圆柱瘤基因（CYLD）的增强表达介入Fas/FasL途径诱导人肺癌细胞株A549/H460坏死的研究

目的探讨圆柱瘤基因（CYLD）在肺癌细胞株A549/H460的表达,及增强其表达对Fas/FasL途径诱导肺癌细胞死亡的影响,并探讨其可能的机制。方法选取5例肺腺癌手术患者癌组织标本作为实验组,自身癌旁正常组织作为对照组,进行CYLD基因实时荧光PCR定量分析;肺癌细胞株A549/H460分别进行CYLD基因增强质粒转染,同样采取实时荧光定量PCR及统计学检验对转染效果进行分析;转染前后的A549/H460细胞株FasL膜蛋白刺激进行培养24 h,通过流式细胞分析细胞死亡情况。结果 5例癌组织和癌旁正常组织均有CYLD表达,且差异有统计学意义（P〈0.05）,对照组为实验组的2.53倍;CYLD基因在A549细胞的表达较转染前高1.40倍,在H469细胞则比转染前高1.83倍;A549/H460细胞的坏死率较转染前增加,尤其是在提前加入zVAD-fmk后进行Fas信号诱导坏死率增加显著。结论肺癌细胞CYLD基因的表达较正常肺组织下调,有效增强肺癌细胞株A549/H460的CYLD基因表达后,FasL膜蛋白可通过Fas信号诱导其显著坏死。

The research of Fas/FasL pathway in human lung cancer A549/H460 cell lines with up-regulated gene expression of CYLD inducing necrosis

Objective To investigate the expression level of tumor-suppressing gene CYLD in human lung carcinoma and the effect of programmed cell death induced by Fas/FasL pathway in human lung cancer NCI-A549/H460 cell lines complemented with exogenous FLAG-CYLD.Methods The expression level of endogenous CYLD gene with cancerous tissue of five operative specimens in lung cancer was assessed by real-time QPCR,and adjacent normal lung tissue as control.The recombinate vector pcDNA3.0-FLAG-CYLD was transfected into NCI-A549/H460 cell lines.FLAG-CYLD+/+ NCI-A549/H460 cells were selected by culture media containing G418 and continued to perform culturing.The express level of CYLD gene after transfection to untransfected NCI-A549/H460 by real-time QPCR were compared.Mem-FasL was added and incubated respectively with FLAG-CYLD+/+ and FLAG-CYLD-/-A549/H460 for 24 h.In addition to above design,caspase inhibitor z-VAD-FMK was mixed in cell culture fluid for 30 min prior to induction by mem-FasL.The programmed cell death rates were assessed by flow cytometry(FCM).Results Endogenous CYLD gene expression was higher in cancerous tissue than adjacent normal lung tissue by 2.53 times;the differences were statistically significant(P < 0.05).After exogenous FLAG-CYLD transfection,the value of CYLD expression was higher than before by 1.40 times and 1.83 times each in A549/H460 cell lines;the rate of necrosis induced by Fas signal was increse after transfecting FLAG-CYLD in A549/H460 cell lines.Conclusion The gene expression of CYLD with adjacent cancerous tissue of the operative specimens in lung cancer is down-regulated.The change in gene expression is distinctly upregulated after the FLAG-CYLD plasmid transfected into NCI-A549/H460 cell line.The necrosis is observed obviously and enhanced induced by Fas signal with mem-FasL in FLAG-CYLD(+) A549/H460 cell lines.