转化生长因子β_1诱导大鼠原代肝星状细胞上皮间质转化模型的建立  

Establishment of the rat primary hepatic stellate cells epithelial-mesenchymal transition model induced by transforming growth factor-β_1

作　　者：安祯祥[1] 何远利[1] 王敏[1] 黄丹[1] 陈玲 陈昱江[1] AN Zhen - xiang1, HE Yuan - li1, WANG Min1, HUANG Dan1, CHEN Ling2, CHEN Yu- jiang1(1. Department of Gastroenterology, The First Affiliated Hospital of Guiyang College of Traditional Chinese Medicine, Guiyang 550001, China; 2. The Administer Not Disease Centre, The Traditional Chinese Medicine Hospital of Qiannan , Duyun 558000, Guizhou Province, Chin)

机构地区：[1]贵阳中医学院第一附属医院消化科,贵阳550001 [2]黔南州中医医院治未病中心,贵州都匀558000

出　　处：《中国临床药理学杂志》2018年第14期1634-1636,共3页The Chinese Journal of Clinical Pharmacology

基　　金：国家自然科学基金资助项目(81460726;81760865);贵州省卫生计生委科学技术基金资助项目(gzwjkj2015-1-058);贵州省教育厅自然科学研究基金资助项目(黔教合KY字[2012]041号);贵州省科技计划课题基金资助项目(黔科合SY字[2012]3146)

摘　　要：目的研究转化生长因子β_1(TGF-β_1)诱导大鼠原代肝星状细胞发生上皮间质转化的条件。方法分离培养大鼠原代肝星状细胞,用TGF-β_1诱导建立上皮间质转化细胞模型,将细胞分为空白对照组和模型组,空白对照组加入培养基处理72 h,模型组加入4个质量浓度(1.25,2.5,5.0,10.0 ng·mL^(-1))TGF-β_1处理72 h,分别命名为模型-Ⅰ、-Ⅱ、-Ⅲ、-Ⅳ组;模型Ⅲ组诱导大鼠原代肝星状细胞24,48,72 h;免疫印迹法检测上皮细胞钙黏蛋白和α-平滑肌肌动蛋白(α-SMA)表达水平,实时定量PCR检测细胞snail基因水平。结果模型组-Ⅱ、-Ⅲ、-Ⅳ组的钙黏蛋白表达水平分别为0.29±0.07,0.28±0.03,0.34±0.10,与空白对照组的0.94±0.05比较,差异均有统计学意义(均P<0.001)。模型组-Ⅱ、-Ⅲ、-Ⅳ组的α-SMA表达水平分别为1.39±0.25,1.23±0.14,1.06±0.13,模型组-Ⅱ、-Ⅲ组与空白对照组的0.91±0.11比较,差异均有统计学意义(P<0.01和P<0.05)。同时,在诱导HSC 72 h,模型Ⅲ组的snail基因水平为5.00±1.59,与空白对照组的1.17±0.11比较,差异有统计学意义(P<0.01)。结论 5.0 ng·mL^(-1)TGF-β_1诱导细胞72 h,能成功诱导大鼠肝星状细胞上皮间质转化模型。Objective To investigate the conditions of the rat primary hepatic stellate cells（ HSC） epithelial-mesenchymal transition induced by transforming growth factor-β1（ TGF-β1）. Methods Isolating and culturing the rat primary HSC,established the cells epithelial-mesenchymal transition（ EMT） model by using TGF-β1. Cells were divided into blank control group and model group. The normal control group was placed into blank medium for 72 h. Model group was placed into the medium adding the different levels TGF-β11. 25,2. 5,5. 0,10. 0 ng·mL^-1 for 72 h,namely model-Ⅰ,-Ⅱ,-Ⅲ,-Ⅳ groups. Induced the rat HSC for 24,48 and 72 h by using the dose of 5 ng·mL^-1 TGF-β1（ model Ⅲ group）. Western Blot was used to test E-cadherin and α-smooth muscle actin protein expression level. The snail gene level of cells was tested by real-time quantitative PCR. Results The expression of the E-cadherin protein in model-Ⅱ,-Ⅲ,-Ⅳ groups were 0. 29 ± 0. 07,0. 28 ± 0. 03,0. 34 ± 0. 10,compared with theblank group（ 0. 94 ± 0. 05）,the difference of model groups was statistically significant（ all P〈0. 001）. The level ofα-SMA protein in model-Ⅱ,-Ⅲ,-Ⅳ groups were 1. 39 ± 0. 25,1. 23 ± 0. 14,1. 06 ± 0. 13,compared model-Ⅱ,-Ⅲ groups with the blank group（ 0. 91 ± 0. 11）,the difference had the statistical significant（ P〈0. 01 or P〈0. 05）. While,the level of snail gene was 5. 00 ± 1. 59,compared with the blank control group 1. 17 ± 0. 11,the difference was statistically significant（ P〈0. 01）. Conclusion Using the dose of 5 ng·mL^-1 TGF-β1 to induce the rat HSC for 72 h could induce the rat HSC EMT model.

关 键 词：肝星状细胞 上皮间质转化 转化生长因子β1 上皮细胞钙黏蛋白 Α-平滑肌肌动蛋白 

分 类 号：R97[医药卫生—药品]