【摘要】 目的探讨非小细胞肺癌（NSCLC）中Neuritin的异常表达对肺血管内皮细胞的生物学作用及其作用机制。方法选择2017年9月至12月新疆维吾尔自治区人民医院NSCLC患者手术切除的癌组织5～10例,提取原代人NSCLC血管内皮细胞（NSCLC-VECs）,原代培养经病理确诊的NSCLC患者NSCLC-VECs细胞,选择CD34及FactorⅧ经免疫组化鉴定。利用荧光定量PCR（QPCR）及Western-blotting检测NSCLC-VECs及人肺微血管内皮细胞株（HPMECs）中Neuritin的表达水平。构建过表达/敲低Neuritin的NSCLC-VECs、HPMEC细胞系及阴性对照组,MTT、细胞划痕、Transwell小室、流式细胞术分别检测转染干扰/过表达Neuritin对细胞增殖及转移能力、细胞周期、细胞凋亡的影响;扫描电镜观察转染后细胞形态的改变。结果 NSCLC-VECs细胞中的Neuritin表达显著高于HPMEC细胞（P <0. 01）。过表达Neuritin后HPMEC和NSCLC-VECs细胞内Notch1、VEGFR蛋白和mRNA表达均显著升高（P <0. 01）;细胞体外增殖、划痕愈合及体外迁移能力较阴性对照组显著增强（P <0. 05）;电镜下观察细胞表面伪足明显,细胞间粘连增加;同时,过表达Neuritin促进了HPMEC和NSCLC-VECs细胞周期进程,并抑制细胞凋亡（P <0. 001）。反之,干扰HPMEC和NSCLC-VECs细胞内Neuritin表达后,胞内Notch1、VEGFR蛋白和mRNA表达均受到显著抑制（P <0. 01）;电镜下观察细胞表面光滑,细胞体外增殖活性、划痕愈合能力及迁移能力均较阴性对照组显著降低（P <0. 01）; HPMEC和NSCLC-VECs细胞周期阻滞于G₀/G₁期,细胞凋亡率显著升高（P <0. 001）。结论Neuritin可能作为NSCLC潜在的生物标记物,在NSCLC的发生发展中发挥一定的生物学功能。更多还原

【Abstract】 Objective To investigate the biological effects and mechanism of neuritin abnormal expression on pulmonary vascular endothelial cells in non-small cell lung cancer（ NSCLC）. Methods 5-10 patients with NSCLC underwent surgery in the Xinjiang Uygur Autonomous Region People’s Hospital from September to December 2017 were selected to extract primary human non-small cell lung cancer vascular endothelial cells（ NSCLC-VECs）,the primary cultured vascular endothelial cells of lung cancer patients with pathologically confirmed NSCLC were identified by immunohistochemistry with CD34 and Factor Ⅷ. The expression levels of neuritin in NSCLC-VECs and human pulmonary microvascular endothelial cells（ HPMECs） were detected by quantitative PCR（ QPCR） and Western-blotting. NSCLC-VECs and HPMEC cells were constructed to knock down and over express neuritin cells. MTT,cell scratch,Transwell chamber and flow cytometry were used to detect the effects of neuritin on cell proliferation and metastasis,cell cycle and apoptosis. The morphological changes of cells after transfection were observed by scanning electron microscope. Results The expression of neuritin in NSCLC-VECs cells was significantly higher than that in HPMEC cells（ P < 0. 01）. After overexpression of neuritin,the Notch1 and VEGFR protein and mRNA expression in HPMEC and NSCLC-VECs cells increased significantly（ P < 0. 01）. Cell proliferation,scratch healing and migration in vitro were significantly enhanced compared with the negative control group（ P < 0. 05）. Cell surface pseudo foot was obvious,and the adhesion between cells increased. At the same time,overexpression of neuritin promoted cell cycle progression and inhibited cell apoptosis of HPMEC and NSCLC-VECs（ P < 0. 001）. Conversely,after interfering with si-neuritin expression in HPMEC and NSCLC-VECs cells,the Notch1,VEGFR protein and mRNA expression was significantly inhibited（ P <0. 01）. Under electron microscopy,the cell surface was smooth,the cell proliferation activity,scratch healing ability and migration ability in vitro were significantly lower than those in the negative control group（ P < 0. 01）. HPMEC and NSCLC-VECs cell cycle arrested in G₀/G₁ phase,and apoptosis rate increased significantly（ P < 0. 001）. Conclusion Neuritin may be a potential biomarker of NSCLC,and it play a certain biological role in the genesis and development of NSCLC.更多还原