胍丁胺对实验性自身免疫性脑脊髓炎的治疗作用及机制

发布时间：2018-05-29 18:32

本文选题：实验性自身免疫性脑脊髓炎 + 多发性硬化　；参考：《吉林大学》2016年博士论文

【摘要】：多发性硬化(multiple sclerosis,MS)是中枢神经系统一种自身免疫性疾病,以炎症,脱髓鞘,轴索缺失,神经胶质细胞过度增生为特征。MS仍无法治愈,目前治疗主要在于减轻疾病的复发和延缓疾病的进展。实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)是MS的经典动物模型。胍丁胺(Agmatine,Agm)是一种内生性胺。EAE发病过程中骨形成蛋白(bone morphogenetic protein,BMP)的表达升高。研究表明Agm可通过调节BMP2/4/7的表达可以改善脊髓损伤后的神经功能。Agm对MS/EAE将会是一种潜在的治疗方法,应用Agm治疗MS/EAE的研究目前较少。研究目的:观察Agm对谷氨酸诱导损伤性神经元的保护作用;用髓鞘少突胶质细胞糖蛋白和完全弗式佐剂混合乳液皮下注射C57BL/6小鼠建立EAE动物模型;用Agm腹腔注射EAE小鼠,观察EAE症状的变化,并阐述其可能治疗机理。研究方法:(1)建立谷氨酸损伤模型及星型胶质细胞与神经元共培养体系。(2)Agm刺激星型胶质细胞和神经元,ELISA检测BMP的表达。(3)Agm刺激谷氨酸诱导的共培养的星型胶质细胞与神经元。(4)用MOG35-55加完全弗氏佐剂(CFA)混合乳液皮下注射C57BL/6小鼠,腹腔注射百日咳毒素(pertussis toxin,PTX)建立EAE模型。(5)EAE动物,分不同时间,不同剂量腹腔注射Agm,对照组PBS腹腔注射,观察各组的治疗效果。(6)发病高峰期(第20天)分离治疗有效组,对照组和正常组小鼠脊髓,通过免疫荧光,Western blotting和PCR检测各组BMP2、BMP4、BMP7,p Smad1/5/8,GFAP,Olig2,MAP2,CD11b/c,NF-κBp65,p-IκB-α,IκB-α的表达量。研究结果:(1)Agm对神经元的保护作用与剂量有关。(2)Agm刺激星型胶质细胞和神经元后BMP7表达明显上升(P 0.01)。(3)MOG抗原和CFA乳液皮下注射,PTX腹腔注射能够有效诱导EAE模型。(4)与PBS组相比,Agm治疗组脊髓内BMP2/4/7,NF-κBp65,p-IκB-α的表达降低(P 0.01)。(5)与PBS组相比,Agm治疗组脊髓内IκB-α的表达增高(P 0.01)。(6)与PBS组相比,Agm治疗组脊髓内小胶质细胞和的数量明显减少了(P 0.01),神经元和少突胶质细胞的数量明显增加了(P 0.01)。(7)所有实验均重复3次以上,所得数据用均数±标准差表示,两组之间的计量资料的均数比较用t检验,三组之间的均数比较用单因素方差分析,用所有实验均重复3次以上,所得数据用均数±标准差表示,两组之间的计量资料的均数比较用t检验,三组之间的均数比较用单因素方差分析,用重复测量数据的方差分析比较各EAE治疗组之间的差异是否具有统计学意义,P 0.05时差差异具有统计学意义。研究结论:(1)Agm可通过调节BMP7的表达保护谷氨酸引起的神经元损伤。(2)C57BL/6小鼠应用MOG35-55加CFA可成功诱导EAE模型。(3)Agm能有效减轻EAE症状。(4)Agm可通过调节BMP2、7的表达减轻EAE症状。(5)Agm能有效减轻EAE小鼠脊髓内小胶质细胞的表达,增加少突胶质细胞和神经元的表达。
[Abstract]:Multiple sclerosis (MS) is an autoimmune disease of the central nervous system, characterized by inflammation, demyelination, axonal deletion and glial hypertrophy. Current treatment mainly aims at reducing the recurrence of disease and delaying the progress of disease. Experimental autoimmune encephalomyelitis (EAE) is a classical animal model of MS. The expression of bone morphogenetic protein was increased during the pathogenesis of agmatine agm, an endogenic amine. Studies have shown that Agm can improve the neural function after spinal cord injury by regulating the expression of BMP2/4/7. AGM will be a potential treatment for MS/EAE. There are few studies on the treatment of MS/EAE with Agm. Objective: to observe the protective effect of Agm on glutamate induced injured neurons, to establish EAE animal model by injecting C57BL/6 mice subcutaneously with oligodendrocyte glycoprotein and complete Freund adjuvant mixed emulsion, and to inject EAE mice intraperitoneally with Agm. To observe the changes of EAE symptoms and explain its possible therapeutic mechanism. Methods: to establish the model of glutamic acid injury and the co-culture system of astrocytes and neurons. AGM stimulated astrocytes and neurons were used to detect the expression of BMP in astrocytes and astrocytes induced by glutamate by Elisa. Neurons. 