嗅鞘细胞移植对大鼠脊髓挫伤后急性期巨噬细胞向M2亚型极化的影响及意义

Effect of olfactory ensheathing cells transplantation on polarization of M2 macrophages and its significance in rats with spinal cord contusion at acute phase

目的观察嗅鞘细胞（OECs）移植对脊髓挫伤后急性期损伤区巨噬细胞向M2亚型极化的调控作用，探讨OECs移植促进脊髓损伤修复的机制。方法分离培养原代OECs，移植备用。暴露SD大鼠T10脊髓，并用NYU-Ⅱ撞击机将撞击棒（10g）从25mm高处垂直落下挫伤脊髓。伤后即刻将24只脊髓挫伤模型大鼠按随机数字表法分为对照组和OECs移植组，每组12只，分别将DMEM／F12培养基或OECs悬液（浓度3×10^4个／μL，1μL×3次）注入损伤脊髓处。伤后1-9周时每周采用BBB评分法对大鼠进行运动功能评分。伤后1周时采用免疫荧光染色标记并计数损伤区M2型巨噬细胞及髓鞘相关抑制因子Nogo-A阳性细胞，同时采用Western blotting检测炎症因子白介素（IL）-4、IL-6蛋白的表达。伤后9周时采用HE染色观察损伤脊髓的病理改变。结果伤后1-9周时，OECs移植组每周BBB评分均明显高于对照组，差异均有统计学意义（P〈0．05）。伤后1周时，OECs移植组M2型巨噬细胞数量明显高于对照组（3.24％±0．56％ vs．0．63％±0．21％1，Nogo—A阳性细胞数量、荧光强度均明显低于对照组[（43±24）个／视野 vs．（207±88）个／视野；0．042±0．006 vs．0．062±0．011]，抗炎因子IL-4蛋白表达量明显高于对照组（0．717±0．152 vs.0．183±0．063），促炎因子IL-6蛋白表达量明显低于对照组（0．550±0．124 vs. 1．060±0．209），差异均有统计学意义（P〈0．05）。伤后9周时，HE染色显示OECs移植组囊性空洞面积[（1．511±0．581）mm^2]较对照组[（2．939±0．823）mm^2]明显减小，差异有统计学意义（P〈0．05）。结论OECs移植可促进脊髓损伤区巨噬细胞向M2亚型极化，并改善炎症微环境、抑制Nogo-A表达，从而促进脊髓结构和功能的恢复。

Objective To observe the regulatory effect of olfactory ensheathing cells （OECs） on polarization of M2 macrophages after acute contusion of spinal cords in rats, and explore the mechanism of OECs transplantation in promoting repair of spinal cord injury. Methods The primary OECs were isolated and cultured for transplantation. The T10 spinal cords of SD rats were exposed and impacted by impact stick （10 g） dropping vertically from a height of 25 mm with a NYU-II impactor. Twenty-four rats with contusion of spinal cord immediately after injury were randomly divided into DMEM/F12 control group and OECs transplantation group according a random number table. DMEM/F12 culture medium or OECs suspension （3×10^4μL, 1μL×3 times） was implanted into the injured spinal cords. At one to 9 weeks after injury, Basso Beattie Bresnahan （BBB） scale was used to evaluate the motor functions in rats. At one week after injury, M2 macrophages and Nogo-A positive cells were labeled by immunofluorescence staining; the expressions of interleukin （IL）-4 and IL-6 were detected by Western blotting. At 9 weeks after injury, the pathological changes of injured spinal cords were observed by HE staining. Results One and 9 weeks after injury, BBB scores in OECs transplantation group were significantly higher than those in control group at each time point after injury （P〈0.05）. One week after injury, the number of M2 macrophages in OECs transplantation group was significantly larger than that in control group （3.24% ±0.56% vs. 0.63% ±0.21%）, and the number of Nogo-A positive cells was significantly smaller and fluorescence intensity was significantly lower in OECs transplantation group than those in control group （[43±24] field vs. （[207±88]/field; 0.042±0.006 vs. 0.062±0.011, P〈0.05）; the IL-4 protein level was significantly higher and the IL-6 protein level was significantly lower in OECs transplantation group than those in the control group （0.717±0.152 vs. 0.183±0.063; 0.550±-0.124 vs. 1.060±0.209; P〈0.05）. Nine weeks after injury, the cytoplasmic cavity area in OECs transplantation group （[1.511 ±0.581] mm^2） was significantly lower than that in control group [2.939±0.823] mm^2, P〈0.05）. Conclusion OECs transplantation may promote macrophages polarizing to subtype M2, improve the microenvironment of inflammation, reduce Nogo-A secretion, thus promote the structure and spinal cord function recovery.