Napsin A基因转染干预A549细胞的上皮-间质转化  

Napsin A transfection interferes the ephethlial-mesenchymal transition of A549 cells

作　　者：管淑红[1] 郑金旭[2] 汤艳[2] 宋萍[2] 许清[2] 刘继柱[2] 

机构地区：[1]江苏大学基础医学与医学技术学院,江苏镇江212013 [2]江苏大学附属医院呼吸科,江苏镇江212001

出　　处：《解剖学报》2011年第3期350-355,共6页Acta Anatomica Sinica

基　　金：卫生部科研资助项目(wkj2006-2-026);江苏省“333工程”资助项目(苏人才办2007-16-09)

摘　　要：目的探讨Napsin A基因转染至A549细胞对其上皮-间质转化(EMT)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A549细胞染色体中并鉴定。用转化生长因子-β1(TGF-β1)刺激A549细胞构建体外EMT模型,倒置显微镜下动态观察细胞形态学的变化,观察Napsin A基因转染对A549细胞在体外EMT模型中细胞EMT和表达黏着斑激酶(FAK)的影响。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蛋白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,E钙蛋白的mRNA和蛋白表达水平明显下调(P<0.01),相反Ⅰ型胶原则显著上调(P<0.01),提示体外构建EMT模型获得成功。转Napsin A基因A549细胞在TGF-β1干预后,其细胞形态间质改变、E钙蛋白和Ⅰ型胶原的表达量也发生相似变化趋势,但变化幅度显著变小(其中E钙蛋白:P<0.01,Ⅰ型胶原:P<0.05)。体外EMT模型中,细胞FAK蛋白表达量增多(P<0.01),但转基因细胞上调趋势明显小于未转基因细胞(P<0.01)。结论转染Napsin A基因至A549细胞可以部分阻滞细胞EMT进程,其作用机制可能与抑制整合素信号转导通路有关。Objective To study the effect and mechanism of Napsin A gene transfeetion into A549 cells on ephethlial-mesenchymal transition （EMT） in vitro. Methods A recombinant lentiviral plasmid PLJM1-Napsin A was constructed, then transfected into A549 cell and identified. A549 cells EMT model was established by transforming growth factor beta-1 （TGF-β1） treatment in vitro. The morphology change was observed under inverted microscopy successively. To observe the degree of EMT by TGF-β1 intervening A549 cells, the expression of E-eadherin and collagen type I was detected by reverse transcription-polymerase chain reaction（RT-PCR） and Western blotting. Finally, in order to investigate the mechanism, the protein expression of focal adhesion kinase （FAK） was detected by Western blotting. Results The result of sequencing the recombinant lentiviral plasmid PLJM1-Napsin A was predicted as designed. The expression of Napsin A protein in transgenic A549 cells was more than that in A549 ceils and A549-PLJM1 cells（P 〈 0.01 ）. After TGF-β1 treatment, the cells changed from epithelial-shaped into interstitial-shaped; TGF-β1 induced EMT in A549 cells, as demonstrated by significant reduction of E-eadherin mRNA and protein levels （P 〈 0.01 ） as well as up-regulation of collagen type I （P 〈 0. 01 ）. Transfection of Napsin A in A549 cells could not only restrain the TGF-β1-induced morphological changes, but also partially block the TGF-β1-indueed expression of E-caherin（ P 〈0. 01 ） and collagen type I （P 〈0. 05） expression. In EMT model in vitro, TGF-β1 treatment significantly induced the expression of FAK（P 〈 0.01 ） , but this induction was significantly blocked by Napsin A overexpression （ P 〈 0.01 ）. Conclusion It could partially block EMT by Napsin A gene transfection into A549 ceils, and the mechanism might be associated with the inhibition of integrin signaling pathway.

关 键 词：NAPSIN A 基因转染 A549细胞 上皮一间质转化 反转录一聚合酶链反应 免疫印迹法 

分 类 号：R563.9[医药卫生—呼吸系统]