NLRP3炎症小体活化对肺腺癌细胞A549增殖、迁移、侵袭的影响及其作用机制

发布时间：2018-09-08 21:30

【摘要】：【背景和目的】:原发性支气管肺癌是当今世界发病率和死亡率最高的恶性肿瘤之一,严重威胁人类的健康和生命。随着环境污染尤其是空气污染的加剧,我国肺癌发病率持续快速上升,且有低龄化趋势。尽管近年来手术、化疗、靶向治疗等综合治疗效果明显改善,但其5年生存率仍较低。因此除了传统的治疗手段外,肿瘤的免疫治疗以及肿瘤微环境的影响受到越来越多学者的关注。肿瘤相关性炎症已经成为肿瘤的“无限复制的潜能、促血管生成、对程序性细胞死亡的逃逸、生长信号的自给自足、对生长抑制剂的不敏感、组织侵犯和转移”之外的“第七大特征”[1]。炎症(inflammation)是机体抵御外界病原体侵袭以及各种化学、物理性损伤的免疫反应。慢性炎症可以促进肿瘤的发生和发展,并参与肿瘤细胞的发生、生长和转移的各病理过程。炎症小体(inflammasome)是由胞浆内模式识别受体(pattern recognition receptors,PRRs)参与组装的多蛋白复合物,能启动和参与炎症反应的核心环节。NLRP3(NOD-like receptor family pyrin domain containing 3)炎症小体是目前研究较多且能被多种刺激物活化的炎症小体。文献显示NLRP3炎症小体参与多种肿瘤(如黑色素瘤、肝癌、胃癌等)的进展过程,但在不同的肿瘤中发挥的作用不同甚至相反,具有一定的细胞或组织特异性。因此,本实验将研究NLRP3炎症小体活化对肺腺癌细胞株A549生物学特征的影响及其可能的作用机制,为临床治疗肺腺癌提供新的思路及理论基础。【方法】:1.我们用细胞免疫荧光、western blot、酶联免疫吸附试验(Enzyme-linked immunosorbent assay,ELISA)方法检测在A549细胞中NLRP3炎症小体是否能够活化。2.用Ed U增殖d{入方法检测NLRP3炎症小体活化后对A549细胞增殖的影响。用Annexin V/PI染色流式细胞仪检测NLRP3炎症小体活化后对A549细胞凋亡的影响。此外,用慢病毒干扰(sh NLRP3)方法稳定下调NLRP3表达,caspase-1抑制剂z-YVAD-FMK抑制caspase-1,IL-18结合蛋白(interleukin-18binding protein,IL-18BP)、IL-1受体拮抗剂(IL-1receptor antagonist,IL-1Ra)阻断IL-18、IL-1β信号通路,从这三个方面抑制NLRP3炎症小体的活性后,用Ed U增殖d{入方法观察细胞增殖情况的变化。用western blot方法检测p-Akt、p-GSK-3β、p-ERK1/2、p-CREB等信号通路的变化。3.用细胞划痕实验、transwell小室迁移实验检测NLRP3炎症小体活化后对A549细胞迁移的影响。用Matrigel侵袭实验检测NLRP3炎症小体活化后对A549细胞侵袭的影响。此外,从这三个方面抑制NLRP3炎症小体的活性:即用慢病毒干扰(sh NLRP3)方法稳定下调NLRP3表达,caspase-1抑制剂z-YVAD-FMK抑制caspase-1,IL-18结合蛋白(IL-18BP)、IL-1受体拮抗剂(IL-1Ra)阻断IL-18、IL-1β信号通路后,观察细胞迁移、侵袭情况的变化。用western blot方法检测Snail、E-cadherin等信号通路的变化。【结果】:1.使用经典的NLRP3炎症小体刺激剂LPS+ATP后,细胞免疫荧光显示在LPS+ATP组有较多的点状沉积代表NLRP3和接头蛋白ASC的结合,western blot显示活性形式的capspe-1 p10明显增高,ELISA显示细胞外IL-18、IL-1β明显增高,而在control组、LPS组、ATP组没有出现这些结果,说明LPS联合ATP可以在A549细胞中激活NLRP3炎症小体。2.LPS+ATP活化NLRP3炎症小体后促进了A549细胞增殖,p-Akt、p-GSK-3β、p-ERK1/2、p-CREB明显增高。在sh Ctrl细胞中,LPS+ATP同样促进细胞增殖及上调p-Akt、p-GSK-3β、p-ERK1/2、p-CREB,但在sh NLRP3细胞中,LPS+ATP的上述作用没有显现,说明通过sh NLRP3稳定下调NLRP3而抑制炎症小体活性后可以逆转LPS+ATP的作用。Caspase-1抑制剂z-YVAD-FMK部分抑制LPS+ATP的促增殖作用,使上调的p-Akt、p-GSK-3β、p-ERK1/2、p-CREB下降但仍高于control水平。单独使用IL-18BP或IL-1Ra均可部分抑制LPS+ATP的促增殖作用,IL-18BP对Akt/GSK-3β信号通路的影响相对更大,IL-1Ra对ERK1/2信号通路的影响相对更大,联合使用IL-18BP和IL-1Ra可以阻断LPS+ATP的促增殖作用。3.Annexin V/PI染色流式细胞仪检测结果显示NLRP3炎症小体活化对A549细胞的凋亡无明显影响。4.NLRP3炎症小体活化促进了A549细胞迁移、侵袭。5.NLRP3炎症小体对A549细胞迁移和侵袭的影响,可能的信号通路包括E-cadherin、Snail。LPS+ATP组,细胞E-cadherin表达下降,Snail表达升高。在sh Ctrl细胞中,LPS+ATP同样促进细胞迁移、侵袭及下调E-cadherin、上调Snail,但在sh NLRP3细胞中,LPS+ATP的上述作用没有显现,说明通过sh NLRP3稳定下调NLRP3而抑制炎症小体活性后可以逆转LPS+ATP的作用。Caspase-1抑制剂z-YVAD-FMK部分抑制LPS+ATP的促迁移、促侵袭作用,但使下降的E-cadherin上调至control水平、升高的Snail下调至control水平,而只是部分抑制了p-JNK、p-p38、p-ERK1/2的丝裂原活化蛋白激酶(Mitogen activated protein kinases,MAPK)信号通路。单独使用IL-18BP或IL-1Ra均可部分抑制LPS+ATP的促迁移、促侵袭作用,使下降的E-cadherin上调、升高的Snail下调,但仍与control组有明显差异,而联合使用IL-18BP和IL-1Ra可以阻断LPS+ATP的促迁移、促侵袭作用,使E-cadherin、Snail变化至基础水平。【结论】:1.NLRP3炎症小体活化可以促进A549细胞增殖、迁移、侵袭,可能通过调节p-Akt、p-GSK-3β、p-ERK1/2、p-CREB、E-cadherin、Snail、MAPK等信号通路而发挥作用。2.NLRP3炎症小体可以作为调节肺腺癌增殖、迁移、侵袭的细胞内信号,可能成为肺癌潜在的治疗靶点。
