【作者】 陈黎燕；

【导师】 秦新月；

【作者基本信息】 重庆医科大学 ， 神经病学， 2009， 硕士

【摘要】 目的多发性硬化是一种致残率较高的中枢神经系统炎性脱髓鞘性疾病。在病理上主要表现为位于中枢神经系统白质的多发性炎性脱髓鞘和轴突损伤,而后者又被认为是多发性硬化致残的主要原因。中枢神经再生是近年来神经康复的难点和热点,而轴突生长抑制因子被认为是导致中枢神经再生困难的主要原因。近年来通过在脊髓损伤和脑损伤模型中的研究发现RGM (repulsive guidance molecule)不仅是一种轴突导向分子,还是一种对中枢神经系统轴突再生具有抑制作用的膜蛋白。RGM参与了中枢神经系统损伤后轴突生长抑制性信号的传递,在轴突再生过程中起关键作用。近年来研究发现,多发性硬化早期存在轴索损伤,RGM在多发性硬化的轴索损伤及修复中是否扮演着重要的角色?迄今尚未见文献报道。本实验拟通过建立急﹑慢性多发性硬化模型,检测RGM在实验性自身免疫性脑脊髓炎(EAE)大鼠脑和脊髓中的表达规律,从而推测RGM在多发性硬化疾病的发生发展和转归中的可能作用和机制。方法运用豚鼠脊髓匀浆和完全弗氏佐剂混合制成油包水抗原乳剂,并注射百日咳杆菌建立EAE大鼠模型。通过HE染色来判断模型成功与否。分别设立正常对照组﹑EAE急性组﹑EAE慢性完全缓解组和EAE慢性部分缓解组,通过神经功能缺损评分判断大鼠临床症状。通过RT-PCR来检测RGMa mRNA在各个组中的表达规律,并通过免疫组化来检测膜蛋白RGMa在各个组脑和脊髓中的表达规律。结果在正常对照组和EAE各组,RGMa mRNA在急性组表达量最高,在慢性组表达量较急性组降低,但仍高于正常对照组。RGMa蛋白在正常对照组主要表达在脑皮质、脊髓灰质﹑白质纤维束、少突胶质样细胞、脉络丛、海马和一些内皮细胞;在EAE急性组RGMa蛋白阳性表达明显较对照组增高,RGMa阳性表达主要聚集在病灶和病灶周围的区域,如血管周围浸润的炎性细胞和病灶周围增生的星形胶质细胞;在EAE慢性组,RGMa蛋白的表达量较急性组稍有降低,但仍高于正常组,主要表达在病灶周围大量增生的星形胶质细胞。结论RGMa mRNA和RGMa蛋白在EAE急性期组的表达明显增高,在EAE慢性组其表达量降低但仍高于正常对照组,提示RGMa与多发性硬化疾病的发生发展密切相关。RGMa蛋白主要表达在少突胶质细胞,星形胶质细胞等几种大量表达轴突生长抑制因子的组织和细胞上,提示RGMa很有可能作为一种轴突生长抑制因子在多发性硬化中起作用。更多还原

【Abstract】 Objective Multiple sclerosis (MS) is a kind of inflammatory demyelinating diseases in central nervous system (CNS) with high disability. In pathology, it presents as multiple inflammatory demyelinating lesions and axonal injury in white matter of CNS and the latter is the main reason of disability of MS patients. In recent years, the regeneration of CNS has been the focus of nervous rehabilitation. The axonal growth inhibitors are considered to be one of the main factors causing the difficulty of CNS regeneration. Recently, the analyses of repulsive guidance molecule (RGM) expression in the model of spinal cord injury and brain injury indicate that RGM is not only an axonal guidance molecue and also an membrane protein limitating axonal growth of CNS. RGM participates and plays an important role in the transfer of axonal growth inhibitory signal after injury of CNS . However, no study has been reported about the RGM expression in multiple sclerosis. In our study, we analyzed the expression pattern of RGM in spinal cord and brain of rats in acute and chronic model of multiple sclerosis and speculated the possible function and mechanism of RGM in the occurrence, progress and prognosis of multiple sclerosis.Methods Experimental autoimmune encephalomyelitis (EAE) was induced by a single 400-μl subcutaneous injection of the following emulsions in the footpad: guinea pig spinal cord homogenate (GPSCH) (50% w/v in saline) emulsified in an equal volume of Freund’s complete adjuvant (FCA) and 0.1 ml of Bordetella pertussis in the instep (approximately 4.0×109 live bacteria). The judgment of the model was done with HE stain. The rats were divided into four groups: the control, EAE acute group, EAE chronic group with complete remission and EAE chronic group with partial remission. Neurological deficits score was used to evaluate EAE. The Immunohistochemistry and double labeling of immunofluorescence were used to analyze the RGMa expression pattern in spinal cord and brain of rats in control and EAE groups. Reverse transcriptase Polymerase Chain Reaction (RT-PCR) was done to detect the expression of RGMa mRNA in the four groups.Results In control group and EAE groups, the expression of RGMa mRNA was highest in EAE acute group and decreased in EAE chronic groups. In brain and spinal cord of control rats, RGMa immunoreactivity was detected on cortex, grey matter, white matter fibers, oligodendrocyte, choroid plexus, hippocampus and few endothelial cells. In EAE acute group, the RGMa immunopositive cells significantly elevated as compared to control and accumulated in lesions and peri-lesional areas such as inflammatory cells aroud vessels and astrocytes. In EAE chronic group, the RGMa immunopositive cells decreased as compared with EAE acute group but still higher than the control and mainly expressed in the hyperplastic astrocytes surrounding the lesions.Conclusion The RGMa mRNA and RGMa protein were increased significantly in EAE acute group and decreased in EAE chronic groups but still higher than control group. It indicated that RGMa may play an important role in the onset and development of multiple sclerosis. RGMa Protein mainly expressed in the tissue or cells such as oligodendrocytes and astrocytes which greatly express axonal growth inhibitor. It indicated that RGMa may act as an axonal growth inhibitor in multiple sclerosis patients.更多还原