摘要
目的通过使用不同浓度的棕榈酸(PA)、转化生长因子-β1(TGF-β1)分别诱导人肝星状细胞(LX-2)与人肝癌细胞(HepG2)探讨制备脂肪性肝纤维化细胞模型的方法。方法选用人LX-2细胞与人HepG2细胞,分别给予100、200、400μmol·L^(-1) PA处理及2、5、10 ng·mL^(-1) TGF-β1处理,两组均处理24 h后利用MTT细胞毒性实验检测细胞存活率,通过油红O染色检测细胞脂肪变性,采用qRT-PCR与Western blot实验检测细胞α-平滑肌动蛋白(α-SMA)及Ⅰ型胶原蛋白(CollagenⅠ)的mRNA及蛋白表达水平,了解细胞纤维化情况。结果MTT结果显示,不同实验组与对照组比较,PA分别诱导HepG2细胞与LX-2细胞24 h后,两组细胞存活率在100、200、400μmol·L^(-1) PA的诱导下出现不同程度降低(P均<0.01);TGF-β1分别诱导两组细胞24 h后,细胞存活率在5、10 ng·mL^(-1) TGF-β1诱导下出现不同程度降低(P均<0.05)。油红O染色结果显示,与对照组相比,不同浓度PA诱导24 h后,HepG2、LX-2细胞均随PA浓度的增大,脂滴形成增多,具有明显的浓度依赖性;不同浓度TGF-β1诱导24 h后,HepG2细胞内未见明显脂滴,2、5 ng·mL^(-1) TGF-β1诱导后LX-2细胞内可见少量脂滴,10 ng·mL^(-1) TGF-β1诱导后可见LX-2细胞内脂滴明显增多。qRT-PCR结果显示,200、400μmol·L^(-1) PA诱导HepG2细胞内、400μmol·L^(-1) PA诱导LX-2细胞内α-SMA、CollagenⅠmRNA相对表达水平均上调(P均<0.01);10 ng·mL^(-1) TGF-β1诱导HepG2组细胞内CollagenⅠmRNA相对表达水平上调,5、10 ng·mL^(-1) TGF-β1诱导LX-2细胞内α-SMA、CollagenⅠmRNA相对表达水平均上调(P均<0.05)。Western blot结果显示,200、400μmol·L^(-1) PA及5、10 ng·mL^(-1) TGF-β1诱导后两组细胞内α-SMA及CollagenⅠ的蛋白相对表达水平均升高(P均<0.05)。结论200μmol·L^(-1) PA诱导HepG2细胞24 h、10 ng·mL^(-1) TGF-β1诱导LX-2细胞与HepG2细胞24 h是成功构建脂肪性肝纤维化细胞模型的适宜条件。
Objective Construction of fatty liver fibrosis cell model by using different concentrations of palmitic acid(PA)and transforming growth factor-β1(TGF-β1).Methods Human hepatic stellate cells LX-2 and human hepatocarcinoma cells HepG2 were selected to be treated with 100,200,400μmol·L^(-1) palmitic acid(PA)and 2,5,10 ng·mL^(-1) TGF-β1,both groups were treated for 24 h,then MTT cytotoxicity test was used to detect cell viability,oil red O staining was used to detect cell fatty degeneration,Western blot and qRT-PCR experiment were used to detect cellα-smooth muscle activity protein(α-SMA)and typeⅠcollagen(CollagenⅠ)protein and mRNA expression levels to detect cell fibrosis.Results The results of MTT showed that the cell survival rate of the two groups after 100,200,400μmol·L^(-1) PA induction decreased with the increase of PA concentration,and the two groups of cells survived after 5,10 ng·mL^(-1) TGF-β1 induction.The rate did not change significantly with the increase of TGF-β1 concentration,and the difference was statistically significant(P all<0.05).The oil red O staining results showed that the lipid droplets content in the two groups of cells increased significantly with the increase of PA concentration after the induction of different concentrations of PA,and there were no obvious lipid droplets in HepG2 cells after induction of different concentrations of TGF-β1.The lipid droplet content in LX-2 cells increased with the increase of TGF-β1 concentration.The results of qRT-PCR showed that the relative expression levels ofα-SMA and CollagenⅠmRNA in HepG2 cells were up-regulated in 200 and 400μmol·L^(-1) PA groups;the relative expression levels ofα-SMA and CollagenⅠmRNA in LX-2 cells were up-regulated by 400μmol·L^(-1) PA,10 ng·mL^(-1) TGF-β1 induced an increase in the relative expression of CollagenⅠmRNA in HepG2 cells;5,10 ng·mL^(-1) TGF-β1 induced an increase in the relative expression ofα-SMA and CollagenⅠmRNA in LX-2 cells,and the differences were statistically significa
作者
梁涵子
李雨涵
李家瑞
李建宁
宋辉
杨怡
LIANG Hanzi;LI Yuhan;LI Jiarui;LI Jianning;SONG Hui;YANG Yi(Department of Biochemistry and Molecular Biology,School of Basic Medical Sciences,Ningxia Medical University,Yinchuan 750004,China;Institute of Endocrinology,Ningxia Medical University,Yinchuan 750004,China;Department of Cell Biology and Genetics,School of Basic Medical Sciences,Ningxia Medical University,Yinchuan 750004,China)
出处
《宁夏医科大学学报》
2021年第6期549-555,共7页
Journal of Ningxia Medical University
基金
国家自然科学基金(81670798)。
