【摘要】：【背景和目的】:原發(fā)性支氣管肺癌是當今世界發(fā)病率和死亡率最高的惡性腫瘤之一,嚴重威脅人類的健康和生命。隨著環(huán)境污染尤其是空氣污染的加劇,我國肺癌發(fā)病率持續(xù)快速上升,且有低齡化趨勢。盡管近年來手術(shù)、化療、靶向治療等綜合治療效果明顯改善,但其5年生存率仍較低。因此除了傳統(tǒng)的治療手段外,腫瘤的免疫治療以及腫瘤微環(huán)境的影響受到越來越多學者的關注。腫瘤相關性炎癥已經(jīng)成為腫瘤的“無限復制的潛能、促血管生成、對程序性細胞死亡的逃逸、生長信號的自給自足、對生長抑制劑的不敏感、組織侵犯和轉(zhuǎn)移”之外的“第七大特征”[1]。炎癥(inflammation)是機體抵御外界病原體侵襲以及各種化學、物理性損傷的免疫反應。慢性炎癥可以促進腫瘤的發(fā)生和發(fā)展,并參與腫瘤細胞的發(fā)生、生長和轉(zhuǎn)移的各病理過程。炎癥小體(inflammasome)是由胞漿內(nèi)模式識別受體(pattern recognition receptors,PRRs)參與組裝的多蛋白復合物,能啟動和參與炎癥反應的核心環(huán)節(jié)。NLRP3(NOD-like receptor family pyrin domain containing 3)炎癥小體是目前研究較多且能被多種刺激物活化的炎癥小體。文獻顯示NLRP3炎癥小體參與多種腫瘤(如黑色素瘤、肝癌、胃癌等)的進展過程,但在不同的腫瘤中發(fā)揮的作用不同甚至相反,具有一定的細胞或組織特異性。因此,本實驗將研究NLRP3炎癥小體活化對肺腺癌細胞株A549生物學特征的影響及其可能的作用機制,為臨床治療肺腺癌提供新的思路及理論基礎�！痉椒ā�:1.我們用細胞免疫熒光、western blot、酶聯(lián)免疫吸附試驗(Enzyme-linked immunosorbent assay,ELISA)方法檢測在A549細胞中NLRP3炎癥小體是否能夠活化。2.用Ed U增殖d{入方法檢測NLRP3炎癥小體活化后對A549細胞增殖的影響。用Annexin V/PI染色流式細胞儀檢測NLRP3炎癥小體活化后對A549細胞凋亡的影響。此外,用慢病毒干擾(sh NLRP3)方法穩(wěn)定下調(diào)NLRP3表達,caspase-1抑制劑z-YVAD-FMK抑制caspase-1,IL-18結(jié)合蛋白(interleukin-18binding protein,IL-18BP)、IL-1受體拮抗劑(IL-1receptor antagonist,IL-1Ra)阻斷IL-18、IL-1β信號通路,從這三個方面抑制NLRP3炎癥小體的活性后,用Ed U增殖d{入方法觀察細胞增殖情況的變化。用western blot方法檢測p-Akt、p-GSK-3β、p-ERK1/2、p-CREB等信號通路的變化。3.用細胞劃痕實驗、transwell小室遷移實驗檢測NLRP3炎癥小體活化后對A549細胞遷移的影響。用Matrigel侵襲實驗檢測NLRP3炎癥小體活化后對A549細胞侵襲的影響。此外,從這三個方面抑制NLRP3炎癥小體的活性:即用慢病毒干擾(sh NLRP3)方法穩(wěn)定下調(diào)NLRP3表達,caspase-1抑制劑z-YVAD-FMK抑制caspase-1,IL-18結(jié)合蛋白(IL-18BP)、IL-1受體拮抗劑(IL-1Ra)阻斷IL-18、IL-1β信號通路后,觀察細胞遷移、侵襲情況的變化。用western blot方法檢測Snail、E-cadherin等信號通路的變化。【結(jié)果】:1.