摘要
目的探讨虎杖苷对转化生长因子-β1(transforming growth factor-β1,TGF-β1)诱导的人类肺泡上皮A549细胞生长及上皮-间质转化(epithelial-mesenchymal transition,EMT)过程的作用机制。方法体外培养A549细胞,随机分成5组:空白组、对照组、低剂量组、中剂量组、高剂量组,分别加入普通培养液、含5ng/mL TGF-β1的培养液、含浓度为50μmol/mL的虎杖苷加TGF-β1的培养液、含浓度为100μmol/mL的虎杖苷加TGF-β1的培养液、含浓度为150μmol/mL的虎杖苷加TGF-β1的培养液进行干预,观察并记录不同时间段细胞形态变化;MTT法检测虎杖苷最佳药物浓度;蛋白质印迹法(Western blot)、实时PCR(Real-time PCR)检测上皮标志物E-cad、间质标志物Vimentin蛋白及mRNA表达水平。结果倒置相差显微镜观察到虎杖苷和TGF-β1干预后A549细胞由原来的鹅卵石状上皮形态转变成梭形,且细胞间隙变大,细胞间的连接变得松散,对照组较为明显,低、中、高剂量组比对照组细胞形态稍饱满;48h中剂量组虎杖苷对TGF-β1诱导的A549细胞EMT抑制作用最明显。在虎杖苷和TGF-β1作用下,E-cad蛋白及mRNA表达总体呈下调趋势(P<0.05),与对照组比较,各药物组E-cad蛋白及mRNA表达下调幅度较小,且48h比24h明显。而Vimentin蛋白及mRNA表达总体呈上调趋势,与对照组比较,各药物组Vimentin蛋白及mRNA表达上调的幅度较小,且48h比24h明显,差异有统计学意义(P<0.05)。结论虎杖苷能抑制TGF-β1诱导的A549细胞EMT过程,并存在时间和剂量依赖性,具有抑制肺纤维化的作用。
Objective To explore the effect of polydatin on the growth of TGF-β1induced human alveolar epithelium A549 cells and the mechanism of polydatin for inhibiting the process of epithelial-mesenchymal transition（EMT）.Methods A549 cells in vitrocultured were randomly divided into five groups,i.e.,the blank group,the control group,the low dose polydatin group,the middle dose polydatin group,the high dose polydatin group.Common culture fluid was added in A549 cells of the blank group.Five ng/mL TGF-β1contained culture fluid was added in A549 cells of the control group.50,100,and 150μmol/mL of polydatin plus 5 ng/mL TGF-β1contained culture fluid was added in A549 cells of low,middle,and high dose polydatin groups,respectively.Morphological changes were observed and recorded at different time points.The optimal concentration of polydatin was determined by MTT method.Protein and mRNA expressions of E-cad（epithelial cell marker）and Vimentin（mesenchymal cell marker）were detected by Western blot and Real-time PCR.Results Under inverted phase contrast microscope,A549 cells turned from previous pebble shape to fusiform shape after intervened by poly-datin and TGF-β1.The intercellular space was enlarged and the intercellular connection became loose.These phenomena were more obviously seen in the control group.A549 cells were more satiated in low,middle,and high dose polydatin groups than in the control group.The EMT inhibition was most obviously seen in the middle dose polydatin group at 48 h.Protein and mRNA expressions of E-cad showed an overall descending tendency after intervened by polydatin and TGF-β1（P〈0.05）.But compared with the control group,protein and mRNA expressions of E-cad were down-regulated in a lesser amplitude in each intervened group.Besides,the tendency was more obviously seen at 48 h than at 24 h.Protein and mRNA expressions of Vimentin showed an overall up-regulating tendency.But compared with the control group,protein and mRNA expressions of Vimentin were down-regulated in a lesser
出处
《中国中西医结合杂志》
CAS
CSCD
北大核心
2016年第4期466-470,共5页
Chinese Journal of Integrated Traditional and Western Medicine
基金
国家自然科学基金资助项目(No.81302937)
浙江省自然科学基金资助项目(No.LY13H270009)
