摘要
目的探讨人参皂苷Rh2抗肝癌作用机制。方法 HE染色观察HepG2和HepG2-β-catenin荷瘤裸鼠肿瘤组织的细胞形态;免疫组化检测GSK-3β、β-catenin和MMP-3表达;ELISA法检测肿瘤细胞GSK-3β活性;PCR检测GSK-3β、β-catenin、Bcl-2、Cyclin D1、Bax和MMP-3基因的表达;Western blotting蛋白印迹法检测GSK-3β和β-catenin的表达。结果 HepG2组和HepG2-β-catenin组荷瘤裸鼠肿瘤细胞核成异型性,占整个细胞比例大,但HepG2-β-catenin组更明显。HepG2-β-catenin+人参皂苷Rh2组和HepG2+人参皂苷Rh2组肿瘤细胞胞核固缩,出现大量破碎细胞,而HepG2+人参皂苷Rh2组肿瘤细胞核固缩及破碎细胞更明显。免疫组化结果显示人参皂苷Rh2诱导HepG2及HepG2-β-catenin荷瘤裸鼠后,GSK-3β表达增强,β-catenin、MMP-3表达降低;HepG2+人参皂苷Rh2组中β-catenin、MMP-3表达弱于HepG2-β-catenin+人参皂苷Rh2组,而GSK-3β表达则无明显差异。ELISA结果显示,人参皂苷Rh2诱导HepG2及HepG2-β-catenin荷瘤裸鼠后,GSK-3β的活性均升高。PCR结果显示,HepG2+人参皂苷Rh2组中β-catenin、Cyclin D1、Bcl-2基因表达弱于HepG2-β-catenin+人参皂苷Rh2组,Bax基因表达增强更明显,而GSK-3β基因表达无明显差异。Western blotting结果显示,HepG2+人参皂苷Rh2组中β-catenin蛋白表达弱于HepG2-β-catenin+人参皂苷Rh2组,而GSK-3β蛋白表达无明显差异。结论人参皂苷Rh2对肝癌的抑制作用是通过激活GSK-3β降解β-catenin而实现的,且能抑制肿瘤的转移。
Objective To investigate the anticancer effect and the mechanism of ginsenoside Rh2 on animal model of HepG2 liver carcinoma. Methods HE staining was used to observe cell morphology. Immunohistochemical staining was used to detect the expression of GSK-3β, β-catenin, and MMP-3 in isolated single cells. The activity of GSK-3β was checked by ELISA kit. The expression levels of GSK-3β, β-catenin, Bax, Bcl-2, Cyclin D1, and MMP-3 genes were measured by q RT-PCR. The expression of β-catenin and GSK-3β proteins were determined by Western blotting. Results HE staining showed that the nucleus was atypia and account for a large proportion of the whole cell in HepG2 group and HepG2-β-catenin group. But nucleus atypia in HepG2-β-catenin group was more obvious. Condensation nuclei and a lot of broken cells were observed in HepG2-β-catenin ＋ ginsenoside Rh2 group and HepG2 ＋ ginsenoside Rh2 group. However, condensation nuclei and broken cells in HepG2 ＋ ginsenoside Rh2 group were more obvious. Immunohistochemical results indicated the expression of GSK-3β increased, while β-catenin and MMP-3 expression decreased in HepG2-β-catenin ＋ ginsenoside Rh2 group, compared with HepG2-β-catenin group. The expression of β-catenin and MMP-3 in HepG2 ＋ ginsenoside Rh2 group was lower than that in HepG2-β-catenin ＋ ginsenoside Rh2 group, while GSK-3β was no significant difference. The ELISA results indicated that the activity of GSK-3β was increased in HepG2 ＋ ginsenoside Rh2 group and HepG2-β-catenin ＋ ginsenoside Rh2 group. Compared with HepG2-β-catenin ＋ ginsenoside Rh2 group, the expression of Bax gene in HepG2 ＋ ginsenoside Rh2 group increased significantly, and the expression levels of Bcl-2, Cycli D1, and MMP-3 genes were also significantly lower, the difference was statistically significant（P 0.01）. The Western blotting results showed that compared with HepG2-β-catenin ＋ ginsenoside Rh2 group, the expression of β-catenin protein in HepG2 ＋ ginsenoside Rh2 group was also significantly lower, the difference was statistically significant（P 0.01）. Conclusion In vivo experiment shows that weight of tumor is decreased by ginsenoside Rh2 through activating GSK-3β to degrade β-catenin and could inhibit the ability of HepG2 cells metastasis.
出处
《中草药》
CAS
CSCD
北大核心
2016年第18期3231-3238,共8页
Chinese Traditional and Herbal Drugs
基金
国家自然科学基金资助项目(31271368)
重庆市教委基金项目(KJ110308)