使用經(jīng)典的NLRP3炎癥小體刺激劑LPS+ATP后,細胞免疫熒光顯示在LPS+ATP組有較多的點狀沉積代表NLRP3和接頭蛋白ASC的結(jié)合,western blot顯示活性形式的capspe-1 p10明顯增高,ELISA顯示細胞外IL-18、IL-1β明顯增高,而在control組、LPS組、ATP組沒有出現(xiàn)這些結(jié)果,說明LPS聯(lián)合ATP可以在A549細胞中激活NLRP3炎癥小體。2.LPS+ATP活化NLRP3炎癥小體后促進了A549細胞增殖,p-Akt、p-GSK-3β、p-ERK1/2、p-CREB明顯增高。在sh Ctrl細胞中,LPS+ATP同樣促進細胞增殖及上調(diào)p-Akt、p-GSK-3β、p-ERK1/2、p-CREB,但在sh NLRP3細胞中,LPS+ATP的上述作用沒有顯現(xiàn),說明通過sh NLRP3穩(wěn)定下調(diào)NLRP3而抑制炎癥小體活性后可以逆轉(zhuǎn)LPS+ATP的作用。Caspase-1抑制劑z-YVAD-FMK部分抑制LPS+ATP的促增殖作用,使上調(diào)的p-Akt、p-GSK-3β、p-ERK1/2、p-CREB下降但仍高于control水平。單獨使用IL-18BP或IL-1Ra均可部分抑制LPS+ATP的促增殖作用,IL-18BP對Akt/GSK-3β信號通路的影響相對更大,IL-1Ra對ERK1/2信號通路的影響相對更大,聯(lián)合使用IL-18BP和IL-1Ra可以阻斷LPS+ATP的促增殖作用。3.Annexin V/PI染色流式細胞儀檢測結(jié)果顯示NLRP3炎癥小體活化對A549細胞的凋亡無明顯影響。4.NLRP3炎癥小體活化促進了A549細胞遷移、侵襲。5.NLRP3炎癥小體對A549細胞遷移和侵襲的影響,可能的信號通路包括E-cadherin、Snail。LPS+ATP組,細胞E-cadherin表達下降,Snail表達升高。在sh Ctrl細胞中,LPS+ATP同樣促進細胞遷移、侵襲及下調(diào)E-cadherin、上調(diào)Snail,但在sh NLRP3細胞中,LPS+ATP的上述作用沒有顯現(xiàn),說明通過sh NLRP3穩(wěn)定下調(diào)NLRP3而抑制炎癥小體活性后可以逆轉(zhuǎn)LPS+ATP的作用。Caspase-1抑制劑z-YVAD-FMK部分抑制LPS+ATP的促遷移、促侵襲作用,但使下降的E-cadherin上調(diào)至control水平、升高的Snail下調(diào)至control水平,而只是部分抑制了p-JNK、p-p38、p-ERK1/2的絲裂原活化蛋白激酶(Mitogen activated protein kinases,MAPK)信號通路。單獨使用IL-18BP或IL-1Ra均可部分抑制LPS+ATP的促遷移、促侵襲作用,使下降的E-cadherin上調(diào)、升高的Snail下調(diào),但仍與control組有明顯差異,而聯(lián)合使用IL-18BP和IL-1Ra可以阻斷LPS+ATP的促遷移、促侵襲作用,使E-cadherin、Snail變化至基礎水平�！窘Y(jié)論】:1.NLRP3炎癥小體活化可以促進A549細胞增殖、遷移、侵襲,可能通過調(diào)節(jié)p-Akt、p-GSK-3β、p-ERK1/2、p-CREB、E-cadherin、Snail、MAPK等信號通路而發(fā)揮作用。2.NLRP3炎癥小體可以作為調(diào)節(jié)肺腺癌增殖、遷移、侵襲的細胞內(nèi)信號,可能成為肺癌潛在的治療靶點。
[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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R734.2

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