预知子籽提取物抑制HepG2肝癌细胞增殖作用及对SEPP1等分子表达的影响

Effect of Akebia trifoliate Koidz Seed Extract on the proliferation of HepG2 Hepatocellular Carcinoma Cells and protein expression of SEPP1

目的:观察中药预知子籽乙醇提取物(ATKS)对HepG2肝癌细胞增殖及对SEPP1等分子表达的影响。方法:将不同浓度ATKS作用于HepG2细胞24h、48h、72h,MTT法检测HepG2细胞的细胞存活率;相差倒置显微镜下观察药物作用72h后的细胞形态;RT-qPCR方法检测SEPP1等6个基因表达量的变化;Western Blot方法检测SEPP1蛋白水平变化。结果:ATKS作用后,HepG2细胞增殖被抑制,并存在量效和时效关系,其中72h细胞半数抑制浓度为2.0mg/ml;较低浓度对细胞形态影响较小,随着浓度增加细胞数量逐渐减少,2.4 mg/ml以上细胞数量锐减,状态变差,药物细胞毒作用凸显;2.0 mg/ml作用72h后,SEPP1基因表达被显著下调,其上游基因CAPNS1、ITGA2、ITGAV、ITGB1、ITGB5却一致上调;SEPP1蛋白表达量也出现显著下降。结论:ATKS能抑制HepG2肝癌细胞增殖,并具有量效和时效关系;SEPP1等基因和蛋白表达量发生变化,可能与ATKS的抑制作用有关。

Objective： To observe the Effect of Akebia trifoliate（ Thunb.） Koidz Seed Extract（ ATKS） on the proliferation of HepG2 Hepatocellular Carcinoma Cells and protein expression of SEPP1 and other 5 genes. Methods： ATKS was extracted with alcohol. The inhibiting effect was observed by MTT method post 24,48 and 72 hours treatment with different concentration ATKS; the morphology of HepG2 cells was observed under inverted phase contrast microscope post 72 hours treatment; the mRNA expression was measured with RTq PCR method;the protein expression was measured with Western-Blot method. Results： ATKS could significantly inhibit the malignant proliferation of HepG2 cells. This inhibiting effect existed time-effect and dose-effect relationship. The Half inhibitory concentration is 2. 0 mg/ml post 72 h treatment; the cells＇ morphology were scarcely changed post treated with lower concentration,the number of cells dropped as the increasing concentration,and was sharply reduced in the concentration over 2. 4 mg/ml,and drug cytotoxicity highlighted. mRNA expression SEPP1 was significantly down-regulated post 72 h treatment at the concentration of 2. 0 mg/ml,while its upstream genes,CAPNS1,ITGA2,ITGAV,ITGB1 and ITGB5 were consistent up-regulated; protein level of SEPP1 was down-regulated as well. Conclusion： ATKS could inhibit the proliferation of HepG2 cells,the effect appears time-effect and dose-effect relationship. Expression changes of SEPP1 and other 5 genes at mRNA or protein level,which partially may be the mechanism of ATKS.