Yangjinhua Alkaloids Role Of TGF-β1-induced EMT In Human Lung Adenocarcinoma Cell A549 Cells For Research

Objective To investigate the role of transforming growth factor Yangjinhua alkaloids in-β1(TGF-β1)in vitro induced human lung adenocarcinoma cell line A549 mesenchyme of the process.Methods A549 cells were cultured in vitro were divided into three groups,normal control group,TGF-β1 group and the drug group.Normal control group without adding TGF-β1;TGF-β1 group added 5 ng/mL TGF-β1;drug group was divided into low-dose group(50ug/ml),the concentration group(100ug/ml)and high concentration(200ug/ml)(concentration derived from the previous experiment MTT assay),respectively,in each group plus 5 ng/mL TGF-β1 and the corresponding concentration of the drug,24 hours after the intervention morphological changes under an inverted microscope,respectively,for 24 hours 48 hours,72 hours later,by Western-Blot and RT-PCR to detect the epithelial A549 cells markers CK-19 and mesenchymal cell marker a-smooth muscle actin(α-SMA)protein and gene expression changes.Results 1.Inverted phase contrast microscope at 5 ng/ML of TGF-β1-induced cell morphology cobblestone-like control group was gradually transformed by the spindle,similar to fibroblasts,the connection between the cells become loose,TGF-β1-induced A549 cells to mesenchymal cell morphology transformation.2.Western-blot Show:normal control group,the expression of a-SMA expression of CK-19 and traces;TGF-β1 group CK-19 expression was significantly reduced,a-SMA expression increased,compared with the control group(P<0.01)the difference was statistically significant;later to Yangjinhua alkaloids intervention group compared with the drug group TGF-β1,upregulation of the expression of CK-19(P<0.01),but still lower than the control group(P<0.01);α-SMA expression has been suppressed(P<0.01);their differences and the role of time and the drug concentration was positively correlated,when the concentration of drug in 72 hours,and most obvious difference is 200ug/ml3 RT-PCR Show:TGF-β1 group compared with the control group,CK-19mRNA expression decreased,increased mRNA expression of a-SMA,the difference was statistically significant(P<0.01).Drug group compared,CK-19 mRNA expression in the three groups were higher than the drug TGF-β1 group(P<0.01),mRNA expression of a-SMA group of TGF-β1 were lower(P<0.01).Compared to the drug group CK-19mRNA expression gradually increased role between three time points(P<0.01),no significant difference of a-SMA mRNA expression(p>0.05).Conclusion 1.TGF-beta 1 can induce epithelial mesenchymal changes of A549 cells,Morphological changes to the spindle by a pebble-like,after Yangjinhua alkaloids intervention in the form can not be fully reversed.2.Yangjinhua alkaloids in protein levels can inhibit a-SMA expression,increased expression of epithelial markers CK-19,and the time-dependent and concentration-dependent manner,at the time of 72 hours and most obvious effect of the concentration of 200ug/ml.3.Yangjinhua alkaloids in gene expression levels can increase CK-19mRNA reduce the expression of a-SMA mRNA’s.4.Yangjinhua alkaloids in TGF beta 1-induced EMT in A549 cells can inhibit the expression of alpha SMA expression,increase the expression of CK-19,but can be resistance to pulmonary fibrosis by EMT,remains to be further in-depth study.