辅助性T细胞在脱髓鞘性视神经炎发病中的作用

Mechanism of T helper cell for demyelinated optic neuritis

背景 视神经炎是神经眼科临床常见疾病之一,易反复发作并可造成视神经轴索损害及不可逆性视力损伤,且无有效的防治方法,其发病机制被认为与机体免疫调节失衡有关. 目的 研究辅助性T细胞（Th）亚群调节性T细胞（Treg）、Th17特异性细胞因子及其转录因子在脱髓鞘性视神经炎早期发病中的表达,以明确实验性自身免疫性脑脊髓炎（EAE）小鼠脱髓鞘性视神经炎的免疫反应启动及免疫炎症维持机制. 方法 采用随机数字表法将6～9周龄清洁级C57BL/6（H-2b）雌性小鼠84只随机分为正常对照组12只和免疫后7、11、14、19、23、28 d组（模型组亚分）各12只.模型组小鼠分别于背部两侧皮下注射相应抗原200 μg,免疫当天记作第0天,于免疫后第0、2天给予模型组小鼠腹腔内注射百日咳疫苗400 ng,对照组注射生理盐水.观察视神经炎小鼠的眼部表现及闪光视觉诱发电位（F-VEP）变化;苏木精-伊红染色观察视神经的组织学改变;免疫组织化学法检测视神经轴索损伤标志物β-淀粉样前体蛋白（β-APP）表达量的变化;逆转录PCR（RT-PCR）法检测视神经组织中转化生长因子-β（TGF-β）、白细胞介素-1β（IL-1β）、IL-17、IL-10及叉头蛋白3（Foxp3）等的动态表达.结果 免疫后11d,模型组小鼠陆续出现神经系统症状,苏木精-伊红染色可见模型组视神经组织内出现炎性因子浸润,免疫组织化学法检测发现模型组轴索损伤标志物β-APP阳性染色增强;F-VEP检测结果显示,免疫后7、11、14、19、23、28 d组小鼠P1波潜时值较正常对照组小鼠延长,差异均有统计学意义（t=4.487、15.203、16.364、11.540、11.959、16.163,P＜0.05）;免疫后7d,正常对照组与模型组小鼠N1-P1振幅的差异无统计学意义（t=-0.992,P=0.378）;而免疫后11、14、19、23、28 d组小鼠N1-P1振幅明显低于正常对照组,差异均有统计学意义（t=-13.161、-31.401、-16.109、-7.025、-7.257,P＜0.05）.小鼠视神经中TGF-β、IL-1β、IL-17、Foxp3、IL-10 mRNA表达各时间点间整体比较差异有统计学意义（F=12.721、15.015、14.343、69.374、68.290,均P=0.000）,与正常对照组比较,免疫后11d、14d组,TGF-β mRNA水平明显升高,免疫后19d、23 d组,IL-1β mRNA水平明显升高,免疫后7d、11d组IL-17 mRNA水平明显升高,免疫后7、11、14、19、23、28 d组Foxp3 mRNA水平均明显降低,免疫后19、23、28 d组IL-10 mRNA水平明显降低,差异均有统计学意义（P＜0.05）.结论 Th17细胞亚群参与了脱髓鞘性视神经炎免疫损伤的启动,而Th1细胞亚群维持了炎症损伤,Treg亚群较Th2亚群更早出现异常.Th17/Treg比例失衡可能参与了脱髓鞘性视神经炎的发生.

Background Optic neuritis is one of the common clinical neuro-ophthalmic diseases.Recurrent optic neuritis can cause irreversible axonal damage and visual impairment,and no prevention method is effective up to now.Its pathogenesis is considered to be associated with the body＆#39;s immune imbalance.Objective This proposal tried to establish experimental autoimmune encephalomyelitis （EAE） and explore the immunomodulatory effects of T helper （Treg）,Th17 cell in the model of optic neuritis.Make the immune mechanism of demyelinating optic neuritis clear.Methods EAE model was established to observe the clinical symptoms and visual electrophysiological changes of optical neuritis in mice;hematoxylin-eosin staining to observe the histological changes in the mouse models of EAE; immunohistochemical staining to detect optic nerve axon damage markers β-amyloid peptide precurso （β-APP） protein expression quantity changes;reverse transcription PCR （RT-PCR） to detect the dynamic expression of forkhead/winged helix transcription factor p3 （Foxp3）,transforming growth factor-β （TGF-β）,interleukin-1β （IL-1β）,IL-17,IL-10 in optic nerve tissue.Results After immunization 11 days,neurological symptoms of EAE mice started to appear,hematoxylin-eosin staining showed optic nerve tissue infiltration of inflammatory cytokines; immunohistochemical staining showed enhancement of axonal injury markers β-APP positive staining; flash visual evoked potential（F-VEP） test showed 7,11,14,19,23,28 days,compared with the normal mice,P1 prepatents of model group were prolonged after modeling,the differences were statistically significant （t =4.487,15.203,16.364,11.540,11.959,16.163,all at P〈0.05）.When 7 days after modeling,the difference of N1-P1 amplitude between normal group and model group was no statistical significance （t =-0.992,P =0.378）; while 11,14,19,23,28 days after modeling,compared with normal mice,N1-P1 amplitudes of model mice were significantly lower,the differences were statistically significant（all at P〈 0.05）.The expressions of TGF-β,IL-1β,IL-17,Foxp3,IL-10 mRNA among different time points had stastistie significant differences （F =12.721,15.015,14.343,69.374,68.290,all at P =0.000）,compared with the normal group,when 11 days,14 days after modeling,TGF-β mRNA levels were significantly increased ; 19 days,23 days after modeling,IL-1β mRNA levels were significantly increased;7 days,11 days after modeling,IL-17 mRNA levels were significantly increased ;7,11,14,19,23,28 days after modeling,Foxp3 mRNA levels were significantly lower; 19,23,28 days after modeling,IL-10 mRNA levels were significantly lower,the difference were statistically significant （all at P〈 0.05）.Conclusions Th17 cells launch demyelinating optic neuritis immune injury,and Th1 subgroup performs the function of maintaining inflammatory lesions,Treg cell subgroup is abnormal much earlier than that of the Th2 subgroup.The imbalance of Th17/Treg cell may be involved in the pathogenesis of demyelinating optic neuritis.