LKB1基因转染对人肺腺癌细胞生物学行为影响.doc

LKB1基因转染对人肺腺癌细胞生物学行为影响 　 作者：梁璇, 南克俊, 李春丽, 姚煜, 田涛, 王淑红 【摘要】 　　目的: 探讨外源性LKB1基因表达对人肺腺癌细胞生物学行为的影响。方法: 应用巢式PCR方法扩增LKB1基因编码区, 利用脂质体转染技术将真核表达重组体pcDNALKB1质粒和空载体pcDNA3.1质粒分别导入A549细胞, 经G418筛选后获得稳定转染细胞克隆, Western blot检测LKB1和STAT3在A549细胞中的表达变化。通过绘制生长曲线和克隆形成试验检测获得性表达LKB1基因后肺癌细胞的增殖变化, 流式细胞术(FCM)分析其细胞周期及其凋亡的变化。结果: 成功获得了稳定表达外源性LKB1基因的A549细胞系, 与转染空白载体组比较, 转染LKB1基因的细胞生长速度明显减慢, 细胞周期中G1/G0期比例明显增加, S期比例减少, 细胞凋亡率升高; 同时STAT3蛋白的磷酸化水平明显降低。 结论: LKB1可能通过下调STAT3蛋白磷酸化水平的表达从而抑制A549细胞的恶性生物学行为。 　　AIM: To determine the effects of exogenous LKB1 gene on biological behavior of lung carcinoma cells. METHODS: Nest PCR was used to clone LKB1 gene pcDNALKB1 and pcDNA3.1 were introduced into A549 cell line by lipofectin transfection, and the A549 cells stably expressing LKB1 gene were established by G418 selection. The expression of LKB1 and STAT3 protein was detected by Western blot. Cell proliferation was observed by growth curve and clone formation. Cell cycle and apoptosis were assayed by flow cytometry(FCM). RESULTS: A549 cells stably expressing LKB1 protein were established. Compared with the vector transfected cells, the positive clone cells grew more slowly. FCM showed that more positive clone cells went into phase G0/G1 and fewer cells went into phase S. Moreover, there was significant difference on apoptosis between the two group cells. CONCLUSION: LKB1 protein might inhibit malignant biological behavior of A549 cells partly by downregulating the tyrosinephosphorylated level of STAT3 protein. 【关键词】 LKB1; 转染; 增殖; 凋亡; 肿瘤; STAT3 　　LKB1/STK11 (serine theronine protein kinase 11) 基因是一种丝氨酸/苏氨酸激酶编码的蛋白, 于1998年首次在PeutzJeghers肿瘤综合症（PJS）患者中发现, 其胚系突变是PJS患者的主要致病因素, 也是易于罹患肿瘤的重要遗传学基础［1-3］。LKB1作为抑癌基因, 可能在肺癌的发生发展过程中发挥着重要作用, 但其机制尚未明确。为了对LKB1的功能进行更深入的研究, 我们以不表达LKB1蛋白的肺腺癌细胞株A549为研究对象, 成功地建立了表达外源性LKB1基因的细胞模型, 针对获得性表达外源性LKB1基因后A549细胞的生物学行为进行了研究, 进一步探讨其可能的作用机制, 为临床防治提供一定的线索。 　　1 材料和方法 　　1.1 材料 TRIzol、 逆转录试剂盒、 OptiMEMI培养基、 转染试剂Lipofectamine 2000、 G418购自Invitrogen公司; 质粒提取试剂盒购自Promega公司; 蛋白裂解液购自PI