人参总皂苷增强小鼠黑色素瘤B16细胞缝隙连接功能的实验研究

Effects of Total Ginsenosides on Gap Junction Function in Melanoma B16 cells

目的探讨人参总皂苷（total ginsenosides，TGs）对小鼠黑色素瘤B16细胞缝隙连接（Gapiunction，GJ）功能的影响及其作用机制。方法MTT法测TGs对B16细胞生长的影响，荧光显微镜结合荧光示踪法分析GJ功能变化，以各试验组受体细胞均数与对照组的比值作为评价GJ功能的指标，流式细胞仪检测对照组和实验组的绿色荧光供体细胞（G4）与双阴性受体细胞（G3）比值（G4／G3）变化分析CJ功能改变，Westernblot分析连接蛋白（Connexin，Cx）表达。结果1～8μmol·L^-1 TGs处理B16细胞48h对其生长状态无明显影响；用1，2，4，8μmol·L^-1 TGs处理细胞48h后，荧光显微镜下观察，TGs能明显提高B16细胞荧光染料Calcein传递，对照组和实验组G4／G3比值分别是0．06±0．01，0．09±0．02，0．10±0．01，0．12±0．03和0．13±0．02，各实验组G4／G3值明显高于对照组（P〈0．01）；Westernblot分析结果显示，用1，2，4，8μmol·L^-1 TGs处理B16细胞48h对其连接蛋白Cx32表达有明显的增强作用，但对Cx43和Cx26表达无影响。结论体外较低浓度TGs能够有效促进B16细胞GJ功能，并具有一定的剂量一效应关系。1～8μmol·L^-1 TGs虽能显著促进B16细胞Cx32蛋白的表达，但在本试验浓度范围内并无明显的量效关系，且对Cx43和Cx26表达无明显影响。

Objective To investigate the effects of total ginsenosides （TGs） on the gap junction （GJ） function in melanoma B16 ceils, and to explore the possible mechanism. Methods Effects of TGs on the proliferation of B16 cells were determined by MTT. The gap junction function was analyzed by fluorescent tracer method under fluorescent microscope. The ratio of average count of the receptor cells in each test group to that in the control group was used as an evaluation index of GJ function, and flow eytometry was used to detect the ratio of G4 to G3 in Calcein labeled cells （G4） group and double negative cells （G3） group for the analysis of the changes of the gap junction function. The expression of connexins（Cx） was detected by Western blotting method. Results TGs at 1-8 μmol·L^-1 had no effect on the growth of B16 cells after co-culture for 48 h. After co-cuhure with 0, 1, 2, 4 and 8 μmol·L^-1 of TGs for 48 h, the transfer of the Calcein in B16 cells was obviously enhanced under fluorescence microscope, and the G4/G3 ratio was 0.06＋0.01 in the control group, and was 0.09±0.02, 0.10±0.01, 0.12±0.03 and 0.13 ±0.02 in 1, 2, 4 and 8μmol·L^-1 TGs experimental groups, respectively, the G4/G3 ratio in the experimental groups was significantly higher than that in the control group（P 〈 0.01 ）. The results of Western blotting showed that the expression of Cx32 in B16 cells was obviously increased after co-culture within 0, 1, 2, 4 and 8μmol·L^-1 of TGs for 48 h, but TGs at the above concentrations had no effect on the expression of Cx43 and Cx26. Conclusion Low concentrations of TGs can effectively promote the gap junction function of B16 cells in a dose-dependent way in vitro. TGs at 1-8μmol·L^-1 can significantly improve Cx32 protein expression in B16 cells, but show no obvious concentration-response relationship, and has no obvious influence on the expression of Cx43 and Cx26 Keywordls： Total ginsenosides; Fluorescent tracer; B16 cells; Gap junction; connexin; Flow cytometry