【作者】 张艳；

【导师】 秦新月；

【作者基本信息】 重庆医科大学 ， 神经病学， 2010， 硕士

【摘要】 目的多发性硬化是中枢神经系统(Central nervous system, CNS)常见的自身免疫性疾病,以慢性炎性脱髓鞘为主要病理改变。在青壮年发病率相对较高,其具体的发病机制不明,缺乏有效的特异性治疗,具有较高的致残率。实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis, EAE)是研究多发性硬化常用的动物模型,该模型在临床、病理、生化等方面均能很好地模拟多发性硬化的发病过程。Nogo受体(nogo receptor,NgR)是轴突再生抑制因子Nogo-A,髓鞘相关糖蛋白(Myelin-associated glycoprotein, MAG),少突胶质细胞髓鞘糖蛋白(Oligodendrocyte Myelin glycoprotein, OMgp)的共受体,因而在CNS损伤后轴突再生障碍中起重要作用。近来有研究表明在周围神经损伤的动物模型中,NgR可能可引导损伤部位巨噬细胞的清除,从而起到调节炎症的作用。此外,有研究表明在多发性硬化病人的脑组织中NgR的表达是增高的。这些都提示NgR在CNS免疫性疾病中可能具有免疫调节的作用。近年来,基因沉默技术中的短链RNA干扰(short interfering RNAs, siRNAs)技术可高度特异性降解目的基因,且不引起非特异性干扰反应,因而对揭示某疾病中表达异常增高的基因或蛋白的功能是非常有用。小胶质细胞是中枢神经系统中常驻的单核吞噬细胞之一,其在功能及形态上与巨噬细胞具有许多相似之处,参与脑内的炎症反应,并可能参与髓鞘的损伤与修复过程。因此,通过重组腺病毒为载体的NgR特异性shRNA转染EAE大鼠脑组织,并观察转染前后小胶质细胞及髓鞘的情况,可有助于阐述NgR在EAE中是否具有调节小胶质细胞及髓鞘再生的作用。本研究拟探讨：1在EAE动物模型中,NgR的的表达是否增高,以及小胶质细胞的数量变化及激活等情况；2以重组腺病毒为载体的NgR特异性shRNA转染EAE大鼠的侧脑室,转染后EAE大鼠脑组织NgR基因及蛋白表达的变化,以及转染后EAE脑组织小胶质细胞的数量及激活情况的变化；3该转染对EAE大鼠临床发病及脑组织炎性细胞聚集、髓鞘缺失情况的影响。从而揭示NgR在多发性硬化中可能具有的调节小胶质细胞及髓鞘再生的作用。为阐明多发性硬化等中枢神经免疫性疾病的发病机制提供理论依据,并为其基因治疗的临床应用奠定基础。方法1.建立Wistar大鼠EAE模型,运用MRI扫描、HE染色、Luxol Fast Blue染色等方法鉴定EAE模型制备是否成功；通过免疫荧光染色观察EAE脑组织中NgR表达是否上调,以及巨噬/小胶质细胞的数量是否增加及其活化情况。2.应用人胚肾293细胞扩增重组腺病毒,采用赛多利斯腺病毒纯化试剂盒纯化、收集重组腺病毒,通过TCID50测定病毒滴度；用等体积的高、中、低滴度的重组腺病毒转染EAE大鼠侧脑室,荧光显微镜观察绿色荧光蛋白(green fluorescence protein,GFP)的表达,采用HE染色观察局部炎症反应,从而筛选出最佳的转染滴度。3.用中滴度的Ad-shRNA-NgR、Ad-shRNA-Hk分别转染重组腺病毒载体Ad-shRNA-NgR干预组(特异性RNAi组)及重组腺病毒载体Ad-shRNA-HK干预组(阴性对照组),空白对照组以等量的PBS代替。在转染后第7及14天取材,通过RT-PCR观察转染后脑组织NgRmRNA的表达,免疫荧光染色观察转染后脑组织NgR蛋白的表达情况以及小胶质细胞的数量变化及激活情况,行HE、Luxol Fast Blue染色以观察炎性反应、髓鞘缺失等组织病理学变化,并对各组大鼠的临床转归进行记录。结果1.所制备的EAE模型在MRI及病理学方面的改变符合经典EAE表现。EAE脑组织中NgR的表达上调,小胶质细胞数量增加,并以激活的小胶质细胞为主。2.以GFP标记了的重组腺病毒转染293细胞后,转染后第24-72小时,荧光显微镜下观察发现：GFP表达逐渐增多。经扩增、纯化后得重组腺病毒Ad-shRNA-NgR及Ad-shRNA-Hk的滴度分别为：2.93×1010pfu/ml,2.84×1010pfu/ml。高、中、低滴度组EAE大鼠脑组织均出现GFP阳性表达,且高、中滴度组转染效率明显高于低滴度组,但高滴度组与中滴度组的GFP表达无明显差异。HE染色显示,高滴度组大鼠脑组织转染局部有明显急性炎性反应,其余组转染局部未发现明显炎性反应。3.转染后第7天(d14)：特异性RNAi组脑组织中NgR mRNA及蛋白的表达明显下降(p<0.01),小胶质细胞的表达及髓鞘缺失情况与阴性及空白对照组未见明显差异。转染后第14天(d21)：特异性RNAi组脑组织中NgR mRNA及蛋白表达仍低于空白及阴性对照组(p<0.01),但较该组转染后第7天时有所增加(p<0.05)；小胶质细胞的数量较空白及阴性对照组多(p<0.05),且以活化状态为主；髓鞘缺失的恢复较空白及阴性对照组好(p<0.05)。4.与空白及阴性对照组比较,特异性RNAi组大鼠的发病潜伏期延长(p<0.05)、临床症状评分减轻(p<0.05)；d21时的髓鞘缺失缓解略明显；d21时,病灶部位内的炎性细胞消退较慢。结论EAE模型的制备是成功的。中滴度的腺病毒可高效、安全地转染EAE脑组织。NgR特异性RNAi可明显降低EAE脑组织中NgR基因及蛋白的表达,并有利于髓鞘缺失的修复,但对于激活的小胶质细胞的早期清除不利。该现象可能是通过病灶内激活的小胶质细胞启动了髓鞘再生的修复机制,从而促进髓鞘再生。更多还原

