IL-33/TLRs信号通路在A549细胞上皮-间质转化中的作用
摘要
目的 观察生长转化因子β1（TGF-β1）对上皮来源的A549细胞增殖及相关细胞标记物的影响，探讨IL-33/TLR4信号通路在上皮来源的A549细胞上皮-间质转化中的作用。
方法 培养A549细胞，用5ng/ml的TGF-β1刺激A549细胞；在不同时间点，MTT法检测TGF-β1对A549细胞增殖的影响；Real-time PCR方法检测IL-33/TLR4信号通路中关键因子IL-33，TLR4、PI3K基因的表达改变；western blot方法检测α-SMA， E-cad， p-AKT蛋白的动态表达。
结果 （1）.MTT结果显示，各组细胞生长良好，随时间的延长，细胞数量逐渐增多，但加入5ng/ml TGF-β1刺激12h，24h，48h后，A549细胞的增值与对照组相比，无统计学意义（P>0.05）。
（2）.在5ng/ml的TGF-β1刺激下，A549细胞与对照组相比，E-cad蛋白表达量逐渐下降（P<0.05），而α-SMA的表达呈逐渐上升的趋势（P<0.05）。提示，A549细胞上皮细胞特征逐渐减少，而间质细胞特征逐渐增多，即A549细胞发生了上皮-间质转化。
（3）. Real-time PCR结果显示，经5ng/ml的TGF-β1处理的A549细胞与对照组相比，在12h、24h、48h，IL-33/TLR4信号通路的关键基因IL-33、TLR4均呈先升高后下降的趋势，在24h时表达量最高，差异有统计学意义(P<0.05)。 （4）.western blot结果显示，经5ng/ml的TGF-β1处理的A549细胞，随时间的延长，在12h、24h、48h，p-AKT的表达量逐渐升高，与对照组相比，差异有统计学意义(P<0.05)。
结论 （1）5ng/ml的TGF-β1刺激A549细胞后，上皮细胞标志性蛋白E-Cad表达逐渐降低，间充质细胞标志性蛋白α-SMA表达逐渐升高，即TGF-β1诱导A549细胞过程中存在上皮-间质转化（EMT），提示EMT与纤维化病变密切相关。 （2） 在TGF-β1诱导的A549细胞上皮间质转化过程中，存在IL-33、TLR4等的过表达，提示IL-33/TLR4信号通路发挥了作用。
（3） 在EMT过程中，PI3K/AKT信号通路被激活，TLR4能与PI3K结合，而
TLR4的激活依赖于IL-33，故推测PI3K/AKT信号通路的激活与IL-33的过表达有关。
（4）EMT过程中，存在IL-33、TLR4呈先升高后下降的趋势，提示肺组织受损伤后，IL-33作为警报素，启动肺组织的自我修复，参与了肺纤维化的形成。
关键词 上皮-间质转化 (EMT)；白细胞介素-33（IL-33）； Toll样受体（TLRs）；磷脂酰肌醇-3-激酶（PI3K）。
Abstract
Objective: To investigate the effects of A549 cells on the cell proliferation of epithelial origin and related cell markers by TGFβ1.To investigate the role of IL-33/TLR4 signaling pathway in A549 cells in epithelial mesenchymal transition Methods: Cultured A549 cells were treated by TGFβ1 (5ng/ml).The effect of proliferation of A549 was detected by MTT at different time. The expression of the key factor IL-33、TLR4、PI3K in the IL-33/TLR4 signal pathway was determined by Real-time PCR. The dynamic protein expression of α-SMA、E-cad、p-AKT was determined by western blot analysis. Results:
(1) MTT results showed that the cells grew well in every group. With the prolongation of the time, the cells gradually increased in number. But with the addition of TGFβ1(5ng/ml) stimulates 12h、24h、48h. The proliferation of the A549 cells has no statistical significance compared with the control group（P>0.05）.
(2)With the stimulation of the TGFβ1(5ng/ml).The expression of the epithelial cell marker protein E-cad decreased gradually（P<0.05）.But the expression of α-SMA showed an increasing tendency（P<0.05）.The results suggested that the characteristics of the epithelial cells decreased gradually. But the characteristics of the mesenchymal cells increased gradually. Epithelial mesenchymal transition happened in the A549 cells.
(3)The results of the Real-time PCR showed that the expression of the IL-33 and TLR4 in the IL-33/TLR4 signal pathway of the A549 cells treated by TGFβ1 (5ng/ml) were first increased and then decreased In 12h, 24h, 48h compared with the control group. The expression in the 24h was the highest, the difference was statistically significant (P<0.05).
