HMGB1在实验性自身免疫性脑脊髓炎病程中的作用

发布时间：2018-06-29 21:35

本文选题：高迁移率族蛋白1 + 实验性自身免疫性脑脊髓炎　；参考：《华中科技大学》2015年博士论文

【摘要】：背景：高迁移率族蛋白1(high-mobility group box-1protein, HMGB1)是一种高度保守的非组蛋白染色体结合核蛋白,HMGB1的表达量、细胞定位和亚细胞定位模式决定其作用。近期研究表明,HMGB1参与了自身免疫性脑脊髓炎的发生,同时当脊髓和脑损伤时HMGB1导致机体神经炎症,但在疾病发生、发展过程中脊髓组织HMGB1表达模式的动态变化仍不清楚。本研究为揭示HMGB1在实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis, EAE)病程中的作用提供了新的证据,局部阻断HMGB1缓解EAE临床症状和抑制CNS炎症提供了新的治疗思路。目的：阐明EAE疾病病程中成年小鼠脊髓组织HMGB1数量、细胞定位和亚细胞定位以及给予HMGB1拮抗剂后对EAE疾病的影响。方法：利用MOG35-55抗原肽免疫C57BL/6小鼠建立EAE模型,根据EAE的临床症状和疾病发展趋势将EAE划分为发病前期(Pre-onset)、发病初期(Onset)、高峰期(Peak)和慢性期(Chronic)四个时期。以正常C57BL/6小鼠作为对照。在EAE病程相应的时间点取材进行以下检测： 1.通过蛋白印迹技术和免疫组织化学染色检测EAE病程中脊髓组织HMGB1表达。通过酶联免疫吸附试验检测EAE病程中不同体液(血清、脊髓组织间液和脑脊液)中HMGB1的水平。通过免疫组织化学染色和蛋白印迹技术检验脊髓组织中细胞核和胞浆的内源性HMGB1表达情况。通过免疫荧光双重染色和免疫组织化学染色观察了EAE病程中脊髓组织TLR4的动态表达和TLR4在CNS中细胞定位。同时,通过苏木素伊红染色检测EAE病程中脊髓炎性细胞浸润情况。 2.通过免疫荧光双重染色观察HMGB1在正常成年小鼠脊髓组织、CNS细胞株(小鼠神经母细胞瘤细胞N2a、小鼠小胶质细胞BV2和脑胶质瘤细胞U87)的神经元、小胶质细胞和星形胶质细胞中的细胞核定位,体外原代培养神经元和星形胶质细胞进一步确定胞核HMGB1的表达。通过免疫荧光双重染色观察EAE病程中脊髓组织HMGB1表达模式的动态变化。 3.观察HMGB1拮抗剂(甘草甜素(glycyrrhizin, GL))或中和性抗HMGB1单克隆抗体(neutralizing anti-HMGB1monoclonal antibody, HMGB1Ab))对EAE脱髓鞘的影响,通过预防性和治疗性腹腔注射GL、HMGB1Ab或侧脑室注射HMGB1Ab,评估EAE临床症状、病理特征、不同体液中HMGB1的水平、脊髓组织HMGB1的表达、CNS星形胶质细胞、小胶质细胞和神经元的变化。流式细胞术检测腹腔给予HMGB1Ab处理后对脾脏Th1、Th17细胞数量的影响。结果： 1.EAE病程中脊髓组织表达HMGB1增加：与Normal组相比较,EAE病程四个时期脊髓组织表达HMGB1总蛋白均显著增加,于Onset期达到最高峰。不同体液(血清、脊髓组织间液和脑脊液)中胞外HMGB1的水平增加；EAE高峰期脊髓细胞HMGB1从细胞核向胞浆转位；EAE病程中脊髓组织表达TLR4增加,神经元、星形胶质细胞和小胶质细胞均表达TLR4。组织病理学检测发现脊髓组织中有炎性细胞浸润,特别是在EAE发病初期和高峰期。 2.正常状态下脊髓组织部分星形胶质细胞和小胶质细胞的胞核表达HMGB1,少许神经元的胞核表达HMGB1; CNS细胞株(小鼠神经母细胞瘤细胞(N2a)、小鼠小胶质细胞(BV2)、脑胶质瘤细胞(U87))的胞核表达HMGB1。同时,原代培养的神经元和星形胶质细胞进一步证实了细胞核表达HMGB1。然而,EAE病程中脊髓组织HMGB1总蛋白和胞外HMGB1水平升高,HMGB1阳性的星形胶质和小胶质细胞数量增加,伴随着胞外HMGB1水平升高,定居于脊髓前角和中央管周围区域的神经元核HMGB1向胞浆转位。 3.体内实验结果显示,腹腔预防性给予GL不影响EAE发生、发展,腹腔治疗性给予GL能够缓解EAE临床症状,侧脑室治疗性给予HMGB1Ab处理几乎完全抑制EAE发生、发展。治疗性给予HMGB1拮抗剂GL或HMGB1Ab发挥有效的抗炎作用缓解EAE临床症状,减轻脊髓组织炎性细胞浸润和髓鞘脱失,降低不同体液中HMGB1水平和脊髓组织HMGB1表达,下调外周脾脏组织Th1和Th17细胞数量,抑制EAE脊髓组织中星形胶质细胞、小胶质细胞的活化,同时降低神经元的损伤。结论：本研究观察了EAE疾病发生、发展过程中脊髓组织HMGB1表达模式的动态变化,在疾病起病阶段阻断HMGB1能够缓解EAE临床症状,自身免疫性脑脊髓炎(人类多发性硬化症)疾病可以以HMGB1为潜在的治疗靶点。
[Abstract]:Background: high mobility group protein 1 (high-mobility group box-1protein, HMGB1) is a highly conserved non histone chromosome binding nucleoprotein. The expression of HMGB1, cell location and subcellular localization patterns determine its role. Recent studies have shown that HMGB1 is involved in the occurrence of autoimmune encephalomyelitis, and at the same time as spinal cord and brain damage. HMGB1 causes neuroinflammation in the body, but the dynamic changes in the HMGB1 expression pattern in the spinal cord are still unclear during the development of the disease. This study provides new evidence to reveal the role of HMGB1 in the course of experimental autoimmune encephalomyelitis (experimental autoimmune encephalomyelitis, EAE), partially blocking the slow HMGB1. It provides new therapeutic ideas for solving EAE clinical symptoms and inhibiting CNS inflammation.
Objective: to elucidate the effects of HMGB1 on the number of EAE in adult spinal cord tissue, cell localization and subcellular localization, and the effects of HMGB1 antagonists on EAE disease.
Methods: the EAE model was established by immunization of MOG35-55 antigen peptide in C57BL/6 mice. According to the clinical symptoms of EAE and the development trend of the disease, EAE was divided into four stages of early onset (Pre-onset), early onset (Onset), peak period (Peak) and chronic phase (Chronic). Normal C57BL/6 mice were used as control. The time points were taken at the corresponding time points of EAE course. The following test:
1. the expression of HMGB1 in the spinal cord of EAE was detected by Western blot and immunohistochemical staining. The level of HMGB1 in different body fluids (serum, spinal intertissue fluid and cerebrospinal fluid) in the course of EAE disease was detected by enzyme linked immunosorbent assay. The nucleus and cell in spinal cord tissue were examined by immunohistochemistry and egg white blot. The expression of endogenous HMGB1 in the pulp was observed. The dynamic expression of TLR4 in the spinal cord tissue in the course of EAE and the location of TLR4 in CNS were observed by immunofluorescence double staining and immunohistochemical staining. Meanwhile, the infiltration of inflammatory cells in the course of EAE was detected by hematoxylin staining.