[Abstract]:BACKGROUND AND OBJECTIVE: Primary bronchogenic carcinoma (PBC) is one of the most common malignant tumors with the highest morbidity and mortality in the world today, which seriously threatens human health and life. With the aggravation of environmental pollution, especially air pollution, the incidence of lung cancer in China continues to rise rapidly and tends to be younger. However, the 5-year survival rate is still low. Therefore, in addition to the traditional treatment, tumor immunotherapy and the influence of tumor microenvironment have attracted more and more scholars'attention. Inflammation is the body's immune response to external pathogens and various chemical and physical injuries. Chronic inflammation can promote the occurrence and development of tumors and participate in tumor cells. Inflammatory bodies (inflammasomes) are polyprotein complexes assembled by intracytoplasmic pattern recognition receptors (PRRs), which can initiate and participate in the core of inflammation. Inflammatory bodies of NLRP3 have been shown to be involved in the progression of various tumors, such as melanoma, hepatocellular carcinoma, gastric cancer, etc. However, they play different or even opposite roles in different tumors and have certain cell or tissue specificity. [Methods] 1. We detected NLRP3 inflammation in A549 cells by immunofluorescence, Western blot and enzyme-linked immunosorbent assay (ELISA). The effect of activation of NLRP3 inflammatory bodies on the proliferation of A549 cells was detected by Ed U proliferative d{entry method. The effect of activation of NLRP3 inflammatory bodies on the apoptosis of A549 cells was detected by Annexin V/PI staining flow cytometry. In addition, the expression of NLRP3 was stably down-regulated by lentivirus interference (sh NLRP3), and the expression of Caspase-1 inhibitor z-YVAD-1 was down-regulated by Caspase-1 inhibitor z-YVAD-PI. FMK inhibited caspase-1, IL-18 binding protein (IL-18BP) and IL-1 receptor antagonist (IL-1Ra) blocked IL-18 and IL-1 beta signaling pathways. After inhibiting the activity of NLRP3 inflammatory bodies from these three aspects, the proliferation of NLRP3 cells was observed by Ed U proliferating D {entry method. Changes of Akt, p-GSK-3beta, p-ERK1/2, p-CREB signaling pathways. 3. The effects of activation of NLRP3 inflammatory bodies on the migration of A549 cells were detected by cell scratch test and Transwell chamber migration test. Matrigel invasion test was used to detect the effects of activation of NLRP3 inflammatory bodies on the invasion of A549 cells. Inhibitory effect of Caspase-1 inhibitor z-YVAD-FMK on caspase-1, IL-18 binding protein (IL-18BP) and IL-1 receptor antagonist (IL-1Ra) on IL-18 and IL-1 beta signaling pathways was observed. The changes of cell migration and invasion were detected by Western blot. Changes of pathway. [Results] 1. After the use of LPS + ATP, the classical inflammation body stimulator