肿瘤细胞间质液微环境促肺癌机制和药物治疗

发布时间：2018-04-25 17:19

本文选题：肿瘤细胞间质液 + 微环境　；参考：《河南大学》2016年硕士论文

【摘要】：肿瘤的发生和发展必须有充足的营养供给,一般认为肿瘤的营养供给依赖于新生血管形成。然而,众所周知肿瘤新生血管具有畸形高通透性的特点,这种畸形新生血管很难完成充足的营养供给,临床上抗新生血管形成“饿死”肿瘤的疗法不仅没有达到预期的效果,甚至使肿瘤更加恶化的现象也证实了这个观点。相反,肿瘤细胞间质液因含有丰富的营养和细胞生长因子,恰好能够沐浴和支持肿瘤细胞生长,有可能是肿瘤发展的直接和主要营养来源,因此对肿瘤细胞间质液促癌作用的认识是肿瘤防治成功的关键。本研究以肺癌为研究对象探索肿瘤细胞间质液微环境促进肺癌发生和发展的机制以及相关的药物治疗,以期为肺癌防治提供有效的措施和方法。本研究首先从肿瘤组织中分离肿瘤细胞间质液并进行蛋白定量得到10μg蛋白质/毫升的肿瘤细胞间质液用于体内和体外试验。体外试验我们用肿瘤细胞间质液处理LLC细胞,流式细胞术检测肿瘤干细胞标志物CD133、Nanog和PCNA的表达,软琼脂克隆形成和细胞成球实验检测肿瘤细胞自我更新,MTT法检测肿瘤细胞增殖,免疫荧光技术检测肿瘤细胞上皮间质转化,细胞划痕实验检测肿瘤细胞迁移,Transwell实验检测肿瘤细胞侵袭,AO/EB荧光双染法检测肿瘤细胞自噬和凋亡。接着,我们采用气管滴注方式建立了小鼠原位肺癌共移植模型,通过免疫组化和Western Blot检测肺癌小鼠肺组织中肿瘤干细胞标志物Nanog,细胞增殖标志物PCNA,细胞水运载标志物水通道蛋白1,新生血管形成标志物CD31,低氧标志物低氧诱导因子HIF-1和碳酸酐酶IX,及上皮间质转化标志物N-cadherin、E-cadherin、Vimentin和Cytokeratin 18表达;同时,用伊文斯兰通透性和干湿重法检测肺癌小鼠肺组织血管通透性和肺组织含水量;用ELISA试剂盒检测肺癌小鼠血清中血管紊乱标志物5-HIAA,糖酵解标志物ROS、Lactate和Pyruvate,炎症标志物8-OHdG、TNF-α和hs-CRR。另外,我们用新生血管形成抑制剂Vadimezan、抗炎药物地塞米松、Na-K-ATP酶抑制剂地高辛、以及醛固酮受体阻断剂螺内酯治疗肿瘤细胞间质液共移植肺癌小鼠,观察其对肿瘤细胞间质液形成、新生血管形成、血管通透性及肿瘤生长的影响。体外试验结果表明,肿瘤细胞间质液处理后的LLC细胞肿瘤干细胞标志物CD133、Nanog和PCNA的表达增加,肿瘤细胞软琼脂克隆和细胞成球实验的克隆数增多,上皮间质转化增加,饥饿诱导的肿瘤细胞自噬和凋亡减少。小鼠原位癌实验结果显示,肿瘤细胞间质液与肿瘤细胞共移植后小鼠肺癌发生率升高,肺部肿瘤结节数增多,肺组织血管通透性增强,肺组织含水量增加;肺组织肿瘤干细胞标志物Nanog,细胞增殖标志物PCNA,细胞水运载标志物水通道蛋白1,低氧标志物低氧诱导因子HIF-1和碳酸酐酶IX,及上皮间质转化标志物N-cadherin和Vimentin表达升高,E-cadherin和Cytokeratin18表达降低;炎症标志物、糖酵解标志物和氧化损伤标志物水平升高;新生血管形成标志物CD31的表达没有显著变化。有趣的是,炎症反应抑制剂地塞米松、Na-K-ATP酶抑制剂地高辛和醛固酮受体阻断剂螺内酯能够降低肿瘤细胞间质液诱导的肺癌发生率,减少肺癌小鼠肺部肿瘤结节数,增加新生血管形成标志物CD31表达但降低血清中血管紊乱标志物5-HIAA水平;新生血管形成抑制剂Vadimezan虽然降低新生血管形成标志物CD31表达,却增加血清中5-HIAA水平,提高肺癌发生率,增加肺癌小鼠肺部肿瘤结节数;另外,我们发现肿瘤组织含水量升高时组织中5-HIAA和水通道蛋白1的表达增加,但是组织中CD31的表达无明显变化。综上所述,肿瘤细胞间质液能够促进小鼠肺癌发生和发展,其促肺癌作用与肿瘤细胞间质液诱导小鼠肺组织低氧微环境、糖酵解代谢、炎症反应和新生血管紊乱有密切联系,与正常新生血管形成无关。
[Abstract]:The occurrence and development of tumor must be provided with sufficient nutrition. It is generally believed that the nutritional supply of tumor depends on the formation of neovascularization. However, it is known that the neovascular neovascularization is characterized by high permeability, which is difficult to complete the sufficient nutrient supply and is clinically resistant to the formation of "starving" tumor. Not only does the method fail to achieve the desired effect, even the worsening of the tumor confirms this view. On the contrary, the tumor cell interstitial fluid, which is able to bathe and support tumor cell growth because of its rich nutrition and cell growth factors, may be the direct and main source of tumor development and therefore to the tumor cells. The understanding of the role of fluid promoting cancer is the key to the success of cancer prevention and control. This study aims to explore the mechanism of tumor cell interstitial fluid microenvironment to promote the occurrence and development of lung cancer and the related drug treatment, so as to provide effective measures and methods for the prevention and treatment of lung cancer. In vitro and in vitro, 10 g / ml of tumor cell fluid was used in vivo and in vitro. In vitro, we used tumor cell fluid to treat LLC cells. Flow cytometry was used to detect the expression of tumor stem cell markers CD133, Nanog and PCNA, soft agar Kuron formation and cell formation test for detection of tumor cells Self renewal, MTT assay was used to detect tumor cell proliferation, immunofluorescence technique was used to detect epithelial mesenchymal transition of tumor cells, cell scratch test was used to detect tumor cell migration, Transwell test was used to detect tumor cell invasion, and AO/EB fluorescence double staining method was used to detect autophagy and apoptosis of tumor cells. Cancer stem cell markers