Ⅱ型肺泡上皮细胞转染Napsin A基因对肺纤维化的干预作用

Effect of Napsin A transfection into type Ⅱ alveolar epithelial cells on pulmonary fibrosis

目的 探讨Napsin A基因转染至Ⅱ型肺泡上皮细胞对肺纤维化的作用和机制.方法 采用慢病毒载体质粒PLJM1构建重组质粒PLJM1-Napsin A,将Napsin A基因转染至Ⅱ型肺泡上皮细胞系细胞-A549细胞染色体中并鉴定.予转化生长因子β1（TGF-β1）刺激A549细胞构建体外肺纤维化模型,倒置显微镜下动态观察细胞形态学的变化 用MTT法检测A549细胞转基因前后受TGF-β1刺激情况下增殖能力的变化,并绘制生长曲线 分别用RT-PCR和Western印迹方法检测未转基因和转基因A549细胞被TGF-β1干预后其表达E钙蛋白和纤维连接蛋白基因和蛋白水平的变化,以判断其发生上皮-间质转化（EMT）的情况 用Western印迹方法检测A549细胞转基因前后表达黏着斑激酶（FAK）蛋白量的变化,探讨Napsin A干预肺纤维化的机制.结果 重组质粒PLJM1-Napsin A测序结果与设计序列完全符合,转Napsin A基因A549细胞表达Napsin A基因和蛋白均显著高于非转基因细胞组（P＜0.01）.细胞经TGF-β1刺激后形态上演变为间质细胞,提示体外构建肺纤维化模型成功.MTT法检测转基因细胞在TGF-β1诱导下其增殖速度减慢（P＜0.05） E钙蛋白的基因和蛋白表达水平在体外肺纤维化模型中明显下调（E钙蛋白mRNA：A549-PLJM1：0.77±0.09,A549-PLJM1-Napsin A：0.79±0.03,A549＋TGF-β1：0.41±0.05,A549-PLJM1＋TGF-β1：0.40±0.05 E钙蛋白分别为：0. 76±0.06,0.73±0.09,0.20±0.05,0. 22±0.03,P＜0.01）,相反纤维连接蛋白则明显上调（P＜0.01）,但转染Napsin A基因后其变化幅度减小（P＜0.01,P＜0.05）.体外肺纤维化模型中,细胞FAK蛋白表达量增多（A549：0.49±0.04,A549-PLJM1：0.50±0.06 A549-PLJM1-NapsinA：0.48±0.08 A549＋TGF-β1：3.49±0.61 A549-PLJMI＋TGF-β1：3.54±0.25,P＜0.01）,但转基因细胞上调趋势显著小于未转基因细胞组（P＜0.01）.结论 转染Napsin A基因至Ⅱ型肺泡上皮细胞可以抑制肺纤维化,其作用机制可能与抑制整合素信号传导通路有关.

Objective To study the in vitro effect and mechanism of Napsin A gene transfection into type Ⅱ alveolar epithelial cells on pulmonary fibrosis. Methods A recombinant lentiviral plasmid PLJM1Napsin A was constructed and transfected into human type Ⅱ alveolar epithelial cell line A549. The model of pulmonary fibrosis was established by the in vitro stimulation of A549 cells by transforming growth factor beta-1 （TGF-β1）. The morphological changes were observed continuously under inverted microscopy. The proliferation of transgenic and non-transgenic cells was detected by MTT. To observe the degree of epithelialmesenchymal transition （ EMT ） by TGF-β1 intervening A549 cells, the expressions of E-cadherin and fibronectin were determined by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot.Lastly the protein expression of focal adhesion kinase （FAK） was detected by Western blot to investigate the mechanism. Results The result of sequencing the recombinant lentiviral plasmid PLJM1-Napsin A was the same as the design sequence. Napsin A mRNA and protein were expressed in transgenic A549 cells（ P 〈0. 01 ）. The model of pulmonary fibrosis was established successfully based on the morphology of transformed interstitial cell. As compared with the control group, the proliferation rate of transgenic cells decreased significantly （P 〈0. 05 ）. The mRNA and protein expression of E-cadherin significantly decreased in the model of pulmonary fibrosis （ P 〈 0. 01 ）, while the expression of fibronectin markedly increased （ P 〈 0. 01 ）.But the change rate of transgenic cells decreased （ P 〈 0. 01, P 〈 0. 05 ）. The expression of FAK was significantly elevated after the stimulation of TGF-β1 （ P 〈 0. 01 ）. But the upward trend of the transgenic cells was smaller as compared with the control group （P 〈 0. 01 ）. Conclusion Pulmonary fibrosis may be suppressed by the transfection of Napsin A gene into type Ⅱ alveolar epithelial cells. And the mechanism may be through the inhibition of integrin signal transduction.