of NLRP3, more dot-like deposits in LPS + ATP group represented the binding of NLRP3 and adaptor protein ASC. Western blot showed that the active form of capspe-1 P10 was significantly increased. ELISA showed that extracellular IL-18 and IL-1 beta were significantly increased, while in control group, more dot-like deposits represented the binding of NLRP3 and adaptor protein ASC. LPS + ATP could activate NLRP3 inflammation corpuscle in A549 cells. 2. LPS + ATP activated NLRP3 inflammation corpuscle promoted A549 cell proliferation, p-Akt, p-GSK-3beta, p-ERK1/2, p-CREB increased significantly. LPS + ATP also promoted cell proliferation and up-regulated p-Akt, p-GSK-3beta, p-ERK1/2, p-CREB in SH Ctrl cells. However, the above-mentioned effects of LPS+ATP did not appear in SH NLRP3 cells, suggesting that the inhibition of inflammatory corpuscle activity by stably down-regulating NLRP3 by SH NLRP3 could reverse the effect of LPS+ATP. Caspase-1 inhibitor z-YVAD-FMK partially inhibited the proliferation of LPS+ATP, resulting in the up-regulation of p-Akt, p-GSK-3beta, p-ERK1/2 and p-CREB levels. IL-18BP or IL-1Ra alone can partially inhibit the proliferation of LPS+ATP. IL-18BP has a greater effect on Akt/GSK-3 beta signaling pathway, and IL-1Ra has a greater effect on ERK1/2 signaling pathway. Combined use of IL-18BP and IL-1Ra can block the proliferation of LPS+ATP. 3. Annexin V/PI staining flow cytometry showed that NLRP could inhibit the proliferation of LPS+ATP. Activation of NLRP3 promotes migration of A549 cells. Invasion of NLRP3 promotes migration and invasion of A549 cells. Possible signaling pathways include E-cadherin, Snail.LPS+ATP, decreased expression of E-cadherin and increased expression of Snail. TP also promoted cell migration, invasion and down-regulation of E-cadherin and up-regulation of Snail, but LPS+ATP did not show the above effect in SH NLRP3 cells, suggesting that inhibiting the activity of inflammatory corpuscles by stably down-regulating NLRP3 could reverse the effect of LPS+ATP. Caspase-1 inhibitor z-YVAD-FMK partially inhibited the migration of LPS+ATP and promoted the invasion of SH NLRP3 cells. The decreased E-cadherin was up-regulated to the control level, the elevated Snail was down-regulated to the control level, but only partially inhibited the mitogen-activated protein kinases (MAPK) signaling pathway of p-JNK, p-p38, p-ERK1/2. The combination of IL-18BP and IL-1Ra can block the migration and invasion of LPS+ATP, and make E-cadherin and Snail change to the basic level. [Conclusion] 1. The activation of NLRP3 inflammatory bodies can promote the proliferation, migration and invasion of A549 cells, possibly through the regulation of P. N-Akt, p-GSK-3beta, p-ERK1/2, p-CREB, E-cadherin, Snail, MAPK and other signaling pathways play a role.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R734.2

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