Mmu-miR-27a-5p促进巨噬细胞极化对脊髓损伤恢复的研究

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摘要目的探讨mmu-miR-27a-5p对小鼠脊髓损伤处巨噬细胞极化及后肢运动功能恢复的影响。方法将160只SPF级雄性小鼠随机分为假手术组(A组)、模型组(B组)、mmu-miR-27a-5p移植组(C组)、mmu-miR-27a-5p阴性对照组(D组)，每组40只。A组为假手术组，B组、C组和D组构建脊髓损伤模型。术后第3天开始给予治疗，A组小鼠不予处理，B组给予10 μL生理盐水损伤区域注射，C组和D组分别给予mmu-miR-27a-5p移植物悬液和mmu-miR-27a-5p阴性移植物悬液10 μL于损伤区域注射。观察小鼠行为学改变、巨噬细胞极化的情况及胶质瘢痕的变化。结果实验过程中共死亡11只小鼠。术前四组小鼠行为学评分差异无统计学意义(P＞0.05)；治疗后观察各时间点，C组行为学评分均高于B组和D组，差异有统计学意义(P＜0.01)。脊髓胶质瘢痕HE染色显示A组各时间点脊髓组织正常；C组脊髓损伤后1 d出血均较明显，炎症细胞聚集，在损伤7 d、14 d较B组轻；D组脊髓损伤后HE染色与B组无差别。免疫组化染色：A组棕褐色细胞形态无明显变化，C组中棕褐色细胞数量明显低于B组和D组，差异均有统计学意义(P＜0.01)；B组和D组中胶质纤维酸性蛋白(glial fibrillary acidic protein，GFAP)的表达显著高于C组，差异均有统计学意义(P＜0.01)；A组GFAP的表达为正常形态。Western Blot检测结果显示A组白细胞介素(interleukin，IL)-1β、肿瘤坏死因子(tumor necrosis factor，TNF)-α、IL-10、Arginase-1在各时间点的表达差异无统计学意义(P＝0.96)，C组在术后7 d、14 d较B组和D组的IL-1β、TNF-α表达低，而IL-10、Arginase-1表达高，差异有统计学意义(P＜0.001)。细胞流式检测B组和D组在术后7 d、14 d的CD16标记明显高于C组，差异有统计学意义(P＜0.001)，C组Arginase-1术后7 d、14 d的标记明显高于B组和D组，差异有统计学意义(P＜0.001)。结论脊髓损伤后14 d内，通过移植mmu-miR-27a-5p促进小鼠脊髓损伤局部巨噬细胞向M2型分化，并伴随小鼠后肢运动功能的改善。

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	杨明坤
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	吴登普
	党晓谦

关键词 ：脊髓损伤, 胶质瘢痕, 巨噬细胞, 极化

Abstract：

Objective To investigate the effect of mmu-miR-27a-5p on macrophage polarization at the site of spinal cord injury and the recovery of hindlimb motor function after spinal cord injury in mice.Methods A total of 160 mice were randomly divided into sham (group A)，model (group B)，mmu-miR-27a-5p-mimic (group C)，mmu-miR-27a-5p-negative control (group D).On day 3 after successful establishment of spinal cord injury model，Group A received no treatment.Group B，C，D were injected with 10μL of saline，mmu-miR-27a-5p-mimic suspension and mmu-miR-27a-5p-negative control suspension into the injured site，respectively.The behavioral changes，macrophage polarization and glial scar were observed.Results The behavioral scores in group C were higher in each treatment than in group B and D (P＜0.01).HE staining of the spinal cord glial scar showed that spinal cord tissue was normal in group A at each time point.In group C，the bleeding was more obvious 1 day after spinal cord injury，while the inflammatory cells gathered，which was lighter than that in group B at 7 days and 14 days after spinal cord injury.In the D group，the staining was not different from that in group B.Immunohistochemical staining showed that there was no significant change in the morphology of tan cells in group A，but the number of tan cells in group C was obviously lower than that in group B and group D，and the differences were statistically significant (P＜0.01).The expression of GFAP in group B and group D were significantly higher than those in group C，and the differences had a statistical significance(P＜0.01).The expression of GFAP in group A was normal.The result of Western Blot showed that there was no statistical significance in the expression of IL-1β、TNF-α，IL-10，and Arginase-1 in group A at each time point(P=0.96).The expression of IL-1β and TNF-Α in group C was lower than that in group B and group D at 7 days and 14 days after the operation，while the expression of IL-10 and Arginase-1 was higher，the differences had a statistical significance (P＜0.01).Flow cytometry showed that the CD16 marks in group and group D were obviously higher than that in group C at 7 days and 14 days after the operation，and the differences were statistically significant(P=0.000).The marks of Arginase-1 in group C were significantly higher than those in group B and group C at 7 days and 14 days after the operation(P=0.000).Conclusion Injection of mmu-miR-27a-5p can promote the polarization of macrophages toward the M2 phenotype at the site of spinal cord injury，and improve the recovery of hindlimb motor function within 14 days after spinal cord injury in mice.

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，ymkun1121@163.com

引用本文:

杨明坤，刘泯邑，杜涛，贾新冬，吴登普，党晓谦. Mmu-miR-27a-5p促进巨噬细胞极化对脊髓损伤恢复的研究[J]. 实用骨科杂志, 2023, 29(3): 224-.
Yang Mingkun，Liu Minyi，Du Tao，JiaXindong，WuDengpu，Dang Xiaoqian. Effect of Mmu-Mir-27a-5p Promotes Macrophage Polarization on Recovery after Spinal Cord Injury. sygkzz, 2023, 29(3): 224-.

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http://www.sygkzz.com/CN/ 或 http://www.sygkzz.com/CN/Y2023/V29/I3/224

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