2. the neurons of HMGB1 in normal adult mouse spinal cord, CNS cell line (mouse neuroblastoma cell N2a, mouse microglia BV2 and glioma cell U87), cell nucleus localization in microglia and astrocytes, and primary cultured neurons and astrocytes in vitro were observed by immunofluorescence double staining. The expression of HMGB1 was determined by immunofluorescence double staining. The dynamic changes of HMGB1 expression pattern in spinal cord tissue during EAE course were observed.
3. observe the effect of HMGB1 antagonist (glycyrrhizin, GL) or neutralized anti HMGB1 monoclonal antibody (neutralizing anti-HMGB1monoclonal antibody, HMGB1Ab) on the demyelination of EAE, through the preventive and therapeutic intraperitoneal injection of GL, HMGB1Ab or lateral ventricle injection HMGB1Ab, to evaluate the clinical symptoms, pathological features, different body fluids. Level, expression of HMGB1 in spinal cord tissue, changes in CNS astrocytes, microglia and neurons. Flow cytometry was used to detect the effect of HMGB1Ab treatment on the number of Th1 and Th17 cells in the spleen.
Result:
In the course of 1.EAE, the expression of HMGB1 increased in the spinal cord tissue. Compared with the Normal group, the total HMGB1 protein expression in the spinal cord tissue in the four period of the EAE course increased significantly, and reached the peak in the Onset phase. The level of extracellular HMGB1 in the different body fluids (serum, spinal intertissue fluid and cerebrospinal fluid) increased; HMGB1 from the nucleus to the peak of EAE from the nucleus to the cytoplasm at the peak period of EAE. Transposition; the expression of TLR4 in the spinal cord tissue in the course of EAE was increased, and the neurons, astrocytes and microglia were all expressed by TLR4. histopathological detection and found that there was inflammatory cell infiltration in the spinal cord, especially at the early stage and peak period of the pathogenesis of EAE.
2. the nucleus of astrocytes and microglia in the spinal cord tissue expressed HMGB1, and the nucleus of a few neurons expressed HMGB1, and the nucleus of CNS cell (N2a), mouse microglia (BV2), glioma cells (U87) expressed HMGB1. simultaneously, the primary cultured neurons and astroglia were fine. The cell nucleus expressed HMGB1. further, however, the level of HMGB1 total protein and extracellular HMGB1 increased in the course of EAE, and the number of HMGB1 positive astrocytes and microglia increased. With the increase of extracellular HMGB1 level, the nucleus HMGB1 settled in the anterior horn of the spinal cord and the region around the central canal to the cytoplasm of the cytoplasm.
3. in vivo experimental results showed that the intraperitoneal prophylactic administration of GL did not affect the occurrence of EAE, development, GL could relieve the clinical symptoms of EAE, and the therapeutic effect of HMGB1Ab treatment in the lateral ventricle almost completely inhibited the occurrence of EAE, and the therapeutic effect was given to the HMGB1 antagonist GL or HMGB1Ab volatile anti-inflammatory effects to alleviate the clinical symptoms of EAE and reduce the spinal cord. Inflammatory cell infiltration and myelin loss in medullary tissue decreased HMGB1 level in different body fluids and HMGB1 expression in spinal cord tissue, reduced the number of Th1 and Th17 cells in the peripheral spleen tissue, inhibited the activation of astrocytes and microglia in EAE spinal tissue, and decreased the damage of neurons.
Conclusion: This study observed the dynamic changes in the HMGB1 expression pattern of spinal cord tissue during the development of EAE disease. Blocking HMGB1 in the onset stage of the disease could alleviate the clinical symptoms of EAE, and autoimmune encephalomyelitis (human multiple sclerosis) disease could be used as a potential therapeutic target for HMGB1.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R744.5

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