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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 investigate the effect and mechanism of Napsin A gene transfection into A549 cells on epithelial-mesenchymal transition (EMT). Methods The lentiviral plasmid PLJM1 was used to construct the recombinant plasmid PLJM1-Napsin A. The Napsin A gene was transfected into the chromosome of A549 cells and identified. A549 cells were stimulated with transforming growth factor-β1 (TGF-β1) to construct EMT model. The morphological changes of A549 cells were observed under inverted microscope. The effect of Napsin A gene transfected A549 cells on the expression of EMT and EMT Effect of kinase (FAK). Results The sequence of the recombinant plasmid PLJM1-Napsin A was completely consistent with the designed sequence. The Napsin A protein expression in Napsin A gene A549 cells was significantly higher than that in non-transgenic cells (P <0.01). Cells stimulated by TGF-β1 morphologically evolved into interstitial cells, E-cadherin mRNA and protein expression was significantly decreased (P <0.01), while the type Ⅰ collagen was significantly increased (P <0.01), suggesting that in vitro EMT model Success. After transfection with TGF-β1, the Napsin A gene A549 cells changed their interstitial morphology, the expression of E-calpain and type Ⅰ collagen also showed a similar trend, but the change range was significantly smaller (E-calpain: P < 0.01, type Ⅰ collagen: P <0.05). In vitro EMT model, the expression of FAK protein increased (P <0.01), but the up-regulation of transgenic cells was significantly less than that of non-transgenic cells (P <0.01). Conclusion Transfection of Napsin A gene into A549 cells can partially block EMT progression and its mechanism may be related to the inhibition of integrin signaling pathway.