代谢因素对肺癌细胞侵袭转移能力的影响

发布时间：2018-01-22 09:44

本文关键词： 肺癌高糖高脂侵袭转移　出处：《北京协和医学院》2017年博士论文　论文类型：学位论文

【摘要】：背景肺癌是一种患病率和死亡率均较高的恶性疾病,影响肺癌患者预后的因素包括TNM分期、临床参数、分子学特征等。有临床研究显示代谢因素如合并有糖尿病、血脂异常也可能影响肺癌患者的预后。考虑到代谢性疾病在人群中的高患病率,我们十分有必要明确代谢因素对肺癌细胞侵袭转移能力的影响。材料和方法本研究使用非小细胞肺癌细胞系A549,使用含不同浓度葡萄糖的培养基以及含不同种类不同浓度脂肪酸的培养基以分别模拟高糖和高脂环境来培养A549细胞。通过transwell实验和检测细胞转移相关蛋白MMP9和CD147表达水平的变化来反映高糖或高脂培养后A549细胞侵袭转移能力的变化。以MTS实验检测高糖或高脂对A549细胞活性的影响。之后使用高通量转录组测序技术研究高糖和高脂条件处理后A549细胞转录组的变化,分析这两种条件影响A549细胞侵袭转移能力和细胞活性的背后机制。结果我们通过transwell实验发现高葡萄糖环境使A549细胞侵袭能力增强,western blot实验显示高糖培养使A549细胞转移相关蛋白MMP9、CD147的表达稍有增加,但变化不显著。棕榈酸和油酸会减弱A549细胞的侵袭能力,亚油酸和A549细胞侵袭能力无明显相关。这3种脂肪酸中棕榈酸可使A549细胞转移相关蛋白MMP9和CD147表达显著减少,而不同浓度的油酸和亚油酸未使A549细胞转移相关蛋白的表达发生显著变化。另外棕榈酸培养使A549细胞活性降低,而高糖培养以及油酸和亚油酸在400μmol/L以内培养一代细胞,细胞活性无显著变化。之后我们对不同浓度葡萄糖和不同浓度棕榈酸处理过的A549细胞进行转录组测序,在前者的3个样本中找到梯度表达的差异表达基因396个,主要富集在细胞粘附、应激反应、有丝分裂等过程和NO-cGMP-PKG、肿瘤相关蛋白多糖通路等。在后者的4个样本中找到梯度表达的差异表达基因254个,主要富集在细胞凋亡、细胞迁移、炎症反应等过程和凋亡、炎症反应、MAPK、粘附相关信号通路。结论高糖环境使A549细胞侵袭转移能力增强。使用棕榈酸模拟高脂环境可以使A549细胞侵袭转移能力和细胞活性均下降。油酸也可能使A549细胞侵袭转移能力下降,而亚油酸对A549细胞的侵袭转移能力无显著影响。转录组测序分析显示高糖环境对A549细胞的影响可能是通过细胞粘附、NO-cGMP-PKG、肿瘤相关蛋白多糖通路,棕榈酸对A549细胞的影响可能是通过凋亡、炎症、MAPK、粘附相关通路。
[Abstract]:Background Lung cancer is a malignant disease with high morbidity and mortality. The prognostic factors of lung cancer include TNM staging and clinical parameters. Molecular characteristics. Clinical studies have shown that metabolic factors such as diabetes mellitus, dyslipidemia may also affect the prognosis of lung cancer patients. Considering the high prevalence of metabolic diseases in the population. It is necessary for us to clarify the effect of metabolic factors on the invasion and metastasis of lung cancer cells. Materials and methods the non-small cell lung cancer cell line A549 was used in this study. A549 cells were cultured in medium containing different concentrations of glucose and different kinds of fatty acids to simulate high glucose and high fat environment respectively. Transwell experiments and detection were carried out. The expression levels of MMP9 and CD147 in A549 cells were detected by MTS assay. The changes of invasion and metastasis ability of A549 cells after high glucose or high fat culture were detected by MTS assay. High throughput transcriptome sequencing was used to study the transcriptional changes of A549 cells treated with high glucose and high fat. The underlying mechanism of these two conditions affecting the invasion and metastasis ability and cell activity of A549 cells was analyzed. Results We found that high glucose environment enhanced the invasion ability of A549 cells by transwell assay. . Western blot assay showed that high glucose culture increased the expression of metastasis associated protein MMP9 / CD147 in A549 cells. Palmitic acid and oleic acid decreased the invasion ability of A549 cells. There was no significant correlation between linoleic acid and invasion ability of A549 cells. Palmitic acid could significantly reduce the expression of MMP9 and CD147 in A549 cells. However, different concentrations of oleic acid and linoleic acid did not significantly change the expression of metastasis related protein in A549 cells. In addition, palmitic acid culture decreased the activity of A549 cells. The cells were cultured in high glucose culture, oleic acid and linoleic acid within 400 渭 mol/L. There was no significant change in cell activity. Then we sequenced the transcriptional sequence of A549 cells treated with different concentrations of glucose and palmitic acid. A total of 396 differentially expressed genes were found in the three samples of the former, mainly concentrated in cell adhesion, stress response, mitosis and NO-cGMP-PKG. In the latter 4 samples, 254 differentially expressed genes were found, mainly concentrated in apoptosis, cell migration, inflammatory reaction and apoptosis, inflammatory reaction. MAPK. Conclusion High glucose environment can enhance the invasion and metastasis ability of A549 cells. Using palmitic acid to simulate high fat environment can decrease the invasion and metastasis ability and cell activity of A549 cells. Oleic acid can also increase the invasion and metastasis ability of A549 cells. The ability of invasion and metastasis of A549 cells was decreased. However, linoleic acid had no significant effect on the invasion and metastasis of A549 cells. Transcriptome sequencing showed that the effect of high glucose environment on A549 cells was probably through cell adhesion to NO-cGMP-PKG. The effect of palmitic acid on A549 cells may be mediated by apoptosis, inflammatory MAPK and adhesion pathway.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R734.2

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