本文選題：高遷移率族蛋白1 + 實(shí)驗(yàn)性自身免疫性腦脊髓炎�。� 參考：《華中科技大學(xué)》2015年博士論文

【摘要】：背景：高遷移率族蛋白1(high-mobility group box-1protein, HMGB1)是一種高度保守的非組蛋白染色體結(jié)合核蛋白,HMGB1的表達(dá)量、細(xì)胞定位和亞細(xì)胞定位模式?jīng)Q定其作用。近期研究表明,HMGB1參與了自身免疫性腦脊髓炎的發(fā)生,同時當(dāng)脊髓和腦損傷時HMGB1導(dǎo)致機(jī)體神經(jīng)炎癥,但在疾病發(fā)生、發(fā)展過程中脊髓組織HMGB1表達(dá)模式的動態(tài)變化仍不清楚。本研究為揭示HMGB1在實(shí)驗(yàn)性自身免疫性腦脊髓炎(experimental autoimmune encephalomyelitis, EAE)病程中的作用提供了新的證據(jù),局部阻斷HMGB1緩解EAE臨床癥狀和抑制CNS炎癥提供了新的治療思路。 目的：闡明EAE疾病病程中成年小鼠脊髓組織HMGB1數(shù)量、細(xì)胞定位和亞細(xì)胞定位以及給予HMGB1拮抗劑后對EAE疾病的影響。 方法：利用MOG35-55抗原肽免疫C57BL/6小鼠建立EAE模型,根據(jù)EAE的臨床癥狀和疾病發(fā)展趨勢將EAE劃分為發(fā)病前期(Pre-onset)、發(fā)病初期(Onset)、高峰期(Peak)和慢性期(Chronic)四個時期。以正常C57BL/6小鼠作為對照。在EAE病程相應(yīng)的時間點(diǎn)取材進(jìn)行以下檢測： 1.通過蛋白印跡技術(shù)和免疫組織化學(xué)染色檢測EAE病程中脊髓組織HMGB1表達(dá)。通過酶聯(lián)免疫吸附試驗(yàn)檢測EAE病程中不同體液(血清、脊髓組織間液和腦脊液)中HMGB1的水平。通過免疫組織化學(xué)染色和蛋白印跡技術(shù)檢驗(yàn)脊髓組織中細(xì)胞核和胞漿的內(nèi)源性HMGB1表達(dá)情況。通過免疫熒光雙重染色和免疫組織化學(xué)染色觀察了EAE病程中脊髓組織TLR4的動態(tài)表達(dá)和TLR4在CNS中細(xì)胞定位。同時,通過蘇木素伊紅染色檢測EAE病程中脊髓炎性細(xì)胞浸潤情況。 2.通過免疫熒光雙重染色觀察HMGB1在正常成年小鼠脊髓組織、CNS細(xì)胞株(小鼠神經(jīng)母細(xì)胞瘤細(xì)胞N2a、小鼠小膠質(zhì)細(xì)胞BV2和腦膠質(zhì)瘤細(xì)胞U87)的神經(jīng)元、小膠質(zhì)細(xì)胞和星形膠質(zhì)細(xì)胞中的細(xì)胞核定位,體外原代培養(yǎng)神經(jīng)元和星形膠質(zhì)細(xì)胞進(jìn)一步確定胞核HMGB1的表達(dá)。通過免疫熒光雙重染色觀察EAE病程中脊髓組織HMGB1表達(dá)模式的動態(tài)變化。 3.觀察HMGB1拮抗劑(甘草甜素(glycyrrhizin, GL))或中和性抗HMGB1單克隆抗體(neutralizing anti-HMGB1monoclonal antibody, HMGB1Ab))對EAE脫髓鞘的影響,通過預(yù)防性和治療性腹腔注射GL、HMGB1Ab或側(cè)腦室注射HMGB1Ab,評估EAE臨床癥狀、病理特征、不同體液中HMGB1的水平、脊髓組織HMGB1的表達(dá)、CNS星形膠質(zhì)細(xì)胞、小膠質(zhì)細(xì)胞和神經(jīng)元的變化。流式細(xì)胞術(shù)檢測腹腔給予HMGB1Ab處理后對脾臟Th1、Th17細(xì)胞數(shù)量的影響。 結(jié)果： 1.EAE病程中脊髓組織表達(dá)HMGB1增加：與Normal組相比較,EAE病程四個時期脊髓組織表達(dá)HMGB1總蛋白均顯著增加,于Onset期達(dá)到最高峰。不同體液(血清、脊髓組織間液和腦脊液)中胞外HMGB1的水平增加；EAE高峰期脊髓細(xì)胞HMGB1從細(xì)胞核向胞漿轉(zhuǎn)位；EAE病程中脊髓組織表達(dá)TLR4增加,神經(jīng)元、星形膠質(zhì)細(xì)胞和小膠質(zhì)細(xì)胞均表達(dá)TLR4。組織病理學(xué)檢測發(fā)現(xiàn)脊髓組織中有炎性細(xì)胞浸潤,特別是在EAE發(fā)病初期和高峰期。 2.正常狀態(tài)下脊髓組織部分星形膠質(zhì)細(xì)胞和小膠質(zhì)細(xì)胞的胞核表達(dá)HMGB1,少許神經(jīng)元的胞核表達(dá)HMGB1; CNS細(xì)胞株(小鼠神經(jīng)母細(xì)胞瘤細(xì)胞(N2a)、小鼠小膠質(zhì)細(xì)胞(BV2)、腦膠質(zhì)瘤細(xì)胞(U87))的胞核表達(dá)HMGB1。同時,原代培養(yǎng)的神經(jīng)元和星形膠質(zhì)細(xì)胞進(jìn)一步證實(shí)了細(xì)胞核表達(dá)HMGB1。然而,EAE病程中脊髓組織HMGB1總蛋白和胞外HMGB1水平升高,HMGB1陽性的星形膠質(zhì)和小膠質(zhì)細(xì)胞數(shù)量增加,伴隨著胞外HMGB1水平升高,定居于脊髓前角和中央管周圍區(qū)域的神經(jīng)元核HMGB1向胞漿轉(zhuǎn)位。 3.體內(nèi)實(shí)驗(yàn)結(jié)果顯示,腹腔預(yù)防性給予GL不影響EAE發(fā)生、發(fā)展,腹腔治療性給予GL能夠緩解EAE臨床癥狀,側(cè)腦室治療性給予HMGB1Ab處理幾乎完全抑制EAE發(fā)生、發(fā)展。治療性給予HMGB1拮抗劑GL或HMGB1Ab發(fā)揮有效的抗炎作用緩解EAE臨床癥狀,減輕脊髓組織炎性細(xì)胞浸潤和髓鞘脫失,降低不同體液中HMGB1水平和脊髓組織HMGB1表達(dá),下調(diào)外周脾臟組織Th1和Th17細(xì)胞數(shù)量,抑制EAE脊髓組織中星形膠質(zhì)細(xì)胞、小膠質(zhì)細(xì)胞的活化,同時降低神經(jīng)元的損傷。 結(jié)論：本研究觀察了EAE疾病發(fā)生、發(fā)展過程中脊髓組織HMGB1表達(dá)模式的動態(tài)變化,在疾病起病階段阻斷HMGB1能夠緩解EAE臨床癥狀,自身免疫性腦脊髓炎(人類多發(fā)性硬化癥)疾病可以以HMGB1為潛在的治療靶點(diǎn)。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R744.5

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