小半夏加茯苓方含药血清抑制HepG2细胞增殖及促进凋亡

目的:采用血清药理学的方法,观察小半夏茯苓方对肝癌HepG2细胞增殖的影响,初步探索该方诱导肝癌细胞凋亡相关因子表达的影响。方法:制备含药血清,正常实验用动物Wistar大鼠,采用灌胃或腹腔注射给药途径,连续给药3 d,其中,正常对照组,灌服生理盐水;环磷酰胺(CTX)组,腹腔注射CTX;原方汤剂组,灌服小半夏加茯苓汤;原方汤剂加CTX组,灌服小半夏加茯苓汤的同时腹腔注射CTX;颗粒剂高中剂量组,灌服相当于小半夏加茯苓汤等量、二倍量的颗粒剂,末次给药后12 h,腹主静脉取血,制备含药血清,刺激体外培养的HepG2肝癌细胞,采用MTT法检测其对细胞增殖抑制的影响,流式细胞术检测其对各时相细胞周期分布,免疫组织化学的方法观察其对凋亡相关蛋白半胱天冬酶-3(caspase-3)、半胱天冬酶-9(caspase-9)的影响。结果:小半夏加茯苓方能抑制人肝癌HepG2细胞的生长,在作用24 h时效果最明显。流式检测结果显示,药物作用细胞24 h,小半夏加茯苓方阻滞细胞于S期,环磷酰胺将细胞阻滞于G2期。小半夏加茯苓方作用24 h能诱导HepG2细胞的凋亡,caspase-3表达与药物的剂量呈正相关。结论:小半夏加茯苓方可能通过激活caspase-3蛋白表达,从而诱导肝癌细胞凋亡,阻滞细胞周期于S期,有效抑制细胞增殖,发挥抗肿瘤的作用。

Serum Containing Xiaobanxia Plus Fuling Formulas Inhibits Proliferation of Human Hepatocellular Carcinoma HepG2 Cell and Induces its Apoptosis

Objective: To investigate the inhibitory effects of the serum containing Xiaobanxia plus Fuling formulas on the proliferation of human hepatoma carcinoma HepG2 cell and on the expression of apoptosis associated factors in HepG2. Method: The effects of Xiaobanxia plus Fuling formulas on the proliferation of HepG2 were determined by MTT assay. The cell cyclic retardation was examined by using flowcytometry. Immunohistochemistrywas used to detect the expression of caspase-3 and caspase-9 in the level of protein. Result: MTT showed that Xiaobanxia plus Fuling formulas had an inhibitory effect on the proliferation of HepG2 cells, and the effect was best at 24 h than that at other time. The cell cycle analysis showed that Xiaobanxia plus Fuling formulas increased the percentage of S phase cells, and cyclophosphamide(CTX) reduced the percentage of G₂ phase cells in 24 hour. The results of the immunohistochemistry demonstrated the expression of caspase-3 was increased with dose-time dependent. Conclusion: The serum containing Xiaobanxia plus Fuling decoction has a significant inhibitory effect on the proliferation of HepG2.The mechanism of inducing cell apoptosis may be related with increasing in the level of caspase-3.