Nanog, cell proliferation marker PCNA, water channel protein 1, neovascularization marker CD31, hypoxia inducible hypoxia inducible factor HIF-1 and carbonic anhydrase IX, and N- of epithelial mesenchymal transition marker, were detected by immunohistochemistry and Western Blot in lung cancer mice. Cadherin, E-cadherin, Vimentin and Cytokeratin 18 were expressed, and the permeability of lung tissue and the water content of lung tissue in lung cancer mice were detected by Evans permeability and dry wet weight method. The blood vessel disorder markers in the serum of lung cancer mice were detected by ELISA kit 5-HIAA, the glycolysis markers ROS, Lactate and Pyruvate, and the inflammatory markers 8-OHdG, TN. F- alpha and hs-CRR., we used neovascularization as inhibitor Vadimezan, antiinflammatory drug dexamethasone, Na-K-ATP enzyme inhibitor digoxin, and aldosterone blocker spironolactone in the treatment of tumor cell interstitial lung cancer mice, and observed the formation of interstitial fluid, angiogenesis, vascular permeability and tumor growth of tumor cells. In vitro test results showed that the expression of LLC cell tumor stem cell markers CD133, Nanog and PCNA increased, the number of clones in the soft agar cloning and cell formation of tumor cells increased, the transformation of epithelial mesenchymal transition increased, and the autophagy and apoptosis of the swell cells induced by starvation were reduced. The results showed that the incidence of lung cancer in the tumor cells was increased, the number of lung nodules increased, the permeability of the lung tissue increased, the water content of the lung increased, the marker of the lung cancer stem cell Nanog, the marker of cell proliferation PCNA, the water channel protein 1 of the cell water carrier, the low oxygen marker The expression of oxygen inducible factor HIF-1 and carbonic anhydrase IX, and the expression of epithelial mesenchymal transition markers N-cadherin and Vimentin increased, the expression of E-cadherin and Cytokeratin18 decreased; inflammatory markers, glycolytic markers and oxidative damage markers increased; the expression of CD31 in the neovascularization marker did not change significantly. Interestingly, the inflammatory reaction was suppressed. Preparation of dexamethasone, Na-K-ATP inhibitor, digoxin and aldosterone blocker, spironolactone can reduce the incidence of lung cancer induced by tumor cell interstitial fluid, decrease the number of lung tumor nodules in lung cancer mice, increase the expression of neovascularization marker CD31, but reduce the level of 5-HIAA in blood vessel disorder markers, and inhibit the formation of neovascularization. Although the preparation of Vadimezan decreased the expression of the neovascularization marker CD31, it increased the level of 5-HIAA in the serum, increased the incidence of lung cancer and increased the number of lung tumor nodules in lung cancer mice. In addition, we found that the expression of 5-HIAA and aquaporin 1 increased in the tissue when the water content was elevated, but the expression of CD31 in the tissue was not significantly changed. In summary, the interstitial fluid of tumor cells can promote the development and development of lung cancer in mice. The role of lung cancer is closely related to the hypoxia microenvironment, glycolytic metabolism, inflammatory reaction and neovascular disorder induced by tumor cell interstitial fluid, and is not related to the formation of normal neovascularization.

【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R734.2

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