELL across the membrane co-culture plates to detect the end of chemotherapy treatment cell viability and apoptosis rate were observed in vitro transfection of CX43 in vitro bystander effect of chemotherapy.3. To establish in vivo rat glioma model of tumor chemotherapy, the bystander effect observed with chemotherapy:The transfected pCMV-Cx43cDNA, empty vector and cells were divided into normal group, chemotherapy treatment with Temozolomide Temozolomide untreated cells Then the two cells are mixed in certain proportion, inoculated subcutaneously into nude mice, observation time and the composition of tumor size and tumor bystander effect in vivo situation of human glioma chemotherapy.Results:1.the recombinant plasmid pCMV-Cx43cDNA was successfully constructed2.successfully transfected C6 cells stably transfected with selected cell lines overexpressing CX43 (C6-Cx43) and empty vector transfected cell lines (C6-non).C6-Cx43 cells Cx43mRNA and CX43 protein was significantly increased 3.TRASWELL cells were cultured in vitro model, the observed in vitro bystander effect of chemotherapy, (C6-Cx43) and control group there was statistically significant4.there is significant statistical difference in tumor size on time of(C6-Cx43) group and control group (C6-pCMV) tumor-bearing mice in vivo model of chemotherapy. When Chemotherapy drugs in the performance of Temozolomide treatment cells in a certain proportion, (C6-Cx43) Time and composition of tumors significantly inhibited tumor size in the (C6-Cx43) compared with the control group, and the difference was significantly greater than the chemotherapy treatment Cells accounted for 0%(C6-Cx43) and (C6-non) difference in tumor size.Conclusion:1.C6 cells transfected with plasmid pCMV-Cx43cDNA, increased expression of connexin CX43, C6 cells GJIC enhancements.2.C6-Non existence of relatively weak bystander effect, and C6-Cx43 significant bystander effect of chemotherapy in vitro chemotherapy. That transfection of connexin CX43 can magnify the bystander effect of chemotherapy in vitro.3.transfection of gap junction protein CX43 can also enlarge the bystander effect of chemotherapy in vivo chemotherapy.