天然免疫蛋白CARD9介导单核巨噬细胞记忆促进血压变异性大加重高血压心脏重塑的机制研究

Hypertension is a common hazard to human health, as well as is associated with a low grade chronic inflammation. The target organ damage with exaggerated blood pressure variability is more serious. Recently, several studies found that activation of mononuclear phagocytes is central to the progression of cardiac remodeling by regulating cell memory. We have confirmed the caspase recruitment domain protein 9 (CARD9) with the surface of macrophages in cardiac remodeling induced inflammation plays a key role. Our preliminary results showed that the heart remodeling with exaggerated blood pressure variability rat increased, with protein CARD9 increased. But the mechanism is unclear. We hypothesized: "Exaggerated blood pressure variability caused by heart and spleen monocytes / macrophages memories mediated by CARD9 cause cardiac remodeling." In this study, we plan to validate our proposal in vivo through using of the established blood pressure variability animal model(sinoaortic denervation) and spontaneously hypertensive rats. We are going to use ultrasound to detect rats cardiac structure，use flow cytometry to check out inflammatory cells, use immunohistochemical detection the expression of CARD9 protein and other inflammatory factors. Through CARD9shRNA interference vector transfused by tail vein and removal of the spleen, we observed above indicators again. Moreover, macrophages cultured by three dimension and mechanical stretch stimulation and flow cytometry analysis of inflammatory cells will be applied to investigate the mechanism underlying our preliminary findings. The final, expression of inflammatory cytokines detected in exaggerated blood pressure variability in hypertensive patients. Within this study, we anticipate to explore a novel therapeutic target of exaggerated blood pressure variability leading to cardiac remodeling.

高血压是危害人类健康的常见病，也是一种慢性低度炎症。而血压变异性大时靶器官损害更严重。新近报道，单核巨噬细胞通过细胞记忆调控炎症反应；我们已证实巨噬细胞表面胱天蛋白酶募集域蛋白9 (CARD9)在炎症引发的心脏重塑中起关键作用；前期实验，发现血压变异模型大鼠心脏重塑加重、CARD9蛋白表达增多；但机制不清。我们提出假设：“血压变异性大引起心脏及脾脏CARD9介导的单核/巨噬细胞记忆导致心脏重塑”。本课题拟以自发性高血压大鼠去窦弓神经复制血压变异模型，超声检测心脏结构，细胞流式检测炎症细胞，免疫组化等检测CARD9蛋白及炎症因子；尾静脉回输CARD9shRNA干扰载体和切除脾脏后观察上述指标；三维培养巨噬细胞机械拉伸刺激，探索CARD9对巨噬细胞记忆功能的调控；最后在血压变异性大的高血压患者中检测CARD9蛋白、炎症因子表达水平，验证我们的假说。为血压变异导致心脏重塑的治疗提供新靶点。

血压变异性大会加重高血压患者靶器官损害，炎症反应是导致心脏损伤的重要机制。近期发现，单核巨噬细胞具有细胞记忆功能，它在血压变异性大所致的心脏重塑中可能起关键作用。但是血压变异如何通过炎症反应促进心脏重塑的具体机制仍不清楚。胱天蛋白酶相关的募集域蛋白9（CARD9）是一种天然免疫蛋白可激活下游的炎症信号通路调节炎症反应。我们前期研究发现CARD9是高血压导致心脏重塑的重要信号通路，在此过程中巨噬细胞是重要的效应细胞。据此我们提出假设：早期血压升高所导致的应激反应可招募循环中的单核细胞穿透血管内皮进入心脏及脾脏组织，引发局部的巨噬细胞炎症记忆，反复血压升高可激活脾脏内单核巨噬细胞系统内CARD9介导的细胞记忆，脾脏来源单核巨噬细胞将释放炎症因子并作用于心脏局部，激活下游的炎症信号通路，导致心脏重塑。本课题以CARD9敲除小鼠为基础，利用血管紧张素II（Ang II）灌注建立小鼠高血压模型，利用去窦弓神经术建立血压变异模型，利用脾脏切除手术模拟脾脏记忆模型，通过流式检测、病理分析研究CARD9蛋白通过单核巨噬细胞介导心脏重塑；通过体外细胞变量拉伸试验模拟血压变异应力环境，探索CARD9调节心脏重塑的分子机制。结果显示：血压变异组在Ang II 灌注7天时室间隔厚度、左室后壁厚度和左室质量均显著增加（P<0.001）；巨噬细胞内CARD9 蛋白表达明显上调（P<0.05）；胶原沉积及心脏组织中巨噬细胞浸润明显增加（均为P<0.01），促炎因子及促纤维化因子表达显著升高（均为P<0.05）。流式细胞仪发现血压变异可使外周血中促炎单核细胞、M1型巨噬细胞的数量增多，脾脏中促炎单核细胞数量减少；CD4 +辅助T细胞和CD8 +效应性T细胞在脾脏高表达。以上结果提示脾脏内单核巨噬细胞系统内CARD9作为重要的信号蛋白，通过介导炎症细胞记忆功能调节炎症反应，在血压变异导致的心肌重塑中发挥重要作用。我们的研究为深入探讨高血压心脏重塑的分子机制及防治策略提供新靶点。

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