烟草烟雾对实验性自身免疫性脑脊髓炎大鼠中枢性炎症及T细胞亚型的影响

Effects of tobacco smoke on central imflammation and differentiation of T cells subtype in rats with experimental autoimmune encephalomyelitis

目的探讨烟草烟雾对实验性自身免疫性脑脊髓炎(EAE)中枢性炎症及T细胞亚型分化的影响。方法将44只SD雄性大鼠分为空气对照组与烟草烟雾对照组(各6只)、空气组与烟草烟雾组(各16只),其中烟草烟雾组与空气组用豚鼠脊髓匀浆与弗氏完全佐剂混合乳液免疫诱导EAE模型,并给予烟草烟雾暴露或假暴露处理;空气对照组与烟草烟雾对照组仅用0.9%氯化钠溶液与弗氏完全佐剂乳液进行免疫诱导,同样给予烟草烟雾暴露或假暴露处理。共暴露20 d,连续观察并记录各组大鼠行为学评分,于实验后采用HE染色、快蓝染色(LFB)及甘氨酸银染观察脑脊髓组织病理改变,采用流式细胞术检测脾脏CD3、CD4、CD8及调节性T细胞(Treg)百分比,采用荧光定量PCR检测脑组织T-bet/CD3、FoxP3/CD3转录水平。结果烟草烟雾组大鼠EAE发病比例高于空气组,Treg细胞比例低于空气组[分别为(3.69±0.24)%和(1.76±0.16)%],中枢FoxP3/CD3转录水平低于空气组[分别为(1.07±0.08)和(0.61±0.08)],差异均有统计学意义(P<0.05)。空气组、烟草烟雾组大鼠第20天行为学评分与外周Treg比例、中枢FoxP3/CD3水平均呈负相关(r值分别为-0.497,-0.485),差异均有统计学意义(P<0.05)。结论烟草烟雾暴露可加重EAE,并可能与Treg细胞分化受限有关。

Objective To understand the effects of tobacco smoke on experimental autoimmune encephalomyelitis and the differentiation of T cell subtypes in rats.Methods A total of 44 SD rats were divided into four groups,air control group,tobacco smoke control group,air group and tobacco smoke group.The tobacco smoke group and air group were immunized with guinea pig spinal cord homogenate and complete Freund’s adjuvant emulsion to develop the rat EAE models,then tobacco smoke exposure or sham exposure were conducted for 20 d.The air control group and the tobacco smoke control group were immunized with 0.9%sodium chloride solution and complete Freund’s adjuvant emulsion,and the same treatments as the above were conducted for 20 d.The clinic scores of the rats were recorded by continuous observation.Pathological examinations of brain and spinal cord were done by HE staining,LFB staining and glycine silver staining.The percentage of spleen CD3,CD4,CD8 and Treg cells was detected by flow cytometry.The transcriptional level of T-bet/CD3 and FoxP3/CD3 in brain tissue was detected by real time quantitative PCR.Results Compared with the air group,the incidence of EAE increased(P<0.05),and the condition was more serious in tobacco smoke group,and the proportion of Treg cells in the tobacco smoke group was lower[(1.76±0.16)%,(3.69±0.24)%,P<0.05],and the transcription level of central Foxp3/CD3 was also lower(0.61±0.08,1.07±0.08,P<0.05).There was a negative correlation between behavioral score and Treg ratio(r=-0.497,P<0.05)and FoxP3/CD3 transcription level(r=-0.485,P<0.05).Conclusion Exposure to tobacco smoke may aggravate experimental autoimmune encephalomyelitis and Treg cell differentiation restriction may be involved in it.