益气清毒消瘤方诱导肠癌细胞凋亡的机制研究

Study on the mechanism of Yiqi Qingdu Xiaoliu Recipe inducing intestinal cancer cell apoptosis

目的初步分析熊墨年教授益气清毒消瘤方诱导大肠癌细胞凋亡的机制。方法采用清洁级雄性昆明小鼠40只,按随机数字表法分为高、中、低剂量组和对照组,每组各10只。对照组给予0.2 mL的生理盐水灌胃,其余三组给予不同浓度的益气清毒消瘤方汤药灌胃,四组小鼠均给药3 d,2次/d,末次给药后1 h下腔动脉取血,制备含药血清。采用等体积不同浓度的小鼠含药血清处理人大肠癌Wide细胞系,每组设4个复孔,培养12、24、48 h后测定各组细胞OD值,计算抑制率;观察各组细胞形态与凋亡情况,检测各组Bcl-2、Bax、Fas的蛋白表达。结果各组细胞培养12、24、48 h后的细胞抑制率呈现时间和剂量依赖关系;高剂量组坏死细胞比例高于对照组、低剂量组和中剂量组,差异有统计学意义(P<0.05);24 h后细胞凋亡率呈现明显的剂量依赖关系(P<0.05);药血清干预组细胞凋亡调控基因Bax、Fas表达高于对照组,Bcl-2表达低于对照组,差异有统计学意义(P<0.05);随着剂量的升高Bax、Fas表达升高,Bcl-2表达下降,差异有统计学意义(P<0.05)。结论益气清毒消瘤方能够提高Bax、Fas表达,抑制Bcl-2表达抑制大肠癌细胞的生长和增殖,有效促进细胞凋亡。

Objective To analyze the mechanism of Professor XIONG Mo-nian′s Yiqi Qingdu Xiaoliu Recipe in inducing apoptosis mechanism of intestinal cancer cells.Methods A total of 40 clean-grade male Kunming mice were used.They were divided into high-dose,medium-dose,and low-dose groups and the control group according to the random number table method,with 10 rats in each group.The control group was given 0.2 mL of normal saline by gavage,and the other three groups were given different concentrations of Yiqi Qingdu Xiaoliu decoction.Each group was intragastric administration twice every day for 3 days.One hour after the last administration,blood was collected from the inferior vena cava to prepare drug-containing serum.Using equal volumes of different concentrations of mouse drug-containing serum to treat human colorectal cancer Wide cell line,each group has 4 multiple wells.After culturing for 12,24,and 48 hours,the OD value of each group of cells was measured,and the inhibition rate was calculated;the morphology and apoptosis of each group were observed,and the protein expression of Bcl-2,Bax,and Fas of each group was detected.Results The cell inhibition rate of each group was presented as a time-and dose-dependent relationship after 12,24,and 48 hours of cell culture.The proportion of necrotic cells in the high-dose group was higher than those in the control group,the low-dose group and the middle-dose group,the differences were statistically significant(P<0.05).After 24 hours,the apoptosis rate showed a significant dose-dependent relationship(P<0.05).The expression of apoptosis regulating genes Bax and Fas in the drug serum intervention group was higher than that of the control group,and the expression of Bcl-2 was lower than that of the control group,the differences were statistically significant(P<0.05);and with the increase of the dose,the expression of Bax and Fas increased,and the expression of Bcl-2 decreased significantly,the differences were statistically significant(P<0.05).Conclusion Yiqi Qingdu Xiaoliu Recipe can increase the expression of Bax and Fas,inhibit the expression of Bcl-2,inhibit the growth and proliferation of intestinal cancer cells,and effectively promote cell apoptosis.