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目的 评价单纯疱疹胸苷激酶 更昔洛韦 (HSV TK GCV)系统杀伤前列腺癌细胞PC 3m的“旁观者效应” ,探讨连接蛋白 (Cx)介导的细胞间隙连接交流 (GJIC)在该系统“旁观者效应”中的作用 ,观察apigenin对PC 3m细胞连接蛋白 43(Cx43)表达及GJIC状况的调控。方法 MTT法评价TK基因系统杀伤PC 3m细胞的“旁观者效应” ,划痕标记染料示踪技术 (SLDT)比较几种代表细胞系GJIC功能状况 ,检测HSV TK GCV对其“旁观者效应”。逆转录 -聚合酶链式反应 (RT PCR)法和SLDT检测PC 3m细胞Cx43表达和GJIC状况 ,并观察apigenin对Cx43表达、GJIC状况及“旁观者效应”的调控作用。结果 HSV TK GCV系统杀伤PC 3m细胞时“旁观者效应”程度较弱 ,TK基因阳性细胞比例约为1 0 %的混合细胞经 1 0 0 μmol LGCV处理 72h后 ,仅 2 3 .5 %± 3 .2 1 %细胞被杀灭。该自杀基因系统对GJIC功能强大的NIH 3T3、Cos 7和L 0 2细胞“旁观者效应”程度远较GJIC功能低下的ACHN和HeLa细胞者强 (P <0 .0 0 1 )。PC 3m细胞Cx43mRNA表达降低 ,GJIC功能低下 ,apigenin不仅可上调PC 3m细胞Cx43表达、促进GJIC功能 ,还可使其“旁观者效应”明显改观 (P <0 .0 0 1 )。结论 细胞内在的GJIC功能与HSV TK GCV对其“旁观者效应”程度有正向关系 ,PC 3m细胞Cx43
Objective To evaluate the “bystander effect” of HSV TK GCV system on prostate cancer cell line PC 3m and to explore the role of connexin (Cx) -mediated gap junctional intercellular communication (GJIC) Bystander effect “, we observed the effect of apigenin on the expression of Cx43 and the regulation of GJIC in PC 3m cells. Methods MTT method was used to evaluate ”bystander effect“ of TK gene system killing PC 3m cells. Scratch-mark dye tracer technique (SLDT) was used to compare the functional status of GJIC on several representative cell lines and to detect ”bystander effect“ of HSV TK GCV. The expression of Cx43 and GJIC in PC 3m cells were detected by reverse transcriptase polymerase chain reaction (RT PCR) and SLDT. The effects of apigenin on Cx43 expression, GJIC status and bystander effect were also observed. Results The bystander effect of HSV TK GCV system was weaker when PC 3m cells were killed. The mixed cells with TK gene positive cells of about 10% were treated with LGCV of 100 μmol for 72 h and only 23.5% ± 3 .2 1% of the cells are killed. The suicide gene system is far more ”bystander effect“ to GJIC-potent NIH 3T3, Cos 7 and L 0 2 cells than GJIC-deficient ACHN and HeLa cells (P <0.001). PC3m cells Cx43mRNA expression decreased, GJIC dysfunction, apigenin PC3m cells can not only up-regulate Cx43 expression, promote GJIC function, but also to make ”bystander effect“ significantly improved (P <0.001). Conclusion The intrinsic function of GJIC in cells is positively correlated with the degree of ”bystander effect" of HSV TK GCV. The expression of Cx43