实验性变态反应性脑脊髓炎大鼠视神经内髓鞘碱性蛋白表达的变化

Expression of myelin basic protein in rat optic nerve with experimental allergic encephalomyelitis

背景脱髓鞘性视神经炎是与多发性硬化（MS）密切相关的一种疾病，发病机制不详，目前相关的基础研究尚少。目的从分子水平揭示髓鞘碱性蛋白（MBP）在实验性变态反应性脑脊髓炎（EAE）大鼠视神经内的表达变化。方法采用随机数字表法将50只清洁级Wistar大鼠分为正常对照组，免疫后8、12、18和25d组。用5只豚鼠以过量麻醉法处死后收集脑脊髓制备匀浆并与完全弗氏佐剂（CFA）等体积混匀后于Wistar大鼠4只脚垫皮下分别一次性注射抗原乳化剂0．5ml，同时于即刻和48h足背皮下分别注射百日咳杆菌0．2ml诱导Wistar大鼠EAE模型，注射当天记为免疫后第0天。造模后观察模型鼠的行为，对各组大鼠进行神经功能评分。在上述相应时间点以过量麻醉法处死大鼠并制作视神经组织切片，经苏木精一伊红染色法观察各组大鼠视神经的组织病理学改变，采用免疫组织化学法和Western blot法观察各组大鼠视神经组织中MBP的表达情况。结果Wistar大鼠免疫后12d左右出现运动功能障碍，神经功能评分开始上升，免疫后18d神经功能评分最高，随后逐渐下降。视神经组织病理学检查结果显示，免疫后12d视神经组织的神经纤维排列不均匀，免疫后18d视神经组织细胞结构紊乱，神经纤维变细小，轴束明显肿胀并有脱髓鞘现象，免疫后25d，异常细胞大量减少。免疫组织化学法检测表明，MBP主要表达于视神经纤维的髓鞘中，免疫后12d视神经组织中MBP阳性染色细胞为（115．75±26．49）个／5个视野，明显低于正常对照组的（167．44±22．49）个／5个视野，差异有统计学意义（t=4．537，P〈0．05），免疫后18dMBP阳性细胞最低，为（75．57±34．54）个／5个视野，与正常对照组比较差异有统计学意义（t=6．362，P〈0．01），免疫后95dMBP阳性细胞数开始回升，但仍明显低于正常对照组，差异有统计学意义（t=4．068，P〈0．05）。Westernblot检测显示，随着免疫时间的延长，MBP／β-actin值在免疫组大鼠视神经组织中的表达逐渐减少，免疫后12d，MBP／β-actin值小于正常对照组，差异有统计学意义（t=4．639，P〈0．05），免疫后18dMBP／β-actin值最低，与正常对照组比较差异有统计学意义（t=8．427，P〈0．01）。结论EAE大鼠视神经组织存在MBP的降解，提示视神经炎是一种视神经的原发性脱髓鞘病变。

Background Optic neuritis is closely associated with multiple sclerosis （MS）. Its pathogenesis is uncompletely clear,and less basic researches are carried out at home and abroad. Objective This study was to reveal the expression of myelin basic protein （MBP） in the optic nerve of rat with experimental allergic encephalomyelitis （EAE） and to provide a theoretical evidence for the research of the relationship of optic neuritis with MS. Methods Fifty clean Wistar rats were randomized into the control group and immune 8,12,18 and 25 days groups. Myelencephalon was collected from 5 guinea pigs to prepare the homogenate and mixed with the isovolumetrie complete Freud＇ s adjuvant （ CFA）. The O. 5 ml mixed antigen emulsifier was subcutaneously injected into the 4 maps together with Bordetella pertussis 0.2 ml under the cutaneous of dorsalis pedis at 0 and 48 hours to induce the EAE. Behavior of the rats was evaluated to score the neurological function. The optical nerve sections were prepared 8,12,18 and 25 days after immunology for the histopathological examination, and immunochemistry and Western blot were used to detect the expression of MBP in optic nerve. The use of the animals complied with the Regulation for the Administration of Affairs Concerning Experimental Animals by State Science and Technology Commission. Results The disorder of motor nerve was seen 12 days following the immune,and the clinical neural functional scores were significantly higher 12 day and peaked on 18 days myelination and then gradually reduced. The histopathological examination showed that the irregular alignment of neural fiber was found at 12 days,and changes of cellular structure, edema of neural shaft bunch were observed at 18 days. However, the abnormal ceils were significantly less 25 days following the immune. Immunochemistry showed that the MBP was expressed mainly in the myelination of optic nerve fibers. The numbers of positive ceils for MBP were （ 115.75 ±26.49） cells/5 fields at the 12th day, showing a significant lowing in comparison with （167.44±22.49） cells/5 fields of control group （ t = 4. 537, P〈0.05）. The positive ceils were lest at the 18th day with the values （75.57 ± 34. 54 ） cells/5 fields （ t = 6. 362, P〈0.01 ）. At the 25th day, positive ceils increased to （ 117.63± 13.78 ）cells/5 fields, which still was lower than those of the control group （t=4. 068 ,P〈0.05 ）. Western blot assay illuminated that with the prolong of immune time,MBP/ β-actin ratio in optic nerve was gradually reduced and followed the same pattern at the 12th day（ t=4. 639 ,P〈0.05）. At 18 days after immune in comparison with the control group,the expression of MBP/β-actin ratio in optic nerve was the least （t= 8. 427,P〈0.01 ）. Conclusions MBP can be degraded in rat optic nerve. This is a further evidence that optic neuritis is a severe demyelination disease. It is clearly related to MS.