巨噬细胞的极化与脊髓胶质瘢痕形成的一般规律研究

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摘要目的探讨巨噬细胞的极化与脊髓胶质瘢痕形成的一般规律。方法将40只SPF级雄性小鼠随机分为实验组和对照组，实验组分为脊髓损伤后1 d、7 d、14 d、28 d组，各组5只，对照组20只。实验组通过手术造模，对照组不造成脊髓损伤。分别于脊髓损伤后1 d、7 d、14 d、28 d取标本，行HE染色、免疫组化染色观察胶质瘢痕形成的过程；通过实时荧光定量聚合酶链式反应(realtimequantitative polymerase chain reaction，RT-qPCR)、酶联免疫吸附测定(enzyme linked immunosorbent assay，ELISA)检测M1型巨噬细胞标记物肿瘤坏死因子(tumor necrosis factor，TNF)-a、白细胞介素(interleukin，IL)-1β和M2型巨噬细胞标记物Arginase-1、IL-10的表达量。结果实验组死亡3只小鼠，及时予以补充，对照组无死亡小鼠。HE染色、免疫组化染色可见实验组脊髓胶质瘢痕形成，对照组无脊髓胶质瘢痕形成。RT-qPCR检测实验组M1型巨噬细胞的标记物IL-1β、TNF-α在脊髓损伤后各时间点较对照组高，差异有统计学意义(P<0.01)；脊髓损伤后第7天表达最高，随后逐渐下降，至28 d趋于稳定；对照组各时间点的IL-1β、TNF-a表达无明显变化(P＞0.05)。实验组M2型巨噬细胞的标记物IL-10、Arginase-1在脊髓损伤后各时间点较对照组高，差异有统计学意义(P<0.01)；脊髓损伤后第14天表达最高，随后逐渐下降，至28 d趋于稳定；对照组各时间点的IL-10、Arginase-1表达无明显变化(P＞0.05)。ELISA检测M1型巨噬细胞的标记物IL-1β、TNF-α在实验组的各时间点表达较对照组明显增高，差异有统计学意义(P<0.01)；实验组IL-1β、TNF-α表达在脊髓损伤后第7天表达最高，随后逐渐下降，至28 d趋于稳定；对照组各时间点的IL-1β、TNF-α表达差异无统计学意义(P＞0.05)。M2型巨噬细胞的标记物IL-10、Arginase-1在实验组的表达较对照组明显增高，实验组各时间点均高于对照组，差异有统计学意义(P<0.01)；实验组IL-10、Arginase-1表达在脊髓损伤后第14天表达最高，随后逐渐下降，至28 d趋于稳定；对照组各时间点的IL-10、Arginase-1表达差异无统计学意义(P＞0.05)。结论胶质瘢痕形成的前7天巨噬细胞主要极化为M1型巨噬细胞，7 d后主要为M2型巨噬细胞，于28d趋于稳定。

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关键词 ：脊髓损伤, 胶质瘢痕, 巨噬细胞, 极化

Abstract：Objective To investigate the relationship between macrophage polarization and glial scar formation in mice model of spinal cord injury(SCI).Methods A total of 40 specific pathogen-free male C57BL/6 mice were randomly divided into the model(n=20)and control(n=20)groups.The model group was divided into 1 d，7 d，14 d，and 28 d post-model groups，with 5 mice in each group.The control group underwent the same procedures without damaging the spinal cord.Spinal cord tissue samples were obtained at 1 d，7 d，14 d，and 28 d after SCI，HE and immunohistochemical staining were used to observe glial scar formation following SCI.RTqPCR and ELISA assay were used to detect the expression of M1 markers TNF-a，IL-1β，and M2 markers Arginase-1，IL-10.Results The staining results showed that the glial scar of spinal cord was formed in the experimental group and no glial scar of spinal cord in the control group.The markers IL-1β and TNF-α of M1-type macrophages in the fluorescence quantitative PCR experimental group were higher than those in the control group at each time point after spinal cord injury，and the difference was statistically significant(P<0.01).The highest expression was observed on the 7th day after spinal cord injury，and then decreased gradually，and tended to be stable on the 28th day.There were no significant changes in the expression of IL-1β and TNF-a in the control group at each time point(P＞0.05).IL-10 and Arginase-1 markers of M2 macrophages in the experimental group were higher than those in the control group at each time point after spinal cord injury，and the difference was statistically significant(P<0.01).The highest expression was observed on the 14th day after spinal cord injury，and then decreased gradually，and tended to be stable on the 28th day.There were no significant changes in the expression of IL-10 and Arginase-1 in the control group at each time point(P＞0.05).The expression of the markers IL-1β and TNF-α of M1-type macrophages detected by ELISA were significantly higher in the experimental group than in the control group at each time point(P<0.01).The expression of IL-1β and TNF-α in the experimental group was the highest on the 7th day after spinal cord injury，and then decreased gradually，and tended to be stable on the 28th day.There were no significant differences in the expression of IL-1β and TNF-α in the control group at each time point(P＞0.05).The expression of markers IL-10 and Arginase-1 of M2-type macrophages in the experimental group was significantly higher than that in the control group，and the difference was statistically significant(P<0.01).The expression of IL-10 and Arginase-1 in the experimental group reached the highest level on the 14th day after spinal cord injury，and then decreased gradually and tended to be stable on the 28th day after spinal cord injury.There was no statistical significance in the expression of IL-10 and Arginase-1 in the control group at each time point(P＞0.05).Conclusion Macrophages were are mainly polarized to M1 phenotype in the first 7 days during glia scar formation after SCI，which were then gradually polarized into M2 phenotype at 7 days，and tended to be stabilized at 28 days after SCI.

Key words： spinal cord injury glial scar macrophage polarization

基金资助:四川省科技厅项目(2018JY0308)

作者简介: 杨明坤(1986— )，男，副主任医师，四川省巴中市中心医院骨科，636000。

引用本文:

杨明坤 1，张旭 1，党晓谦 2，吴登普 1，刘泯邑 1. 巨噬细胞的极化与脊髓胶质瘢痕形成的一般规律研究[J]. 实用骨科杂志, 2021, 27(6): 522-527.
Yang Mingkun 1，Zhang Xu 1，Dang Xiaoqian 2，et al. The Relationship Between Macrophage Polarization and Glial Scar Formation in Mice Model of Spinal Cord Injury. sygkzz, 2021, 27(6): 522-527.

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http://www.sygkzz.com/CN/ 或 http://www.sygkzz.com/CN/Y2021/V27/I6/522

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