白细胞介素6诱导人血管平滑肌细胞成骨样分化的分子机制研究

Cardiovascular comorbidity is the leading cause of death in rheumatoid arthritis (RA), with vascular calcification being the typical pathological feature. However, the mechanism of vascular calcification remains unclear. Our previous study found that IL6 was the independent risk factor of vascular calcification in RA. It could induce the expression of osteoblast-specific regulatory factor BMP2 and the extracellular matrix calcium deposition of human vascular smooth muscle cells (VSMCs). The activation of IL6 soluble receptor mediated trans-signaling and Wnt5a signaling were possibly involved. Therefore, we hypothesize that IL6 may up-regulate the expression of Wnt5a through its soluble receptor, then transcript BMP2 by activating β-catenin/TCF or PKC/NFκB pathway and participate in the calcification of VSMC. In this study, the sgp130Fc fusion protein will be used to detect and verify the receptor pathway of IL6-induced VSMC calcification. The intracellular signaling will be explored by inhibiting or lowering the expression of those involving pathway molecules with small molecule inhibitors or corresponding shRNA lentiviral vectors. Then, the discovered signaling pathways will be studied and confirmed in the IL6 receptor knock out mouse model and human IL6 receptor knock in mouse model. Finally, the molecular mechanism of IL6-induced vascular calcification in RA will be revealed and will provide the basis for the early prevention and treatment of such diseases.

心血管并发症是类风湿关节炎（RA）的首位死因，血管钙化是其典型病理特征，但机制不清。我们前期发现，IL6是RA血管钙化独立危险因子；IL6体外可诱导人血管平滑肌细胞（VSMC）表达成骨细胞分化调控因子BMP2并发生钙化；该作用可能与IL6通过可溶性受体介导反式信号及Wnt5a通路活化有关。推测IL6通过其可溶性受体介导Wnt5a的表达，进而激活β-catenin/TCF或PKC/NFκB通路，启动BMP2的表达，诱发VSMC成骨样分化。本课题拟应用sgp130Fc阻断IL6可溶性受体，明确IL6的受体途径；应用小分子抑制剂及shRNA慢病毒载体阻断或低表达有关通路蛋白，明确IL6诱导VSMC成骨样分化的信号通路；应用IL6受体基因敲除和表达人IL6受体小鼠模型，对上述结果在整体水平进行验证，最终阐明IL6诱导血管钙化的分子机制。该研究将揭示RA血管钙化的机制，为该类疾病的早期防治提供依据。

背景：类风湿性关节炎（RA）是一种常见的风湿病，主动脉钙化是RA患者心血管疾病的典型病理特征，血清IL-6水平升高是RA患者发生血管钙化的独立危险因子，但机制不明。.主要研究内容：从体内实验和体外细胞实验两个部分出发。在细胞水平进行脐动脉平滑肌细胞培养及鉴定，分别给予IL-6、IL-6+可溶性IL-6受体（sIL-6R）、IL-6+sIL-6R+可溶性gp130（sgp130）刺激，茜素红及Von Kossa染色检测钙化水平，免疫印迹检测钙化相关分子TNAP、OPN、BMP2，转录因子Runx2以及Jak2/p-Jak2、Stat3/p-Stat3的表达，免疫荧光检测膜型IL6R（mIL6R）表达，给予JAK2抑制剂CP690550干预，免疫印迹检测Stat3/p-Stat3、Runx2 的表达，探讨IL-6诱导VSMC成骨样分化和钙化的受体途径和信号通路。在动物水平构建重组鼠IL-6 编码 AAV（AAV-IL-6），采用重复尾静脉注射鼠IL-6编码腺相关病毒的方法建立循环持续高表达IL-6 的慢性系统性炎症动物模型，并进行小鼠的一般状态记录、炎症指标、行为学表现测定，干预期结束后对小鼠多器官做苏木精-伊红染色和油红O染色观察组织炎症浸润情况，并在动物体内水平探讨 IL-6 诱导VSMC成骨样分化和钙化的分子机制。.重要结果：在细胞水平明确了IL-6主要通过sIL-6R介导的反式信号通路，激活细胞内Jak2/Stat3信号通路，上调脐动脉平滑肌细胞成骨分化关键分子BMP2、Runx2的表达，诱导脐动脉平滑肌细胞成骨样分化，并且促进骨细胞基质蛋白TNAP、OPN的表达和钙盐沉积，使脐动脉平滑肌细胞成骨分化及钙化。动物水平本研究成功构建了具有长期最佳循环IL-6水平的慢性系统性炎症小鼠模型，表现出基本的SCI相关表型，如病理行为学、急性期反应、骨质疏松症和肌肉减少症等，并在体内水平证明IL-6是诱导VSMC钙化的重要分子，其中Jak2/Stat3/Wnt5a可能是VSMC钙化的相关信号通路。.科学意义：本研究从细胞水平和动物水平，探讨了IL-6诱导VSMC成骨样分化的分子机制，为RA患者血管钙化的治疗提供了新的治疗靶点和思路。成功建立了一种慢性系统性炎症的小鼠模型，出现肌少症、骨质疏松表型，实现了在小鼠中长期循环稳定高表达IL-6，为相关研究提供了可用的模型动物。