Effection And Mechanism Of Truncated β Catenin On Human Embryonic Lung Fibroblast

Recently morbidity and mortality of Interstitial Lung Disease (ILD) obviously presents ascending trend and pulmonary fibrosis is the common outcome in late stage.The disease morbidity is 3 - 6/10⁵ and about 50% patients of IPF can only survive 4-5 years after the diagnosis is confirmed. The disease, whose mortality is similar to that of lung cancer, is a severe threaten to people's health and badly impacts public life-quality.Therefore, extensive research of mechanism and finding new approaches to prevent and cure pulmonary fibrosis has been hotspot and big problem in pulmonology. Although the pathogenesis is not very clear, new understandings about development of pulmonary fibrosis has appeared in company with impenetration of cell biology research and molecular biology research. In a word, research of pulmonary fibrosis gradually appears to be integrated and multidisciplinary crossover.Wnt signal pathway, which acts as a vital role in lung development in mammals, is an important signal molecule in development. Gene knockout experiments confirmed that Wnt2a gene knockout mouse didn't develop lung outline and Wnt5a gene knockout mouse coulde not complete lung mature that was lethal to mouse in late stage of lung development.Many evidence indicated that beta - catenin promoted taking place of fibrosis disease. Aggressive fibromatosis is a disease of benign ,clone hyperplasia of fibrocyte.β-catenin,which starts downstream gene transcription,was found concentrated in cell nuclei in sporadic patients.Documents reportedβ-catenin transfered into endonuclear in basal cell and II type epithelial cell of damaged bronchus in patients who were diagnosed IPF. Reports said downstream effect gene, MMP-7, directly participated in formation of pulmonary fibrosis. Simultaneously, there are many crossover between Wnt /β-catenin signal and TGF -βpathway in addition of TGF-βcould directly activates TCF/LEF to transcribe target genes.Therefore, this research utilized gene transfection, RT - PCR and Western Blot technologies to prove thatβ-catenin have some functions on fibroblast and to investigate its mechanisms.MethodsOn the basis of complete sequence of beta catenin gene which obtained from cDNA library, 89 codon were eliminated at 5' end to form plasmid scat—pcDNA3.0;Then the constructed plasmid was transfected into human embryonic lung fibroblast by Sofast transfection reagent. The transfected cell were collected to analysis the migration ability, level of I ,III type collagen, a smooth muscle actin, epithelial calcium cadherin, MMP -7 level at mRNA and protein level. Interpretation of results was made by integral optical density, which represented the amount of expression product. SPSS10.0 statistical software was used for statistical analysis. Significant test level was 0.05.ResultsPCR product was double cut by enzyme Xho I and Kpn I ,then the fragment was retrieved. The fragment was connected to pcDNA3.0 carrier which was cut by the same two enzymes to form the plasmid which was transformed into Escherichia coli DH5a.Appropriate clone was cultured to extract plasmid.The extract was analysed to confirm that it was constructed correctively.Sequence of reformed plasmid, which is confirmed by sequence test, was identified to that in Genebank of NCBI.The reformed plasmid, pcDNA3.0 plasmid and controll plasmid pEGFP-N1 were transfected into human embryonic lung fibroblasts by Sofast transfection agent,after which green fluorescence was observed in controll group in order to confirm the transfection.RT-PCR and Western Blot analysis showed small quantity expression of beta catenin in control group and carrier transfected HELF,but SCAT-pcDNA3.0 plasmid transfected cell strongly expressed beta catenin. Neomycin resistance gene was not expressed in HELF cell ,but it highly expressed in cells transfected by carrier and SCAT-pcDNA3.0 plasmid.Type I collagen was highly expressed in SCAT-pcDNA3.0 tansfected HELF compared with that in HELF cell and carrier transfected cell.Type III collagen expression was a little difference between carrier transfected cell and HELF.Expression of Wnt/beta catenin downstream gene, MMP-7, cyclin D1 increased accompanied with MMP-9 expression without change.Migration of reformed plasmid transfected cell was markedly increased.Conclusions1. Beta catenin, which promoted collagen synthsis,played a certain role in the formation of pulmonary fibrosis.2. Pulmonary fibrosis took place through the way of beta catenin increasing expression of type I collagen, a smooth muscle actin, MMP-7, cyclin D₁ and decreasing epithelial calcium cadherin expression .3. Beta catenin promoted occurring of pulmonary fibrosis by increasing fibroblast cell motility.