合成病毒多肽诱导实验性自身免疫性脑脊髓炎的行为学,免疫学和病理学研究

Behavioral,immunological and pathological study of induction of experimental autoimmune encephalomyelitis by synthetic viral peptides

目的研究与髓鞘碱性蛋白(MBP)有相似氨基酸序列的4条病毒多肽免疫大鼠后能否诱导出实验性自身免疫性脑脊髓炎(EAE)模型,并探讨其可能的发生机制。方法66只Lewis雌性大鼠,随机分为6组,每组11只。A组用完全弗氏佐剂(CFA)免疫;B组用豚鼠MBP68-86免疫;C组—F组分别用乙型肝炎病毒、JC病毒、EB病毒和HHV-6病毒肽段免疫。EAE评分按国际标准。免疫后第15天处死大鼠,制备淋巴结细胞悬液和腰段脊髓冰冻切片。用MBP68-86和PBS分别刺激6组的淋巴结细胞,测定各组T淋巴细胞对MBP68-86的增殖反应,MBP68-86刺激下的IFN-γ分泌及每条病毒肽与MBP68-86诱导抗体间的交叉反应性。对脊髓切片进行细胞浸润的评级,并用免疫组化法对浸润细胞分类。结果B组(MBP68-86免疫组)和D组(JC病毒肽免疫组)分别有10只鼠和6只鼠出现EAE表现;MBP68-86显著刺激B组和D组大鼠的T淋巴细胞增殖,与CFA组比较有统计学差异(P<0.05);B组和D组的淋巴细胞在MBP68-86刺激下释放的IFN-γ量上升,明显高于CFA组(P<0.05);MBP68-86免疫大鼠的血清与4条病毒肽之间无明显的交叉反应性;发病鼠脊髓内有大量浸润细胞,免疫组化显示为T细胞和巨噬细胞浸润并有小胶质细胞激活。结论JC病毒肽具有与MBP68-86相似、但较弱的致病原性;其可能具有与MBP68-86相似的T细胞表位,而缺乏相似的B细胞表位;提示病毒的分子模拟学说在多发性硬化(MS)发病中可能有一定的作用。

Purpose To observe whether Lewis rats immunized with viral peptides with sequence similarities with myelin basic protein （MBP） could develop experimental autoimmune encephalomyelitis （EAE）. Methods Sixty-six female Lewis rats were randomized into 6 groups with 11 rats each. The rats in groups A to F were immunized with complete FReud Adjuvant （CFA）, guinea pig MBP 68-86, peptides of HBV, JC virus, EBV and HHV-6, respectively. EAE scores were graded and rats weresacrificed at day 15th post immunization. Frozen sections of lumbar spinal cord and lymph node cells suspension were obtained. The lymph node cells of 6 groups were stimulated in vitro with MBP 68-86 and PBS respectively. The lymphocytes proliferation assay and IFN-7 production were tested. Cross-reactivities between the antibodies induced by MBP 68-86 and four synthetic peptides were measured. HE-stained spinal cord sections of each rat were examined for inflammatory cell infiltration and immunohistochemistry methods were used to classify the infiltration cells. Results Ten rats in group B and 6 rats in group D developed EAE. Lymphocytes of rats in group B and D proliferated vigorously to MBP 68-86 and produced higher levels of IFN-7 upon MBP 68-86 stimulation compared to CFA group （P〈0.05）. There were no obvious cross-reactivities among sera antibodies of the rats immunized with MBP 68-86 and four viral peptides. Extensive inflammatory cells in parenchyma and perivascular cuffing were found in spinal cord sections of EAE rats. Immunohistochemistry findings showed that there were many T cells and macrophages, together with microglial cells′ activation. Conclusions JC virus peptide has similar, but weaker immunogenicity,compared with guinea pig MBP 68-86. It might have similar T cell epitopes with MBP 68-86, but not B cell epitopes. Our research provides evidence for molecular mimicry hypothesis in pathogenesis of multiple sclerosis.