摘要
目的探讨临床治疗肺纤维化常方肺痿冲剂方对体外经转化生长因子-β1(transforming growth factor-β1,TGF-β1)诱导肺腺癌A549细胞后发生上皮-间质转化(epithelial mesenchymal transition,EMT)的作用。方法首先应用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,MTT]检测肺痿冲剂方对A549细胞增殖的影响,在3.9062~250μg/mL浓度范围未发现肺痿冲剂方对A549细胞增殖有显著性的影响。下一步将体外培养A549细胞分随机3组:空白组加入F-12培养基,模型组以及中药组分别加入10 ng/mL TGF-β1和62.5μg/mL肺痿冲剂方,继续培养72小时。以光学显微镜观察各组细胞的形态变化,并通过实时荧光定量PCR(reverse transcription-polymerase chain reaction,rt-PCR)检测纤维连接蛋白(fibronectin,FN)、波形蛋白(vimentin,Vim)、E-钙黏素(E-cadherin,E-Cad)mRNA表达水平。结果(1)显微镜下观察结果示:与空白组比较,模型组细胞形态发生明显变化,即从类似椭圆形形转变成伸长的纺锤样的形态,圆形度降低,差异有统计学意义(P<0.01)。与模型组比较,中药组细胞还能保持原形态,细胞圆形度值明显增高,差异有统计学意义(P<0.01);(2)实时荧光定量PCR结果表示:空白组比较,模型组FN mRNA表达升高,差异有统计学意义(P<0.01),E-Cad mRNA表达降低,差异有统计学意义(P<0.01)。与模型组比较,中药组FN mRNA表达降低,差异有统计学意义(P<0.05),E-Cad mRNA表达升高,差异有统计学意义(P<0.05)。结论肺痿冲剂方可提高EMT过程中E-Cad mRNA并抑制FN mRNA的表达,为研究肺痿冲剂方临床治疗肺纤维化提供了可靠的实验依据。
Objective To investigate the effect of clinical treatment of pulmonary fibrosis common Feiwei prescription granules on epithelial-mesenchymal transition(EMT)induced by transforming growth factor-β1(TGF-β1)in lung adenocarcinoma A549 cells.Methods The effect of Feiwei granule on the proliferation of A549 cells was firstly tested by MTT assay.The effect of Feiwei granule on the proliferation of A549 cells was not found in the concentration range of 3.9062~250μg/mL.Next,the A549 cells cultured in vitro were randomly divided into three groups:the blank group was added to the F-12 medium,and the model group and the Chinese medicine group were added with 10 ng/mL TGF-β1 and 62.5μg/mL lung sputum,respectively,and the culture was continued for 72 hours.Morphological changes of cells in each group were observed by light microscopy,and the expression levels of fibronectin(FN),vimentin(Vim)and E-cadherin(E-Cad)were detected by real-time fluorescent quantitative PCR(rt-PCR).Results(1)The results under microscope showed that compared with the blank group,the morphology of the cells in the model group changed significantly,from the elliptical shape to the elongated spindle-like morphology he circularity decreased,and the difference was statistically significant(P<0.01).Compared with the model group,the cells in the traditional Chinese medicine group can still maintain the original morphology,and the cell circularity value significantly increased,statistically significant(P<0.01);(2)Real-time PCR results show:blank group comparison,model group FN mRNA expression difference statistically significant(P<0.01)he expression of E-Cad mRNA decreased,the difference statistically significan(P<0.01).Compared with the model group,the expression of FN mRNA in the Chinese medicine group decreased,difference(P<0.05),the expression of E-Cad mRNA increased,statistically significant(P<0.05).Conclusion Feiwei prescription granule can increase E-Cad mRNA and inhibit the expression of FN mRNA during EMT,and provide a reliable experimental basis for the study of pulmonary fibrosis in the treatment of pulmonary phlegm.
作者
阮越勇
张浩军
疏欣杨
张纾难
RUAN Yueyong;ZHANG Haojun;SHU Xinyang(Graduate School of Beijing University of Chinese Medicine,Beijing 100029,China)
出处
《环球中医药》
CAS
2019年第11期1645-1649,共5页
Global Traditional Chinese Medicine
基金
国家自然科学基金青年项目(81804044)
关键词
肺痿冲剂方
A549细胞
上皮-间质转化
转化生长因子-Β1
Feiwei prescription granules
A549 cells
Epithelial-Mesenchymal transition
Transforming growth factor-β1
作者简介
阮越勇(NGUYEN VIET DUNG)(1988-),越南籍,2016级在读博士研究生。研究方向:中医药防治呼吸系统疾病。E-mail:vietdzung24@yahoo.com;通信作者:张纾难(1963-),博士,主任医师,教授,博士生导师。研究方向:中医药防治呼吸系统疾病。E-mail:shunan_zhang2010@qq.com。
