负性协同刺激分子PD-L1在实验性变态反应性脑脊髓炎中的表达

发布时间：2017-12-31 07:31

本文关键词：负性协同刺激分子PD-L1在实验性变态反应性脑脊髓炎中的表达　出处：《苏州大学》2010年硕士论文　论文类型：学位论文

【摘要】： 背景与目的多发性硬化(multiple sclerosis, MS)是CD4+Th1细胞介导的中枢神经系统(central nervous system, CNS)脱髓鞘为主的自身免疫性疾病。协同刺激分子PD-Ll分子是新近发现的B7家族的负性协同刺激分子之一,该分子通过与主要表达在活化的T细胞、B细胞的抑制性受体PD-l分子结合发挥负性免疫调节作用。实验性变态反应性脑脊髓炎(experimental allergic encephalomyelitis,EAE)的病理变化和发病机制与MS极其相似,是研究人类多发性硬化的理想动物模型。为了进一步分析PD-L1分子在EAE和MS中可能的免疫作用,本实验用周龄和性别匹配的C57BL/6J小鼠成功建立了EAE动物模型,并检测负性协同刺激分子PD-L1在EAE中的表达,以便进一步探讨PD-L1/PD-1途径在发病机制中的作用,为人类MS的临床治疗提供可能的思路。方法第一部分:30只雌性C57BL/6J小鼠随机分为两组,实验组20只和对照组10只。实验组用髓鞘少突胶质细胞糖蛋白(myelin oligodendrocyte glycoprotein, MOG35-55)加完全弗氏佐剂(complete freund adjuvant,CFA)制备抗原佐剂乳化物腋下皮下单点单次注射免疫小鼠,分别在免疫当天和24小时后尾静脉注射百日咳毒素(bordetella pertussis vaccine,BPV),对照组皮下和尾静脉注射PBS,观察两组动物的发病临床表现并按照国际通用的五分制评分法进行症状分级评分,于免疫后12-14天取对照组和症状明显的实验组小鼠的脊髓进行冰冻切片并行HE染色、Luxol Fast-Blue髓鞘染色以鉴定模型成功与否。第二部分:免疫组化检测脊髓组织PD-L1的表达,并用流式细胞仪(Flowcytometry,FCM)、Weston-Blot方法检测两组动物的脾细胞上PD-L1的表达。结果 1.实验组小鼠最早于抗原诱导后的第10天左右开始出现动作迟缓、尾部无力等症状,以后逐渐加重,于免疫后12-14天症状达高峰,有的甚至大小便失禁、死亡,16-19天症状开始缓解,但症状不能完全缓解,对照组小鼠则无任何临床症状,共观察小鼠30天。 2.切片HE染色光镜下见实验组小鼠脊髓组织血管周围大量炎性细胞浸润,呈“袖套”样改变,对照组小鼠未见明显病理改变;LFB髓鞘染色光镜下见实验组急性期小鼠脊髓组织的白质髓鞘脱失明显,对照组小鼠无此改变。 3.免疫组化显示实验组小鼠脊髓组织PD-L1分子的表达明显增加,流式细胞仪(Flow cytometry FCM)、Weston-Blot检测实验组动物的脾细胞上PD-L1分子的表达明显增加。结论 1.本实验建立的小鼠模型在临床表现、病理变化等方面符合复发-非缓解型EAE的表现,模型稳定、可靠,是研究MS的理想动物模型。 2.实验组动物的PD-L1的表达在外周脾细胞和中枢神经系统的脊髓组织都较对照组明显增加。
[Abstract]:Background and purpose Multiple sclerosis multiple sclerosis. S) is a central nervous system mediated by CD4 Th1 cells. Costimulator PD-Ll is one of the newly discovered negative co-stimulators of B7 family. The molecule is expressed mainly in activated T cells. The inhibitory receptor PD-l molecules of B cells play a negative immunoregulatory role. Experimental allergic Encephalomyelitis. Experimental allergic encephalomyelitis. EAE is an ideal animal model for the study of multiple sclerosis. In order to further analyze the possible immunological effects of PD-L1 molecules in EAE and MS, EAE animal models were successfully established in C57BL / 6J mice with week-old and sex-matched C57BL / 6J mice. The expression of PD-L1 in EAE was detected in order to further explore the role of PD-L1/PD-1 pathway in pathogenesis. To provide possible ideas for clinical treatment of human MS. Method Part one: 30 female C57BL / 6J mice were randomly divided into two groups. The experimental group (n = 20) and the control group (n = 10) were treated with myelin oligodendrocyte glycoprotein. MOG35-55) complete freund adjuvant with complete Freund's adjuvant. CFA) was used to prepare antigenic adjuvant emulsifiers for subaxillary subcutaneous single injection to immunize mice. Pertussis toxin Bordetella pertussis vas was injected intravenously on the day and 24 hours after immunization. PBSs were injected subcutaneously and caudally in the control group. The clinical symptoms of the two groups were observed and the symptoms were graded according to the international common five-point scoring method. At 12-14 days after immunization, the spinal cord of the control group and the experimental group with obvious symptoms were taken for frozen sections and HE staining. Luxol Fast-Blue myelin staining was used to identify the success of the model. The second part: immunohistochemistry was used to detect the expression of PD-L1 in spinal cord and flow cytometry was used to detect the expression of FCM. Weston-Blot method was used to detect the expression of PD-L1 on spleen cells of both groups. Results 1. The mice in the experimental group began to appear the symptoms of slow movement and tail weakness at the first 10 days after antigen induction, then gradually aggravated, and reached the peak at 12-14 days after immunization. Some even incontinence, the death of 16-19 days symptoms began to relieve, but the symptoms can not be completely relieved, the control mice without any clinical symptoms, a total of 30 days observation of mice. 2. Under HE staining, a large number of inflammatory cells were infiltrated around the blood vessels of the spinal cord in the experimental group, but no obvious pathological changes were observed in the control group. LFB staining showed that the myelin demyelination of the white matter in the acute phase of the experimental group was obvious, but that in the control group was not. 3.Immunohistochemistry showed that the expression of PD-L1 in the spinal cord of the experimental group was significantly increased, flow cytometry flow cytometry (FCM). The expression of PD-L1 in spleen cells of experimental group was detected by Weston-Blot. Conclusion 1. The mouse model established in this study is stable and reliable in clinical manifestations and pathological changes. It is an ideal animal model for the study of MS. 2. The expression of PD-L1 in peripheral spleen cells and spinal cord of central nervous system in experimental group was significantly higher than that in control group.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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