【Abstract】 Objective Multiple sclerosis (MS) is a inflammatory demyelinating disease in central nervous system (CNS). With the development of magnetic resonance (MRI), The incidence of MS is increasing. The pathogenesis of MS is unclear. Furthermore there is still no good way to treat this disease. So to search for a new method for nerve protection in this disease is very important. EAE is a classical model of MS. It can simulate MS no matter in clinic or in pathology. Nogo receptor is the same receptor for MAG, OMgp and NgR. The three proteins above are the inhibitors for axons regeneration in CNS. Right now some projects indicate that NgR may participate in inflammatory. And in the CNS tissue of MS, the expression of NgR is increased. It seems that NgR may play some role in MS. RNAi can reduce the expression of objective gene. Ad-shRNA-NgR can down regulation the expression of NgR. Microglias is the principal member of macrophage family in CNS. These cells might sustain and propagate inflammation within the CNS during autoimmune inflammation. So by transfecting the Ad-shRNA-NgR into the experimental autoimmune encephalomyelitis (EAE) model’lateral cerebral ventricle and observation the microglias and remyelination could help us to find the effects of NgR on microglias and remyelination.In this research, after transfection the Ad-shRNA-NgR into the experimental autoimmune encephalomyelitis(EAE) model’lateral cerebral ventricle, we investigated:the expression of nogo receptor(NgR)mRNA and protein, the aggregation of inflammation cells, the depletion of myelin sheath and the clinical invasion of the EAE model. By doing this we can find the contribution of NgR myelin regeneration and microglias. This project can provide a therapeutic approach for MS and other inflammation diseases of neurology.Method 1.Established the EAE model and MRI scanning, the dyeing of HE, Luxol fast blue, microglias were carried out for evaluation.2. The recombinated adenovious was amplified in 293 cells(human embroyonic kidney cells)and purified by Sartorius Vivapure AdenoPACK 20 kit.The virus titer was determined by TCID50. Three different titers of recombinated adenovious(high titer, middle titer, low titer) were used to transfect the brain tissue of EAE. Histopathological examination was performed to evaluate the local inflammatory reaction. And green fluorescent protein (GFP) expression was observed under fluorescence microscope. The optimal transfective virus titer was selected;3.48 female Wistar rats were randomed into normal control group、blank control group, Ad-shRNA-HK intervention group(negative control group), Ad-shRNA-NgR intervention group (specific RNAi group). Reverse transcription polymerases chain reaction (RT-PCR) was used to determine the changes of NgR mRNA expression at the 7th and 14th day after transfection.The stain of HE、Luxol Fast Blue、NgR and CDllb/C was also carried out at the 7th and 14th day after transfection.Result 1.The MRI scanning and pathological observations of the rats were consistent with those of classical EAE model.2. After 24 hours of transfection the fluorescence was observed in 293 cells. The titer of amplified recombined adenovirus Ad-shRNA-NgR and Ad-shRNA-HK were 2.93×1010pfu/ml and 2.84×1010pfu/ml respectively. Green fluorescence protein(GFP) could be found in the brain of the high, middle and low titer group at the 7th day of transfection. GFP expression in the high and middle titer group was obviously higher than that in the low titer group. While there was no obvious difference between the high and middle titer group. HE staining showed that acute inflammatory infiltration was only observed in the high titer group;3. NgR protein in the brain of EAE was significantly increased at the 14th and 21th day after induction (p<0.01). NgR mRNA and protein was reduced in the brain of EAE at the 7th and 14th day after RNAi intervention (p<0.01);4.Compared with the blank control group, the Ad-shRNA-NgR specific intervention group had longer latency (p<0.05)、lower clinical score;at the d21,the more significant regeneration of myelin sheath, while the slower extinction of inflamation.Conclusion The EAE model was induced successfully. The middle titers of adenovirus can transfect the brain tissue of EAE model efficiently and safely. The expression of NgR was increased in the brain of EAE. Ad-shRNA-NgR interfering can degrade the expressing of NgR remarkably and enhance the regeneration of myelin sheath, but it’s not good for extinction of activated microglias.更多还原