(4) The results of the western blot showed that the expression of the p-AKT in the A549 cells which is treated by TGFβ1 (5ng/ml) increased gradually compared with the control group with the extension of time(in 12h, 24h, 48h).The difference was
statistically significant(P<0.05). Conclusion:
(1) After the stimulation of the TGFβ1 in the A549 cells. The expression of the epithelial cell marker protein E-cad decreased gradually. But the expression of the Mesenchymal cell marker protein α-SMA increased gradually. The results showed that epithelial mesenchymal transition is induced by TGFβ1 in A549 cell. It confirmed that epithelial mesenchymal transition is closely related to pulmonary fibrosis. (2) In the process of epithelial mesenchymal transition induced by TGFβ1 in A549 cell. The expression of IL-33, TLR4, p-AKT increased, It was inferred that in the process of the epithelial cell injury and the excessive repair of the mesenchymal cell, the over expression of IL-33 and TLR4 played a role in IL-33/TLR4 signaling pathway.
(3)The expression of the p-AKT increased gradually with the extension of the time. So in the process of epithelial mesenchymal transition, PI3K/AKT signaling pathway is activated. TLR4 can bind to PI3K and the activation of TLR4 depended on IL-33.It was inferred that the activation of the PI3K/AKT pathway was associated with the overexpression of IL-33.
(4) With the extension of time, IL-33 and TLR4 increased first and then decreased. Combined with our previous study, we speculated that when lung was injured, IL-33 played a role of warning. It started the repair of lung tissue. With the extension of the time, the effect of the IL-33 decreased. TLR4 which is the downstream of the IL-33 also increased firstly and then decreased, and the activation of PI3K/AKT pathway presents the cascade. The downstream molecules gradually activated, ultimately involved in the process of pulmonary fibrosis.
Keywords:epithelial mesenchymaltransition(EMT);Interleukin-33(IL-33);Toll like receptors (TLRs);phosphatidylinositol -3- kinase(PI3K).
第一章 绪论
1 间质性肺病的概述及研究进展
间质性肺疾病（ILD，interstitial lung disease），又称为弥漫性肺实质性疾病(diffuse parenchymal lung disease，DPLD)，是由多组疾病组成的一类不同性质的疾病，包括200多个病种。基本病理变化为弥漫性肺实质、肺泡炎症和间质纤维化，临床上主要表现为X线胸片弥漫性浸润阴影、进行性加重的呼吸困难、限制性通气障碍、弥散功能降低和低氧血症。目前国际上将ILD/ DPLD分为四类：(1)已知病因的间质性肺疾病：如药物，结缔组织疾病相关的肺部病变等；(2) 少见的间质性肺疾病，如慢性嗜酸性粒细胞性肺炎、朗格罕斯细胞肉芽肿病、淋巴管平滑肌瘤病、肺出血-肾炎综合征即Goodpasture 综合征等； (3) 肉芽肿性间质性肺疾病，如外源过敏性肺泡炎、结节病、Wegenerr肉芽肿等； (4)特发性间质性肺炎（idiopathic interstitial pneumonia，IIP）：寻常性/特发性即UIP/IPF、脱屑性间质性肺炎（DIP）、非特异性间质性肺炎（NSIP）、淋巴细胞性间质性肺炎（LIP）、急性间质性肺炎（AIP）、呼吸性细支气管炎性间质性肺病（RB-ILD）、隐源性机化性肺炎（COP）[1]。
特发性肺纤维化（IPF，idiopathic pulmonary fibrosis）是最常见的肺间质疾病，也是肺间质纤维化的主要原因。2011年美国胸科学会（ATS），欧洲呼吸学会（ERS），日本呼吸学会（JRS），拉丁美洲胸科学会（ALAT）共同发表了关于IPF的最新研究概况的循证指南，为IPF的诊疗提供了更加完善的方案。IPF是一种不明原因的、慢性、进展性的肺部弥漫性疾病，病变部位局限于肺组织，主要表现为呼吸困难和肺功能的障碍。该指南提出诊断IPF时，需排除其他已知原因的引起的ILD，同时强调了高分辨率CT（HRCT）在IPF诊断中的重要性[2]。疾病的发展是一个动态过程，在临床上需结合症状、体征、职业史、用药史、环境因素、影像学检查、肺功能、病理活检、支气管肺泡灌洗等相关检查，对IPF进行全面细致的诊断。由于诊断技术的不断提高，IPF的检出率不断增加，但目前的治疗手段并未获得满意的疗效。在药物治疗治疗方面，指南提出目前尚
IL-33TLRs信号通路在A549细胞上皮-间质转化中的作用
