The Effects And Mechanism Of Napsin A Gene Therapy For Pulmonary Fibrosis

Pulmonary fibrosis is the end of all kinds of interstitial lung diseases. At present, no approach can control or reverse the illness. Lung transplantation is the only way to prolong the survival in patients with pulmonary fibrosis, but it can not be carried out widely because of its expensiveness and donor scarcity. Driven by modern molecular biology and genetic engineering technology, Gene therapy of pulmonary fibrosis is expected to become a breakthrough treatment.Pulmonary fibrosis is considered to be a kind of fibroblast proliferation disease which is confined to the lung. Alveolar epithelial cell damage directly cause pulmonary fibrosis with or without interstitial inflammation and pulmonary fibrosis is classified into epithelial-fibroblast cell disease. According to this view, alveolar epithelial cell damage induce epithelial-mesenchymal transition (EMT), then alveolar epithelial cell transform into fibroblast cell and proliferate, which are the two main links in the formation of pulmonary fibrosis. Inhibiting epithelial cell EMT and proliferation are the two valid targets in the treatment of pulmonary fibrosis.Napsin A is a member of aspartic protease family and expressed significantly in typeⅡalveolar epithelial cell. Research has found that Napsin A can regulate cell biological behavior in addition to its proteolytic activity. Scholars have found that after cell phenotype transformed, such as malignant transformation, the expression of Napsin A is down-regulated, and the lower level of magnitude and phenotype transformation is positively correlated. It suggested Napsin A lost expression may be one of the reasons for cells phenotype transformation. If Napsin A expression restored, we maybe can reverse the process. Some studies have founded Napsin A can inhibit cell proliferation in vitro and vivo experiments. This subject will study the value against pulmonary fibrosis by taking advantage of its biological function in inhibiting cells phenotype transformation and proliferation of Napsin A.The purpose of our research:we transfected Napsin A into typeⅡalveolar epithelial cell line A549 cell stably and constructed pulmonary fibrosis model in vitro, then studied the impact of Napsin A gene on the pulmonary fibrosis model in vitro experiments. At last, we discussed the related intervention mechanisms.Methods:1.A549 cells were cultured with TGF-β1 in vitro and pulmonary fibrosis model built by TGF-β1 stimulated. TGF-β1 concentration gradient set 5 and the optimal concentrations was filtered by MTT assay. The time of model completed was determined by cell morphology observed under inverted microscope. The pulmonary fibrosis model was identified by RT-PCR and western-blot for detecting of the expression of marker proteins in epithelial cell and interstitial cell after model completed.2.Recombinant lentivirus PLJM1-Napsin A plasmid.was constructed To produce virus, plasmid were transfect into packaging cell——293T cell using Lipofectamine 2000TM according to the manufacturer’s instructions. Virus suspension was collected and infected A549 cells. Puromycin was added to the medium. Positive polyclone population was identified based on Napsin A expression.3.To study the impact of Napsin A gene on cell proliferation in the EMT process of pulmonary fibrosis model in vitro experiments, MTT assay and flow cytometry were used to detect. the expression of marker proteins of epithelial cells and interstitial cells were detected by RT-PCR and western-blot in order to identify the effect of Napsin A gene on cell EMT after model completed. 4.Building A549 cell tumor model in nude mouse, the effect of Napsin A gene on cell proliferation in vivo experiments was studied by drawing tumor growth curve, calculating tumor growth inhibition rate according to tumor size and observing the status of A549 cell growth in HE staining pathological section.5.The mechanism of Napsin A intervention in cell proliferation and EMT was explored using Western-blot for detecting the expression of focal adhesion kinase (FAK), the basic molecular of integrin signaling pathway, in A549 cells.Results:1.Establishing in vitro pulmonary fibrosis model, MTT assay was found that A549 cell were in the maximum growth rate induced by TGF-β1 at a concentration of 5μg/L, so the best concentration of TGF-(31 was 5μg/L. On day 7 after TGF-β1 treatment, cells were observed under inverted microscope to change into spindle-shaped slender cells in morphology, so the 7th day was determined to be the time for model completion. The expression of E-cadherin was decrease and the expression of fibronectin and collagen typeⅠwas increased after pulmonary fibrosis model completion.2.Five recombinant lentivirus PLJM1-Napsin A plasmid monoclonal were selected to identify by PCR and Napsin A mRNA was amplified whose bands size were about 1.2 kb. The result of PLJM1-Napsin A plasmid sequenced by automated sequencer was compliant with the design sequence completely. Overexpression of Napsin A was confirmed by RT-PCR and western-blot in A549-PLJM1-Napsin A cells.3.cell proliferation was detected by MTT assay. Napsin A partially but significantly blocked TGF-β1 induced cell proliferation in vitro pulmonary fibrosis model. The results of flow cytometry indicated that Napsin A overexpression can siginificantly induced G0/G1 arrest. The change trend of cell morphology, down-regulated of E-cadherin and up--regulated of fibronectin and collagen type I were also partially inhibited significantly in A549-PLJM1-Napsin A cells.4.A549 cells model in nude mouse was successfully constructed. A549-PLJM1-Napsin A cells tumor grew more slowly and the tumor growth inhibition rate was 39.8%. A549-PLJM1-Napsin A cell changed into round and arraied sparsely and the nuclear chromatin was aggregate and deeply stained in HE staining pathological section.5.The FAK expression of A549 cell was increased detected by Western-blot in vitro pulmonary fibrosis model, but the up-regulated trend could become smaller significantly because of Napsin A gene transfection.So we have results below:1.The pulmonary fibrosis model can be built by TGF-β1-induced A549 cells proliferation and EMT in vitro.2.A549-PLM1-Napsin A cell line who can express Napsin A mRNA and protein stably and permanently can be constructed by using lentivirus medium.3.Napsin A can inhibit cell proliferation in vivo and vitro experiment.4.Napsin A can partially inhibit cell EMT in vitro pulmonary fibrosis model.5.The inhibitory effect of Napsin A on cell proliferation and EMT may be due to inhibition of FAK expression and blocked integrin signaling pathway by Napsin A.