巨噬细胞亚型转变在大鼠肾脏缺血/再灌注损伤中的作用

目的：探讨巨噬细胞在大鼠肾脏缺血/再灌注损伤过程中的亚型转变及意义。方法：将30只雄性SD大鼠随机分成假手术组（Sham，n=6）和缺血/再灌组（IRI，夹闭肾动脉45 min，n=24）。IRI组分别于术后0、6、24和72 h取肾组织，每个时相组6只大鼠。用HE染色观察肾组织损伤程度；免疫组化染色检测细胞增殖核抗原（PCNA）的表达；实时定量RT-PCR检测巨噬细胞移动抑制因子（MIF） mRNA的表达；免疫组织荧光染色检测MIF、单核巨噬细胞趋化蛋白-1（MCP-1）以及活化巨噬细胞标志物CD68的表达，流式细胞分析检测巨噬细胞M1和M2亚型的分布特征。结果：病理结果显示大鼠肾局部损伤情况和炎症细胞浸润程度在24 h时最为严重，之后逐渐恢复。PCNA在再灌后表达明显增加，6 h达峰值，72 h表达下降。相比于正常组，再灌组大鼠肾组织中MIF的mRNA和蛋白表达明显升高；MCP-1表达则在6 h达峰值，随后下降；而CD68阳性的巨噬细胞数量明显增加，24 h达峰值，72 h表达下降。更进一步研究发现缺血/再灌注6 h时，M1亚型分布达最高值；之后随着缺血/再灌注时间延长，M1亚群相对含量开始下调，M2随之升高。结论：在肾脏缺血/再灌注早期，M1巨噬细胞介导的组织损伤发挥主要作用，随后M2型表达逐渐上调，并通过促进细胞增殖修复肾组织损伤。

The role of sub-transform of macrophages in renal ischemia/reperfusion injury in rats

Objective:To investigate the role of sub-transform macrophage in ischemia/reperfusion renal injury in rats, as well as under-lying mechanisms. Methods:Thirty male Sprague-Dawley rats were randomly divided into ischemia/reperfusion (IRI, n=24, renal artery was occluded for 45 min) group and sham-operation (Sham, n=6) group. The kidneys in IRI group were collected at 0, 6, 24 and 72 h after operation (6 rats for each time point). The injury of the kidney was detected with HE staining. Immunohistochemistry staining was performed to evaluate the expression of proliferating cell nuclear antigen (PCNA). Real-time PCR was used to detect the mRNA expression of macrophage migration inhibitory factor (MIF). Moreover, the expression and location of MIF, monocyte chemoattractant protein-1 (MCP-1) and macrophage marker CD68 were examined by immunofluorescence staining. Most importantly, the distribution of macrophage subtypes M1 and M2 was analyzed by flow cytometry. Results:The worst pathologic damage of the renal tissues, as well as infiltration of inflammatory cells, was observed at 24 h after operation in IRI rats, with obvious recovery afterwards. Immunohistochemistry staining showed that the expression of PCNA was significantly increased after the ischemia/reperfusion, peaking at 6 h and reducing at 72 h after operation. Compared with sham group, the levels of MIF at mRNA and protein levels were both significantly increased after the ischemia/reperfusion, while the expression of MCP-1 was peaked at 6 h and decreased afterwards. Moreover, the expression of CD68-positive macrophages were significantly increased in IRI rats, with peaking at 24 h and reducing at 72 h. Furthermore, after 6 h of reperfusion, the percentage of M1 macrophages reached the peak, and thereafter the relative expression of M1 and M2 was reduced and increased, respectively. Conclusion:In the early phase of ischemia/per-fu sion renal injury, M1 macrophage results in renal damage, and afterwards the M2 macrophage is increased and repairs the renal damage by improving the cell proliferation.