鳖甲煎丸通过Wnt/β-catenin信号通路逆转大鼠肝卵圆细胞EMT的机制  

Mechanism of Biejiajian Wan in Reversing EMT in Rat Hetapic Oval Cells via Wnt/β-catenin Pathway

作　　者：招文婷 孙嘉玲 文彬 孙海涛[1] 杨雪梅[1] 陈炜聪 何春雨 钟晓丹 陈冠新 贺松其[1] ZHAO Wen-ting;SUN Jia-ling;WEN Bin;SUN Hai-tao;YANG Xue-mei;CHEN Wei-cong;HE Chun-yu;ZHONG Xiao-dan;CHEN Guan-xin;HE Song-qi(School of Chinese Medicine,Southern Medical University,Guangzhou 510515,China;Air Force Hospital of Southern Theater Command of PLA,Guangzhou 510602,China;Dongguan Hospital of Traditional Chinese Medicine,Dongguan 523000,China)

机构地区：[1]南方医科大学中医药学院,广州510515 [2]解放军南部战区空军医院,广州510602 [3]东莞市中医院,广东东莞523000

出　　处：《中国实验方剂学杂志》2021年第1期38-45,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(81774037)。

摘　　要：目的:研究鳖甲煎丸对转化生长因子-β1(TGF-β1)诱导的大鼠肝卵圆细胞WB-F344上皮间质转化(EMT)的影响,探讨其逆转EMT改变的作用机制。方法:将WB-F344细胞随机分为空白组,TGF-β1模型组(10μg·L-1TGF-β1),鳖甲煎丸低剂量组(10μg·L-1TGF-β1+0.55 g·kg^(-1)鳖甲煎丸),鳖甲煎丸中剂量组(10μg·L-1TGF-β1+1.1 g·kg^(-1)鳖甲煎丸),鳖甲煎丸高剂量组(10μg·L-1TGF-β1+2.2 g·kg^(-1)鳖甲煎丸),除空白组外,均采用TGF-β1诱导WB-F344细胞构建EMT模型,分别加入鳖甲煎丸低、中、高剂量含药血清处理细胞,采用细胞划痕实验检测WB-F344细胞迁移能力的改变;使用蛋白免疫印迹法(Western blot)检测E-钙黏蛋白(E-cadherin),N-钙黏蛋白(N-cadherin),波形蛋白(Vimentin)表达的变化;使用实时荧光定量PCR(Real-time PCR)检测β-连环蛋白(β-catenin)mRNA表达的改变;使用细胞免疫荧光法检测β-catenin的表达。结果:与空白组比较,TGF-β1诱导WB-F344细胞4 d,WB-F344细胞间隙从紧密逐渐变得松散,细胞形态由鹅卵石状向成纤维样细胞转变,且E-cadherin蛋白表达降低,N-cadherin蛋白表达升高(P<0.01),说明成功构建了WB-F344细胞EMT模型。划痕实验结果显示,与空白组比较,TGF-β1模型组WB-F344细胞的迁移能力增强(P<0.01);与TGF-β1模型组比较,鳖甲煎丸中、高剂量组可以明显抑制WB-F344细胞的迁移能力(P<0.05)。Western blot结果显示,与空白组比较,TGF-β1模型组E-cadherin蛋白表达水平降低,N-cadherin,Vimentin蛋白表达水平升高(P<0.01);与TGF-β1模型组比较,鳖甲煎丸中、高剂量组E-cadherin蛋白表达升高,N-cadherin,Vimentin蛋白表达降低(P<0.05,P<0.01)。Real-time PCR结果显示,与空白组比较,TGF-β1模型组中β-catenin mRNA表达升高(P<0.01);与TGF-β1模型组比较,鳖甲煎丸低、中、高剂量组中β-catenin mRNA的表达降低(P<0.01)。细胞免疫荧光结果显示,与空白组比较,TGF-β1模型组β-catenin在细胞核内荧光表达增强Objective: To study the effect of Biejiajian Wan on the epithelial-mesenchymal transition(EMT)of rat hepatic oval cells induced by transforming growth factor-β1(TGF-β1),in order to explore its mechanism in reversing EMT. Method: WB-F344 cells were divided into five groups:blank group,TGF-β1 model group(10 μg·L-1 TGF-β1),low-dose group(10 μg·L-1 TGF-β1+0.55 g·kg^(-1) Biejiajian Wan),medium-dose group(10 μg·L-1 TGF-β1+1.1 g·kg^(-1) Biejiajian Wan),high-dose group(10 μg·L-1 TGF-β1+2.2 g·kg^(-1) Biejiajian Wan). Except blank group,TGF-β1 was used to induce WB-F344 cells in all of the remaining groups to construct an EMT model. After the cells were treated with low,medium and high doses of Biejiajian Wan serum,the changes of migration ability of WB-F344 cells were detected by cell scratching test. The expressions of Ecadherin,N-cadherin and Vimentin were detected by Western blot. Real-time PCR was used to detect the changes in the expression of β-catenin mRNA. The expression of β-catenin was detected by cell immunofluorescence assay.Result: Compared with normal WB-F344 cells,the intercellular space of WB-F344 cells became loose from tight,and the morphology changed from cobblestone to fibroblast after TGF-β1 induced WB-F344 cells for 4 days,and the expression of E-cadherin protein decreased,while the expression of N-cadherin protein increased(P<0.01),indicating that the EMT model of WB-F344 cells was successfully built. Compared with the blank group,the migration ability of WB-F344 cells in TGF-β1 model group was enhanced(P<0.01),compared with TGF-β1 model group, Biejiajian Wan could significantly inhibit the migration ability of WB-F344 cells;specifically,the low-dose group had no statistical significance,and the medium and high-dose groups had statistical significance(P<0.05). Western blot results showed that compared with the blank group, the expression of E-cadherin decreased,whereas those of N-cadherin and Vimentin increased in the TGF-β1 model group(P<0.01),compared with TGF-β1 model gr

关 键 词：鳖甲煎丸 肝卵圆细胞 上皮间质转化 Wnt/β-连环蛋白(β-catenin)通路 肝癌前病变 转化生成因子-β1 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学]