二氢杨梅素联合依托泊苷对绒毛膜癌细胞JAR的抑制作用

Inhibitory effect of dihydromyricetin combined with etoposide on the choriocarcinoma cell JAR

目的探讨二氢杨梅素(DMY)联合依托泊苷(VP16)对绒毛膜癌细胞JAR的抑制作用及其相关机制。方法体外培养JAR细胞,设空白对照组、DMY组、VP16组以及DMY与VP16联合(DMY+VP16)组。MTT法检测各组细胞增殖情况;Annexin V-FITC/PI双染流式细胞术分析各组细胞的凋亡率;克隆形成实验检测细胞生存能力;Western blotting法检测凋亡抑制蛋白Bcl-2、细胞凋亡抑制蛋白2(c-IAP2)表达水平。结果 MTT检测结果显示,与空白对照组比较,DMY组、VP16组及DMY+VP16组在24 h和48 h时间点的细胞存活率均下降,且DMY+VP16组细胞存活率下降最为明显;Annexin V-FITC/PI双染流式细胞术结果显示,与对照组相比各实验组凋亡率均有增高,DMY+VP16组凋亡率明显高于各单独用药组;克隆形成实验结果显示,对于JAR细胞,实验组的克隆球数目均少于对照组,且DMY+VP16组克隆球数目最少;对于正常肝脏HL7702细胞,DMY组与对照组相比无明显差异,VP16组与DMY+VP16相比亦无明显差异;Western blotting实验结果显示,与对照组相比,无论DMY和VP16单独还是联合使用均可下调Bcl-2、c-IAP2蛋白表达,同时DMY+VP16组下调作用最明显。结论二氢杨梅素联合依托泊苷明显增强对绒毛膜癌细胞JAR的生长抑制作用,联合使用可以减少化疗药物VP16的用量,其抑制肿瘤细胞作用可能与下调Bcl-2、c-IAP2蛋白的表达有关。

Objective To investigate the inhibitory effect and related mechanism of dihydromyricetin （DMY） combined with etoposide（VP16） on the choriocarcinoma cell JAR. Methods JAR cells cultured in vitro were devided into control group, DMY group, VP16 group and DMY ＋ VP16 group. The cell proliferation was measured by MTT assay. The apoptosis rate of each group was analyzed by annexin V-FITC/PI double dye flow cytometry. The cell viability was measured by the colony formation capability assay. The protein expression levels of Bcl-2, inhibitor apoptosis protein 2 （c-IAP2） were detected by Western blotting analysis. Results The result of MTT assay showed that compared with the control group, the cell survival ratio of DMY group, VP16 group and DMY ＋ VP16 group reduced at 24 hours and 48 hours. The result of double dye flow cytometry showed that the apoptosis rate of the experimental groups were higher than that of the control group, and the apoptosis rate of DMY ＋ VPI6 group was significantly higher than those treated with DMY or VP16 alone. The result of the colony formation capability assay showed that, for JAR cells, the number of clones in experimental groups were less than that in the control group, and the number of clones in DMY ＋ VP16 group was the least. For normal liver HL7702 cells, there was no significant difference between DMY group and the control group, and there was no significant difference between VP16 group and DMY ＋ VP16. The result of Western blotting showed that the protein expression levels of Bcl-2 and c-IAP2 in experimental group decreased compared with those in the control group, and the decrease of the protein expression levels of Bcl-2 and c-IAP2 in DMY ＋ VP16 group were the most significant. Conclusion Dihydromyricetin combined with etoposide enhanced the inhibitory effect on choriocarcinoma cell JAR, which can reduce the dosage of the chemotherapy drug VP16. The inhibitory effect of tumor cells may be related to the down-regulation of protein expression of Bcl-2, C-IAP2.