小柴胡汤对C6胶质瘤抑瘤作用的实验研究

Experimental study of effect of Xiao Chai Hu Tang on C6 glioma

目的：探讨不同剂量小柴胡汤对体外培养的C6胶质瘤细胞的抑瘤作用及其作用机理。方法：将C6胶质瘤细胞分别接种于96孔、24孔及6孔培养板中,每种培养板均分为4组：对照组、小柴胡汤低剂量组、中剂量组和高剂量组,待细胞铺满培养板底80%~90%,开始加药,培养24小时后终止培养。采用CCK-8、活体染色、ELISA、免疫细胞化学及流式细胞仪检测细胞增殖活性、细胞存活率、ESM-1和VEGF的蛋白含量及蛋白阳性表达强度和细胞的早期、晚期凋亡状况。结果：CCK-8法检测显示小柴胡汤低、中、高剂量组对C6胶质瘤细胞的生长均有抑制作用;ELISA和免疫细胞化学法显示,小柴胡汤低、中、高剂量组均能降低ESM-1和VEGF的蛋白阳性表达强度及蛋白含量水平;流式细胞仪检测显示小柴胡汤低、中、高剂量组均能促进细胞凋亡;倒置显微镜观察可见随剂量增加细胞排列逐渐松散、萎缩,细胞漂浮死亡数量增多;各种检测法均显示随剂量增加抑制作用增强且具有剂量依赖性,组间差异均有统计学意义（P〈0.01）。结论：小柴胡汤对C6胶质瘤细胞的生长具有明显的抑制作用,它可能通过下调ESM-1和VEGF蛋白水平、促进细胞凋亡,从而发挥抑瘤作用。

Objective： To investigate the inhibitory effect and mechanism of different doses of Xiao Chai Hu Tang on C6 glioma cells cultured in vitro. Methods： C6 glioma cells were inoculated in 96 holes,24 holes and 6 holes,each culture plate was divided into4 groups： control group,low dose group,middle dose group and high dose group,when the cells covered the bottom of culture plate80%- 90%,began adding,cultured for 24 hours after the ter mination of training. Cell proliferation activity,cell viability,protein content and protein positive expression intensity of VEGF and ESM-1,cell apoptosis in early and late stage were detected by CCK-8,in vivo staining,ELISA,immunocytochemistry and flow cytometry. Results： CCK-8 assay showed that the growth of C6 glioma cells was inhibited by low,medium and high dose group; ELISA and immunocytochemistry showed that the expression of VEGF and ESM-1 was lower in the lower,middle and high dose groups,and the levels of protein expression and protein levels were decreased. The flow cytometry showed that the low dose of small radix,middle and high dose group could promote the cell apoptosis. Inverted microscope observation showed that with the increase of dose,the number of cells increased gradually,and the number of dead cells increased,and all kinds of detection methods showed that the inhibition effect increased with dose and dose dependence. The difference between the two groups was statistically significant（ P〈0. 01）. Conclusion： The growth of C6 glioma cells was significantly inhibited by Xiao Chai Hu Tang. It may play a role in inhibiting tumor growth by down regulating ESM-1 and VEGF protein level and promoting cell apoptosis.