IL-10-TGF-β/Smads通路在石英粉尘致肾脏纤维化中的作用及机制研究

Studies show that silica-exposed workers have higher mortality from kidney disease than the normal. However, the mechanism that how the inhaled silica induce kidney disease is hardly known. It has shown that no matter where and how the kidney injury happens, kidney fibrosis is almost the final step of all kidney disease. Our study will explore the effects and mechanism of IL-10-TGF-β/Smads in silica-induced kidney fibrosis. We will produce the IL-10, TGF-β and IL-10 together with TGF-β high expression renal mesangial cell models by plasmid transfection, and produce the IL-10, TGF-β and IL-10 together with TGF-β low expression renal mesangial cell models by SiRNA, respectively. Then the cell models are treated by two ways, one is stimulated directly by silica dust, the other one is treated with the culture supernatant of macrophages and silica dust. Then the level of epithelial mesenchymal transition(EMT) and fibrotic responses of these cell models will be measured, the dose-effect and the time – effect relationships of silica exposure and EMT will be definited. In animal experiments, the IL-10, TGF-β and IL-10 together with TGF-β neutralized animal models will be produced and instilled with silica suspension. We then observe and compare the pulmonary fibrosis with the renal fibrosis and verify the role of IL-10-TGF-β/Smads in silica-induced pulmonary and renal fibrosis. We also confirm the effects of IL-10-TGF-β/Smads in the silica-exposed workers with or without kidney disease by molecular epidemiology methods. This study will serve as clues to the mechanisms of the silica-induced extra-pulmonary injury.

石英粉尘导致工人肾脏疾病死亡率明显高于普通人群，但目前对入肺的石英粉尘导致肾损伤的机制研究非常缺乏。间质纤维化通常是肾脏疾病发展的最终环节。本项目拟研究IL-10-TGF-β/Smads在石英致肾纤维化中的作用及机制。采用质粒转染及SiRNA方法分别构建IL-10、TGF-β、IL-10合并TGF-β高表达和低表达肾系膜细胞模型，用不同剂量的石英直接刺激或通过巨噬细胞转染该细胞模型，测定其上皮-间质转化及纤维化程度，明确其剂量和时间-效应关联。分别构建IL-10、TGF-β、IL-10合并TGF-β消减的动物模型，观察石英肺灌注后，小鼠肾脏上皮-间质转化和纤维化程度，验证IL-10-TGF-β/Smads在石英致肾纤维化中的作用。采用分子流行病学方法，验证石英职业接触人群中合并和不合并肾脏疾病者IL-10、TGF-β及Smad的水平，为石英致肺外脏器损伤的机制研究提供线索。

以往研究表明经肺暴露的石英粉尘可以引起肾脏纤维化的发生，但是对于经肺暴露的石英粉尘如何导致肾脏纤维化的机制研究却比较缺乏。通常，间质纤维化是肾脏疾病发展的最终环节。本项目通过细胞实验和动物实验的方法研究了IL-10-TGF-β/Smads在经肺暴露的石英粉尘导致肾脏纤维化中的作用及机制。本项目主要通过细胞实验观察：（1）呼吸性石英粉尘对肾上皮细胞上皮-间质转化的影响；（2）拮抗TGF-β后，呼吸性石英粉尘对肾上皮细胞上皮-间质转化的影响；（3）IL-10和TGF-β重组蛋白对肾上皮细胞上皮-间质转化的影响。通过动物实验观察：（1）不同剂量和不同粒径的石英粉尘经肺暴露后，小鼠肾脏组织的上皮-间质转化以及肾脏纤维化情况；（2）阻断IL-10和TGF-β后，经肺暴露石英粉尘的小鼠肾脏上皮-间质转化以及肾脏纤维化情况。研究发现：（1）石英粉尘刺激肾上皮细胞后，可以通过对TGF-β/Smads 通路的调节导致肾上皮细胞的上皮-间质转化反应，进而促进肾上皮细胞出现纤维化反应；（2）石英粉尘刺激肾上皮细胞后，可以通过抑制TGF-β 来控制肾上皮细胞的上皮-间质转化反应，进而从一定程度上控制肾上皮细胞的纤维化反应；（3）外源性的IL-10和TGF-β可以促进肾上皮细胞上皮-间质的转化和纤维化指标的表达；（4）经肺暴露的石英粉尘剂量较高时，比较容易引发肾脏上皮-间质转化和纤维化形成，而小粒径的石英粉尘经肺暴露后，更容易引起肾脏组织的纤维化形成；（5）阻断IL-10和TGF-β可以从一定程度上控制经肺暴露的石英粉尘导致的上皮-间质转化程度和肾脏纤维化改变。本研究从体外细胞实验和动物体内实验探讨并证明了IL-10-TGF-β/Smads在经肺暴露的石英粉尘导致肾脏上皮-间质转化继而发生肾脏纤维化的过程中具有明显的作用，提示有可能以IL-10-TGF-β/Smads通路为靶点，从一定程度上预防控制石英暴露工人肾脏疾病的发生和发展。