大鼠实验性变态反应性脑脊髓炎CD4^+,CD8^+的改变及意义

Alterations of CD4^+ and CD8^+ in experimental allergic encephalomyelitis and its significance in rats

目的:观察实验性变态反应性脑脊髓炎模型大鼠外周血CD4+,CD8+的改变及免疫调节剂左旋咪唑的作用。方法:实验于2005-05/07在遵义医学院附属医院神经科实验室和贵州省细胞工程重点实验室完成。取Wistar大鼠23只单纯随机分为4组:①正常组(n=5):不干预。②模型组(n=8):每只大鼠一次性注入0.5mL豚鼠脊髓匀浆(相当髓鞘碱性蛋白3.5mg)和完全福氏佐剂混合乳剂,其中0.4mL注入双后足掌皮下,0.1mL注入尾近端。在注入抗原乳剂后即刻、24,48h共3次腹腔注射左旋咪唑10mg/kg。③完全福氏佐剂组(n=5):注射无豚鼠脊髓匀浆生理盐水+完全福氏佐剂混合液0.5mL,用法同模型组,不注射左旋咪唑。④左旋咪唑组(n=5):每间隔24h腹腔注射左旋咪唑,用法同模型组。将注射日规定为第0天,每日观察2次记录大鼠神经体征变化,所有大鼠于注射第16天取材,流式细胞仪检测外周血CD4+,CD8+的变化,苏木精-伊红染色观察病理变化,Loyez氏髓鞘染色法观察髓鞘改变。结果:23只大鼠全部进入结果分析。①神经体征变化:正常组、完全福氏佐剂组和左旋咪唑组均未出现症状,模型组8只大鼠中有7只于第14天出现症状,发病率为87.5%。②CD8+的变化:左旋咪唑组和模型组低于正常组犤(31.24±7.01)%,(20.64±5.52)%,(41.73±7.28)%,P<0.05,0.01犦,且模型组低于左旋咪唑组(P<0.05)。③CD4+的变化:左旋咪唑组和模型组高于正常组犤(45.21±5.35)%,(43.30±5.47)%,(34.44±7.01)%,P<0.05犦,但前2组比较差异不显著。④CD4+/CD8+:左旋咪唑组和模型组高于正常组(1.53±0.49,2.33±1.04,0.86±0.32,P<0.05,0.01)。⑤模型组苏木精-伊红染色见大脑白质及脊髓血管周围有大量炎性细胞浸润;Loyez氏染色见部分脱髓鞘改变。结论:①大鼠实验性变态反应性脑脊髓炎是通过降低CD8+、增高CD4+及CD4+/CD8+介导的免疫性疾病。②左旋咪唑破坏了机体的免疫平衡,是诱导实验性变态反应性脑脊髓炎发生的重要免疫调节剂。

AIM： To observe the changes of CD4^＋, CD8^＋ and effect of levamisole in experimental allergic encephalomyelitis rats. METHODS： The experiment was carried out in the Laboratory of Department of Neurology, Affiliated Hospital of Zunyi Medical College and Key Laboratory of Cell Engineering of Guizhou Province between May and July 2005. Twenty-three Wistar rats were randomly divided into 4 groups ： ①normal group （n=5）, no intervention; ②model group （n=8） which was subcutaneously injected with 0,5 mL homogenate of guinea pig spinal cord （about contained 3.5 mg myelin basic protein, MBP） in complete Freund＇s adjuvant （CFA） 0,4 mL at postpedes-palm and 0.1 mL at proximate of tail, at same time in immunization and after 24 hours and 48 hours, levamisole （10 mg/kg） was intraperitoneally injected. ③CFA group （n=5）： The rats were subcutaneously injected with 0.5 mL saline plus CFA without homogenate of guinea pig spinal cord. The method was the same to that in the model group, no levamisole.④Levamisole group （n=5）： The rats were intraperitoneally injected with levamisole every other 24 hours as the same as in model group. The injected day was considered as the day 0, The symptom of neurology was observed twice a day and rats were killed on day 16 after injection. The CD4^＋, CD8^＋ changes of peripheral blood were detected with flow cytometer. Pathological feature and myelin change were detected with hematoxylin-eosin staining （HE） and Loyez＇s staining, respectively. RESULTS： All the 23 rats were involved in the analysis of resuh.①symptom of neurology： none of the normal group, CFA group and levamisole group. Symptom occurred at day 14 in 7 rats of 8 rats in the model group with the incidence rate of 87.5%. ②change of CD8^＋： The CD8^＋ in levamisole group and model group was lower than that in normal group [（31.24^＋7.01 ）%, （20.64^＋5.52）%, （41.73±7.28）% ,P 〈 0.05,0.01], and CD8^＋ in model group was lower than that in levamisole group （P 〈 0.05）. ③change of CD4^＋： CD4^＋ in levamisole group as same as in model group, which were higher than that in normal group [（45.21±5.35）%, （43.30 ±5.47）%, （34.44^＋7.01）%,P 〈 0.05], but there was no significant difference between the former two groups. ④CD4^＋/CD8^＋： CD4^＋/CD8^＋ in levamisole group and model group was higher than that in normal group （1.53±0.49,2.33±1.04,0.86±0.32,P 〈 0.05,0.01）. ⑤The inflammation cells were observed around vessels with HE staining and demyelination were observed with Loyez staining in cerebral white-matter and spinal cords in model group. CONCLUSION： ①Experimental allergic encephalomyelitis is induced by reducing CD8^＋ and increasing CD4^＋ and CD4^＋/CD8^＋ in Wistar rats. ②Levamisole can± destroy the immunity and is the important immunomodulator of experimental allergic encephalomyelitis.