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采用慢病毒载体质粒PLJM1将NapsinA基因转染到人肺腺癌细胞——A549细胞中,获得稳定表达Napsin A蛋白的特性并鉴定,通过转化生长因子-β1刺激A549细胞发生上皮-间质转化,体外构建上皮-间质转化模型并鉴定。MTT法检测转基因前后A549细胞在上皮-间质转化过程中生长速率的变化;流式细胞术检测其细胞周期的改变,最后予Western blot检测黏着斑激酶的表达情况,探讨Napsin A基因对A549细胞在上皮-间质转化过程中增殖的影响及其机制。结果表明转染后的A549细胞表达Napsin A蛋白明显增加(P<0.01);A549细胞发生上皮-间质转化后细胞E钙蛋白表达下调(P<0.01),Ⅰ型胶原表达上调(P<0.01);转基因细胞在体外上皮-间质转化模型中增殖速度减慢(P<0.05),且细胞周期被阻滞在G_1期(P<0.01),其表达整合素信号传导通路的基础分子——黏着斑激酶的量显著下降(P<0.01)。提示Napsin A基因可以抑制A549细胞在上皮-间质转化过程中的进一步增殖,其机制可能与抑制整合素信号传导通路有关。
The lentiviral vector plasmid PLJM1 was used to transfect NapsinA gene into human lung adenocarcinoma A549 cells. The stable expression of Napsin A protein was identified and identified. Transforming growth factor-β1 stimulated epithelial-mesenchymal transition of A549 cells, The epithelial-mesenchymal transition model was constructed in vitro and identified. MTT assay before and after transfection of A549 cells in the process of epithelial-mesenchymal transition growth rate changes; flow cytometry to detect cell cycle changes, and finally to Western blot detection of focal adhesion kinase expression, explore the Napsin A gene on A549 cells Proliferation in epithelial-mesenchymal transition and its mechanism. The results showed that the expression of Napsin A protein in transfected A549 cells was significantly increased (P <0.01). The expression of E-cadherin in A549 cells was down-regulated after epithelial-mesenchymal transition (P <0.01) (P <0.05). The cell cycle was arrested in G_1 phase (P <0.01), and the expression of integrin signaling pathway basic molecule - The amount of focal adhesion kinase decreased significantly (P <0.01). These results suggest that Napsin A can inhibit the proliferation of A549 cells in the process of epithelial-mesenchymal transition. The mechanism may be related to the inhibition of integrin signaling pathway.