肝癌全方及其不同治法拆方抑制SMMC7721人肝癌细胞增殖的作用  

Inhibitory Effect of Hepatocellular Carcinoma Prescription and Its Different Disassembled Prescriptions on Proliferation of SMMC7721 Hepatoma Cells

作　　者：王晓敏[1] 潘志强[1] 梁龙龙[1] 方肇勤[1] WANG Xiao-min;PAN Zhi-qiang;LIANG Long-long;FANG Zhao-qin(Basic Medical School, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China)

机构地区：[1]上海中医药大学基础医学院

出　　处：《中国实验方剂学杂志》2018年第13期117-123,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(81473562)

摘　　要：目的：探讨肝癌全方及不同治法拆方抗肿瘤作用的分子机制。方法：体外培养SMMC7721人肝癌细胞,分别给予2.5-100 g·L-1肝癌全方、清热方、活血方、健脾方干预24 h后,采用噻唑蓝（MTT）比色法检测细胞增殖;分别采用1.25-20 g·L-1肝癌全方,5-20 g·L-1清热方和活血方及20-120 g·L-1健脾方干预24 h后,运用实时荧光定量聚合酶链式反应（Real-time PCR）检测相关基因的表达;分别采用20 g·L-1肝癌全方和清热方,10 g·L-1活血方及120 g·L-1健脾方干预SMMC7721细胞24 h后,通过蛋白免疫印迹法检测相关蛋白的表达,通过碘化丙啶（PI）/RNase staining solution试剂染色检测细胞周期的改变。结果：与空白组比较,肝癌全方及其不同治法拆方能抑制SMMC7721细胞的增殖,并呈现量效关系（P〈0.05）。与空白组比较,5-20 g·L-1清热方呈现量效抑制细胞周期依赖性激酶阻滞基因1B（CDKN1B）基因表达（P〈0.05）,20-120 g·L-1健脾方也呈现量效抑制CDKN1B基因表达（P〈0.05）;5-20 g·L-1肝癌全方明显抑制糖皮质激素调节蛋白激酶1（SGK1）基因表达（P〈0.05）,且量效显著;5-20 g·L-1清热方量效抑制SGK1基因表达（P〈0.05）;20-120 g·L-1健脾方也抑制SGK1基因表达（P〈0.05）,同时肝癌全方及清热、活血治法方可抑制SGK1磷酸化蛋白的表达（P〈0.05）;1.25,20 g·L-1肝癌全方可促进叉头框蛋白O3（FOXO3）基因表达（P〈0.05）;15,20 g·L-1清热方明显促进FOXO3基因表达（P〈0.05）;20 g·L-1活血方促进FOXO3基因表达（P〈0.05）。与空白组比较,全方组G2期细胞数增加（P〈0.05）,清热组和健脾组G2期细胞数增加更明显（P〈0.05）;肝癌全方及不同治法拆方能够抑制细胞周期蛋白E1（Cyclin E1）蛋白的表达,其中以健脾组抑制效果最显著。结论：肝癌全方及其不同治法拆方通过改变细胞增殖和周期,从而发挥抑制肝癌的作用。Objective： To study the molecular mechanism on anti-tumor effect of hepatocellular carcinoma prescription（ Quanfang） and its different disassembled prescriptions. Method： SMMC7721 human hepatoma cells were cultured in vitro and treated with 2. 5-100 g·L-1 Quanfang,Qingrefang,Huoxuefang and Jianpifang for 24 h. Then cells proliferation was measured by methylthiazolyldiphenyl-tetrazolium bromide（ MTT）assay. After SMMC7721 cells were treated with 1. 25-20 g·L-1 Quanfang,5-20 g·L-1 Qingrefang and Huoxuefang and 20-120 g·L-1 Jianpifang for 24 h,related gene expression levels were detected by Real-time fluorescence quantitative polymerase chain reaction（ Real-time PCR）. After SMMC7721 cells were treated with 20 g·L-1 Quanfang and Qingrefang,10 g·L-1 Huoxuefang and 120 g·L-1 Jianpifang for 24 h,related protein expression levels were detected by Western blot and cell cycle changes were detected by PI/RNase staining solution reagent staining. Result： As compared with blank control group, Quanfang and different disassembled prescriptions inhibited the proliferation of SMMC7721 cells and showed dose-effect relationship（ P〈 0. 05）. As compared with the blank control group,CDKN1 B gene expression was inhibited significantly by 5-20 g·L-1 Qingrefang and 20-120 g·L-1 Jianpifang（ P〈 0. 05） in a dose-effect manner. SGK1 gene expression was inhibited significantly by 5-20 g·L-1 Quanfang and Qingrefang（ P〈 0. 05）; Quanfang and different disassembled prescriptions can inhibited SGK1 phosphorylated protein expression. SGK1 gene expression was inhibited by 5-20 g·L-1 Qingre disassembled prescriptions and 20-120 g·L-1 Jianpi disassembled prescriptions（ P〈 0. 05）. FOXO3 gene expression was promoted significantly respectively by 1. 25,20 g·L-1 Quanfang,15,20 g·L-1 Qingre disassembled prescriptions and 20 g·L-1 Huoxue disassembled prescriptions（ P〈 0. 05）. As compared with blank control group,the number of G2/M cells was increased in Quanfang group（ P〈 0. 05） and w

关 键 词：肝癌 SMMC7721细胞 治法 细胞周期 细胞增殖 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学]