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目的 研究连接蛋白 (Cx)基因对C6胶质瘤细胞的增殖抑制及细胞间隙连接通讯(GJIC)的作用 ,探讨以Cx43基因治疗胶质瘤的可行性。方法 将含Cx43cDNA的质粒以Lipofec tamine介导转染Cx43表达缺失的C6胶质瘤细胞 ,通过Northern印迹杂交、原位杂交及免疫组织化学染色检测Cx43mRNA及蛋白表达 ,划痕标记荧光染料示踪技术 (SLDT)检测GJIC ,MTT法测定细胞增殖率 ,核仁组成区嗜银蛋白 (AgNOR)染色检测细胞增殖活性 ,TUNEL法检测细胞凋亡。结果 转染后C6细胞有不同程度的Cx43mRNA及蛋白表达和GJIC恢复。表达Cx43的克隆细胞增殖明显下降 ,培养 2～ 6d时 ,每天除C6组与空载组差异无显著性外 ,其余各组差异均有非常显著性 (P <0 .0 1) ,但细胞凋亡并未增加。结论 Cx43基因及GJIC在恶性胶质瘤的发生发展过程中起重要作用 ,可能成为恶性胶质瘤基因治疗的优选靶的之一。
Objective To investigate the effect of connexin (Cx) gene on the proliferation inhibition and cell gap junctional intercellular communication (GJIC) in C6 glioma cells and to explore the feasibility of using Cx43 gene to treat glioma. Methods Cx43 cDNA was transfected into C6 glioma cells with Cx43 expression by Lipofectamine. Cx43 mRNA and protein expression was detected by Northern blotting, in situ hybridization and immunohistochemical staining. Scratch-labeling fluorescent dye tracer technique (SLDT). Cell proliferation rate was determined by MTT assay. Proliferation of nucleolar organizer region was detected by AgNOR staining. Cell apoptosis was detected by TUNEL assay. Results C6 cells had different levels of Cx43 mRNA and protein expression and GJIC recovery after transfection. The proliferation of Cx43-expressing clones was significantly decreased. After cultured for 2 ~ 6 days, there was no significant difference between the C6 group and the control group (P <0.01) Death has not increased. Conclusion Cx43 gene and GJIC play an important role in the development of malignant glioma and may be one of the preferred targets for gene therapy of malignant glioma.