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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(Gap junction,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GJ功能改变,Western blot分析连接蛋白(Connexin,Cx)表达。结果 1~8μmol·L-1TGs处理B16细胞48 h对其生长状态无明显影响;用1,2,4,8μmol·L-1TGs处理细胞48 h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);Western blot分析结果显示,用1,2,4,8μmol·L-1TGs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-效应关系。1~8μmol·L-1TGs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 effect of total ginsenosides (TGs) on the function of Gap junction (GJ) in mouse melanoma B16 cells and its mechanism. Methods MTT assay was used to measure the effect of TGs on the growth of B16 cells. Fluorescent microscopy was used to analyze the function of GJ. Fluorescent microscopy was used to evaluate the function of GJ. The ratio of cells in each experimental group to control group was taken as the index to evaluate GJ function. Flow cytometry The changes of GJ function in G4 / G3 / G3 / G3 / G3 / G3 / G3 / G3 / G3 / G3 / G3 / G3 / G3 / G3 / G3 groups were analyzed in control and experimental groups. Connexin (Cx) Results After treated with 1 ~ 8μmol·L-1 TGs for 48 h, the growth of B16 cells was not affected obviously. The cells treated with 1, 2, 4, 8μmol·L-1 TGs for 48 h were observed under fluorescence microscope. G4 / G3 ratio of control group and experimental group were 0.06 ± 0.01, 0.09 ± 0.02, 0.10 ± 0.01, 0.12 ± 0.03 and 0.13 ± 0.02, respectively. The G4 / G3 ratio of the experimental group was significantly higher than that of the control group <0.01). The results of Western blot analysis showed that B16 cells treated with 1, 2, 4, 8μmol·L-1TGs for 48h significantly enhanced the expression of connexin Cx32, but had no effect on the expression of Cx43 and Cx26. Conclusion The lower concentration of TGs in vitro can effectively promote GJ function of B16 cells and has a dose-response relationship. Although 1 ~ 8μmol·L-1 TGs could significantly promote the expression of Cx32 protein in B16 cells, there was no significant dose-effect relationship in the concentration range of this experiment, and had no significant effect on the expression of Cx43 and Cx26.