摘要
目的探讨采用分子克隆技术合成MOC^(Igd)-TrxA融合蛋白免疫C57BL/6小鼠并建立多发性硬化(MS)动物模型实验性自身免疫性脑脊髓炎(EAE)的可行性;通过检测EAE小鼠脾脏中CD4^+CD25^+调节性T细胞数量初步探讨CD4^+CD25^+调节性T细胞在EAE发病机制中的作用。方法 (1)分子克隆技术合成MOG^(Igd)-TrxA融合蛋白,纯化、超滤浓缩后用Bradford法测定蛋白浓度。(2)C57BL/6小鼠分为MOG^(Igd)-TrxA组(MOG组)、豚鼠脊髓匀浆组(GPSCH组)、硫氧还蛋白组(TrxA组)及正常对照组(NC组)4组,12只/组,各组以相应抗原乳剂免疫小鼠制作EAE模型后评估其临床神经功能、组织病理学改变(HE染色和髓鞘Luxol fast blue染色)并评价模型质量。(3)流式细胞仪(FACS)检测小鼠脾脏中CD4^+CD25^+调节性T细胞百分比。结果 (1)纯化浓缩后MOG^(Igd)-TrxA蛋白纯度达98%左右,浓度约2.3 mg/mL。(2)MOG组小鼠与GPSCH组小鼠在临床神经功能评分等方面差异无统计学意义(P>0.05)。两组发病动物组织切片HE染色和髓鞘染色均有不同程度病理改变。(3)CD4^+CD25^+调节性T细胞占CD4^+T细胞比例MOG组为(4.71±1.61)%,GPSCH组为(1.44±0.65)%,均明显低于正常对照组[(9.22±1.24)%]和TrxA组[(8.97±1.20)%](P<0.01)。结论 (1)分子克隆技术合成的MOG^(Igd)蛋白免疫C57BL/6小鼠能够成功诱导出EAE模型,且模型稳定、发病率高,这为进一步研究MS免疫发病机制并采取有效治疗措施提供了一定依据。(2)CD4^+CD25^+调节性T细胞数量与EAE小鼠临床表现呈相关关系。
Objective To investigate the feasibility of inducing experimental autoimmune encephalomyelitis （EAE） models in C57BL/6 mice by MOG^Igd-TrxA fusion protein which was produced through molecular cloning in authors laboratory. Through testing the percentages of CD4 ＋ CD25 ＋ T cells to preliminarily investigate the role of CD4＋CD25＋T cells in the pathogenesis of EAE. Methods （1）The MOG^Igd-TrxA fusion protein was induced and produced by molecular cloning and purified and concentrated, then the protein concentration was measured by Bradford method. （2） Animal experiment： C57BL/6 mice, 12 mice in each group,the mice in MOG group were immunized with MOG^Igd-TrxA fusion protion. Mice in group GPSCH received emulsion of spinal cord homogenate of guinea pigs（GPSCH） and mice received the same volume emulsiom of TrxA and saline/adjuvant were in group TrxA and normal control group （group NC）separately. Clinical neurologic function scores and histopathology were measured to value the models quality. （3） The percentages of CD4＋ CD25＋ regulatory T cells of EAE mice were tested through flow cytometric analysis. Results （1） The MOG^Igd-TrxA fusion protein purity was about 98% after being purified, and its concentration was 2.3 mg/mL. （2） There was no significant differences between the group MOG and group GPSCH in the Clinical neurologie function scores（P〉0.05）. Histologic sections of the brain and spinal cord taken from affected animals of both groups showed pathological change of different levels throughout the central nervous system（CNS）. （3）Percentages of CD4＋CD25＋ T cells in group MOG and group GPSCH were （4.71±1.61）% and （1.44±0.65）% respectively, both of which were significantly lower than those in group NC [（9.22± 1.24） %] and TrxA group [（8.97± 1.20） %] （P〈0.01）. Conclusions （1） The animal models of EAE in C57BL/6 mice induced by MOG^Igd fusion protein, which was produced through molecular cloning in our laboratory, was stable and
出处
《中国神经免疫学和神经病学杂志》
CAS
2009年第2期104-108,共5页
Chinese Journal of Neuroimmunology and Neurology
