骨髓来源单核巨噬细胞对高血压小鼠心脏损伤和重构的影响

目的观察高血压介导骨髓来源单核巨噬细胞在心脏浸润的情况,探讨其对高血压心脏损伤和重构的影响。方法 20只雄性C57BL/6J小鼠随机分为对照组和AngⅡ灌注组,每组10只,采用植入式胶囊渗透压泵分别灌注生理盐水和AngⅡ,采用鼠尾套法监测小鼠动脉血压。于灌注后7 d,采用免疫组织化学、Masson染色和流式细胞技术观察心脏纤维化和心脏中单核巨噬细胞的浸润;采用流式细胞技术和实时荧光定量PCR(rt-PCR)观察循环中单核细胞上CCR2受体表达和心脏中CCR2受体的配体MCP-1表达;16只雄性小鼠随机分为4组,每组4只,采用尾静脉注射脂质体包裹氯膦酸二钠方法清除小鼠体内巨噬细胞,以注射脂质体包裹生理盐水为对照组,分别灌注生理盐水和AngⅡ后观察心脏纤维化。结果与生理盐水灌注对照组相比,AngⅡ灌注后1 d收缩压开始升高,7 d血压显著升高[收缩压:(157.0±13.9)mm Hg比(93.0±11.1)mm Hg,P<0.01],心脏中胶原沉积增加(4.1%±0.7%比0.4%±0.1%,P<0.01),Ⅰ型胶原和肌成纤维细胞的标志物α-SMA的蛋白表达增加。流式检测示心脏中CD45+F4/80+细胞浸润增加;心肌组织半乳糖凝集素2(Mac-2,巨噬细胞标志)和CD68阳性巨噬细胞浸润增加;外周血CD45+CD11B+单核细胞表达CCR2,心脏中MCP-1的mRNA表达增加;小鼠去除巨噬细胞后,AngⅡ灌注7 d的心脏中巨噬细胞浸润减少,心脏纤维化程度减轻。结论高血压促进骨髓来源单核巨噬细胞在心脏的募集,巨噬细胞浸润与心脏纤维化发生相关,去敲除巨噬细胞能够抑制高血压心脏纤维化。

Effect of bone marrow derived macrophages on hypertension induced cardiac injury and remodeling in mice

Objective It was previously proved that the infiltration of bone marrow derived monocyte/macrophage is critical to the inflammatory response.In this study,we aimed to explore the relationship between the infiltration of bone marrow derived monocyte/macrophage and the formation of hypertension-induced cardiac fibrosis. Methods The 20 C57BL/6J mice were infused with a 1500 ng·kg-1·min-1 dose of either 0.9% saline(saline group,n=10) or angiotensin Ⅱ(Ang Ⅱ group,n=10) via an osmotic mini-pump subcutaneously implanted for 7 days.Blood pressure was measured by tail-cuff method to evaluate the effect of Ang Ⅱ infusion.At day 7,Ang Ⅱ or saline infused mice were evaluated by Masson,immunohistochemistry staining and flow cytometry to analyze the cardiac fibrosis and the infiltration of monocyte/macrophage.Flow cytometry and real-time-PCR were used to evaluate the expression of CCR2 on the periphery macrophages and monocyte chemoattractant protein-1(MCP-1),the ligand of CCR2 receptor,in the heart tissue.The 16 mice were divided into 4 groups,the intravenous injection of liposome-encapsulated Clodronate was used to clear off the periphery macrophage,the liposome-encapsulated saline was used as control,then mice were infused with either 0.9% saline or angiotensin Ⅱ to study the fibrosis formation. Results Compared with saline infusion control group,systolic blood pressure began to increase at day 1 and was significantly increased at day 7 after Ang Ⅱ infusion [(93.0±11.1)mm Hg vs.(157.0±13.9)mm Hg,P<0.01].Collagen deposition was significantly increased(4.1%±0.7% vs.0.4%±0.1%,P<0.01) in the Ang Ⅱ infused heart,and the protein expression levels of collagen I and α-SMA were also increased.Flow cytometry results shown that there were more CD45+F4/80+ macrophages infiltrated in the Ang Ⅱ infused heart compared with control heart.Immunhistochemistry staining data shown that the galectin 3(also known as Mac-2) positive and CD68 positive macrophages were also increased in Ang Ⅱ infused heart.The CD45+CD11B+ peripheral monocytes expressed the chemokine receptor CCR2,and in Ang Ⅱ infused heart the mRNA level of MCP-1 was higher than control heart.When macrophages were cleared off by Clodronate,the infiltration of macrophage was reduced in the heart and the degree of cardiac fibrosis was attenuated. Conclusions Hypertension promotes the recruitment of bone marrow-derived monocyte/macrophage to the heart,which is related with the cardiac fibrosis formation.Fibrosis can be reduced when the macrophages were cleared off.