ABSTRACT
Objective To construct the eukaryotic expression vector of Notch1intracellular domain,p3XFLAG?CMV7?NICD1,so as to prepare for the further research and exploration of effect of Notch1 on promoting epithelial?mesenchymal transition of human lens epithelial cells. Methods The cDNA fragment was reversely transcribed by RT?PCR from total RNA extracted from the SRA01/04 cells and was encoded with the specific am?plification?targeted NICD1was obtained from the SRA01/04 cells,then the cDNA fragment was inserted into p3XFLAG?CMV7 to transcribe Esche?richia coli DH5α. And the recombinant plasmid was extracted after bacterial screening by LB plating medium and confirmed by the restriction endo?nuclease digestion and DNA sequencing. Results The target gene obtained had the same molecular size as predicted. It was indicated that recom?bined p3XFLAG?CMV7 plasmid contained correct recombinant human Notch1 sequences and p3XFLAG?CMV7?NICD1 was constructed success?fully. The western blotting showed protein NICD1 expressed in SRA01/04 cells transfected with p3XFLAG?CMV7?NICD1. Conclusion The suc?cessful construction of p3XFLAG?CMV7?NICD1 will provide a foundation for a further study studies in on the effect relationship of Notch signaling pathway and in posterior capsular opacification(PCO)after cataract extraction.
ABSTRACT
Objective To investigate the expression of interleukin (IL)-34/colony stimulating factor(CSF)-1R in the process of transforming growth factor ( TGF)-β1 inducing epithelial-mesenchymal transition ( EMT) of human alveolar epithelial cells A549 cells.Methods A549 cells were cultured in vitro.CCK 8 was used to test the influence of the proliferative rate of A549 cells which were stimulated by TGF-β1 at different concentrations and time points .A549 cells were stimulated by 5μg/L TGF-β1 at 0 hour, the 12th hour, the 24th hour, and the 48th hour.Western blotting was adopted to detect changes of the following proteins: α-smooth muscle actin (α-SMA ) , E-cadherin ( E-Cad ) , matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1(TIMP-1). Real-time PCR was adopted to detect changes of the following genes: IL-34 mRNA, CSF-1R mRNA, MMP-2 mRNA and MMP-9 mRNA.Results TGF-β1 had no significant influence in the proliferation of A 549 cells compared with the control group(P>0.05).TGF-β1(5μg/L)stimulated A549 cells at different time point (0 hour, 12, 24, 48 hours), compared with the control group .The epithelial phenotype E-Cad protein was gradually down-regulated ( P <0.01 ) , while the mesenchymal phenotype α-SMA protein was gradually up-regulated ( P <0.01 ) and the protein of MMP-2 increased gradually (P<0.01).The protein of MMP-9 increased firstly and then was reduced (P<0.01),the peak was at the 24th hour.The protein of TIMP-1 was firstly transiently increased and then reduced (P<0.01), the minimum was at the 48th hour.Compared with the control group , the gene of IL-34 mRNA increased gradually (P<0.01), and the genes of CSF-1R mRNA, MMP-2 mRNA and MMP-9 mRNA increased firstly and then decreased ( P <0.01), which were peaked respectively at the 24th hour, the 24th hour, the 12th hour, respectively.Conclusion In the process of TGF-β1 inducing A549 cells transition,there is accompanied with the expression of IL-34/CSF-1R.