4) C57BL/6 mice were subcutaneously injected with MOG35-55 and complete Freund's adjuvant CFAs. EAE model was established by intraperitoneal injection of pertussis toxinia (PTX). AGM was injected intraperitoneally at different time and dose, and PBS was injected intraperitoneally in the control group. The therapeutic effect of each group was observed. The expression of CD11b- 魏 -p65 p-I 魏 B- 伪 in the spinal cord of mice in control group and normal group was detected by immunofluorescence Western blotting and PCR, and the expression of CD11b- 魏 Bp65 p-I 魏 B- 伪 was detected by BMP2, BMP4, BMP7, Smad1 / 5 / 8, GFAPAPOlig2MAP2map2CD11b- 魏 -p65-p-I 魏 B- 伪. Results the protective effect of AGM on neurons was related to the dose-dependent dose. The expression of BMP7 in astrocytes and neurons was significantly increased after stimulation with AGM and CFA emulsion injection could effectively induce EAE model. 4) and PBS group. Compared with AGM treatment group, the expression of BMP2 / 4 / 7 NF- 魏 Bp65-p-I 魏 B- 伪 in the spinal cord decreased (P 0.01) and the expression of I 魏 B- 伪 in the AGM treatment group was higher than that in the PBS group (P < 0.01); compared with the PBS group, the expression of microglia and the number of microglia in the spinal cord in the AGM treatment group decreased significantly (P 0.01), and the number of neurons and fewer neurons in the spinal cord of the AGM treatment group was significantly lower than that in the AGM group. The number of glial cells increased significantly (P 0.01. 0. 0. 7) all the experiments were repeated more than 3 times. The data were expressed as mean 卤standard deviation, the mean of measurement data between the two groups was compared with t test, and the comparison between the three groups was analyzed by single factor ANOVA, and all the experiments were repeated more than three times. The data were expressed as mean 卤standard deviation, the mean of measurement data between the two groups was compared by t test, and the mean of the three groups was compared by single factor analysis of variance. The variance analysis of repeated measurement data was used to compare the differences between the EAE treatment groups (P < 0. 05). Conclusion the results suggest that the expression of BMP7 can protect the neuronal damage induced by glutamate in the mice with MOG35-55 and CFA. The model of EAE can be induced successfully by MOG35-55 and CFA. It can effectively alleviate the symptoms of EAE. AGM can alleviate the symptoms of EAE by regulating the expression of BMP2O7. 5 AGM can alleviate the symptoms of EAE. 5 AGM can be used to induce EAE model successfully. The effect of AGM can be reduced by regulating the expression of BMP2O7. To reduce the expression of microglia in spinal cord of EAE mice. Increased expression of oligodendrocytes and neurons.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R744.51

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