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目的分析急性CO染毒大鼠T淋巴细胞亚群和Foxp3基因表达水平的变化,初步探讨Foxp3基因在急性CO中毒中枢神经系统免疫损伤中的作用。方法 40只健康雄性SD大鼠随机分为空白对照组和CO染毒组(设立染毒后第3、7、14和21天组)。采用急性静式吸入染毒方法,CO浓度为2 000及3 000 ppm。分别于CO染毒后3、7、14和21 d处死动物,组织病理学显微镜下观察不同时间点大鼠中枢神经元及有髓神经纤维的病理改变;免疫组织化学染色方法检测脑组织中CD4膜分子蛋白的表达水平;流式细胞仪分析外周血T淋巴细胞亚群百分比变化;real-time PCR方法检测外周血中Foxp3基因mRNA的表达情况。结果 CO吸入染毒后大鼠出现明显的中毒症状,陆续出现兴奋躁动直至昏迷,口鼻黏膜樱桃红色。染毒各组大鼠脑组织存在不同程度的病理性改变,主要表现为脑皮质及海马区神经元变性坏死、海马椎体细胞疏松化及少量淋巴细胞浸润等,以14 d病变最为严重。砂罗铬花青染色见大脑白质神经纤维髓鞘肿胀变性,部分髓鞘脱失。免疫组化半定量分析发现急性CO染毒各组大鼠脑皮质及海马区的CD4膜蛋白阳性表达量明显升高。染毒后7和14 d,外周血CD4+T淋巴细胞百分比较对照组明显增加(P<0.05);且伴随T-reg细胞百分比减少(P<0.01)。与对照组比较,染毒各组外周血Foxp3基因mRNA表达水平均下降明显,以第7天下调最为显著(P<0.01)。Pearson相关分析显示外周血Foxp3 mRNA表达水平与CD4+T淋巴细胞数量呈显著性负相关(r=-0.399,P=0.011)。结论急性CO中毒外周血Foxp3基因mRNA表达下调,T-reg细胞数量减少。Foxp3基因转录抑制可能加重T淋巴细胞介导的中枢神经免疫炎症反应,与急性CO中毒神经免疫损伤有关。
Objective To analyze the changes of T lymphocyte subpopulation and Foxp3 gene expression in acute CO poisoning rats and to explore the role of Foxp3 gene in the central nervous system immune injury induced by acute CO poisoning. Methods Forty healthy male Sprague-Dawley rats were randomly divided into blank control group and CO-treated group (groups 3, 7, 14 and 21 after exposure). Acute inhalation of static inhalation method, CO concentrations of 2 000 and 3 000 ppm. The animals were sacrificed at 3, 7, 14 and 21 days after CO exposure respectively. The pathological changes of CNS and myelinated nerve fibers were observed under histopathological microscope at different time points. The expression of CD4 The expression of Foxp3 mRNA in peripheral blood was detected by real-time PCR. The expression of Foxp3 mRNA in peripheral blood was detected by flow cytometry. Results CO inhalation poisoning rats showed obvious symptoms of poisoning, one after another excited restless until coma, nasal mucosa cherry red. The pathological changes of brain tissue were observed in different groups. The main manifestations were neuronal degeneration and necrosis in cerebral cortex and hippocampus, loosening of hippocampus vertebral cells and infiltration of a few lymphocytes. Sara chrysanthemum staining see the white matter nerve fibers myelin swelling degeneration, some demyelination. Semi-quantitative analysis of immunohistochemistry found that the positive expression of CD4 membrane protein in cerebral cortex and hippocampus of rats in acute CO exposure group was significantly increased. The percentages of CD4 + T lymphocytes in peripheral blood were significantly increased (P <0.05) at 7 and 14 d after exposure, and the percentage of T-reg cells was decreased (P <0.01). Compared with the control group, the expression of Foxp3 mRNA in the peripheral blood of all the groups decreased significantly (P <0.01). Pearson correlation analysis showed that there was a significant negative correlation between the expression level of Foxp3 mRNA and the number of CD4 + T lymphocytes in peripheral blood (r = -0.399, P = 0.011). Conclusion The expression of Foxp3 mRNA in peripheral blood of patients with acute CO poisoning is down-regulated while the number of T-reg cells is decreased. Foxp3 gene transcriptional repression may aggravate T-lymphocyte-mediated central nervous system immune-inflammatory reaction, which is related to the neuro-immune injury of acute CO poisoning.