本文選題：多發(fā)性硬化 + 實(shí)驗(yàn)性自身免疫性腦脊髓炎　； 參考：《吉林大學(xué)》2016年博士論文

【摘要】：實(shí)驗(yàn)性自身免疫性腦脊髓炎(EAE)是多發(fā)性硬化(MS)的動(dòng)物模型,是由Th1和Th17細(xì)胞介導(dǎo)的一種中樞神經(jīng)系統(tǒng)自身免疫性炎性脫髓鞘疾病動(dòng)物模型。在中樞神經(jīng)系統(tǒng),滲入的自身活化的Th1/Th17(CD4+T)細(xì)胞,CD8+T細(xì)胞,巨噬細(xì)胞/樹(shù)突狀細(xì)胞,肥大細(xì)胞以及活化的小膠質(zhì)細(xì)胞,會(huì)產(chǎn)生大量的促炎性細(xì)胞因子IFN-γ,IL-17,TNF-α,GM-CSF和IL-23等,促進(jìn)細(xì)胞介導(dǎo)的免疫反應(yīng)。盡管發(fā)病機(jī)理尚不完全清楚,但針對(duì)其炎性反應(yīng)的特點(diǎn),目前臨床上多發(fā)硬化的治療多以抗炎和免疫調(diào)節(jié)為主。IFN-β作為臨床上第一個(gè)批準(zhǔn)的免疫調(diào)節(jié)藥物,對(duì)于MS的治療發(fā)揮了重要作用。但是,迄今為止,內(nèi)源性的IFN-β的來(lái)源以及其具體的作用靶細(xì)胞仍不是十分清楚。中樞神經(jīng)系統(tǒng)的炎癥微環(huán)境、炎癥調(diào)節(jié)以及中樞神經(jīng)系統(tǒng)細(xì)胞對(duì)炎癥反應(yīng)的響應(yīng)程度對(duì)于MS/EAE的疾病進(jìn)展具有重要作用。因此,探索中樞神經(jīng)系統(tǒng)病灶組織在神經(jīng)免疫和炎癥情況下的反應(yīng)對(duì)于闡明MS/EAE的發(fā)病機(jī)制并發(fā)現(xiàn)新的治療靶點(diǎn)具有重要意義。在前期的研究中,基于mi RNA多靶點(diǎn)性的特點(diǎn)以及其在眾多疾病和生理過(guò)程中的重要作用,我們聚焦于EAE疾病中病灶部位mi RNA的表達(dá)變化,并以此為基礎(chǔ)著重從表觀遺傳學(xué)的角度探討CNS特異性mi RNA在MS/EAE炎性脫髓鞘和影響髓鞘再生中的作用和機(jī)制。取得的研究結(jié)果如下:(1)利用200μg MOG35-55免疫8-12周雌性C57BL/6小鼠,發(fā)病高峰期(免疫后20天),麻醉后PBS充分灌注小鼠,取EAE小鼠和同齡同性別小鼠脊髓腰膨大段提取總RNA,反轉(zhuǎn)錄后用SABiosciences公司的小鼠mi RNA PCR array芯片檢測(cè)EAE小鼠脊髓病灶部位mi RNA的表達(dá)差異,發(fā)現(xiàn)EAE小鼠病灶部位mi R-146a、mi R-147、mi R-155、mi R-223、mi R-509-3p的表達(dá)顯著上調(diào),mi R-124,mi R-132,mi R-338的表達(dá)顯著下調(diào)。選取表達(dá)顯著上調(diào)mi R-146a分子為研究對(duì)象,利用RNA原位雜交技術(shù)對(duì)其表達(dá)進(jìn)行驗(yàn)證,發(fā)現(xiàn)EAE小鼠病灶部位的mi R-146a相較于對(duì)照組顯著升高。進(jìn)一步分離純化并體外培養(yǎng)小鼠的原代星形膠質(zhì)細(xì)胞和小膠質(zhì)細(xì)胞,利用IL-17,TNF-α以及IL-17結(jié)合TNF-α等炎癥因子處理,發(fā)現(xiàn)炎癥刺激會(huì)顯著升高兩種細(xì)胞內(nèi)mi R-146a的表達(dá),并且IL-17和TNF-α具有協(xié)同作用效果。(2)構(gòu)建帶有mi R-146a sponge的用以抑制mi R-146a的表達(dá)慢病毒載體,將其命名為mi R-146a inhibitor,包裝慢病毒并進(jìn)行滴度測(cè)定。用PBS將病毒稀釋成5×108IU/ml。利用200μg MOG35-55免疫8-12周雌性C57BL/6小鼠,發(fā)病初期(免疫后第14d)和高峰期(免疫后第20d)利用側(cè)腦室注射的方式分別給予20μl/mouse慢病毒LV-mi R-146a inhibitor或?qū)φ章《?統(tǒng)計(jì)小鼠臨床神經(jīng)功能評(píng)分,發(fā)現(xiàn)EAE中中樞抑制mi R-146a的表達(dá)可以顯著緩解病情。而抑制中樞mi R-146a的表達(dá)對(duì)外周免疫細(xì)胞的比例、數(shù)量、增殖、炎癥因子的分泌并無(wú)影響。免疫組化結(jié)果顯示抑制中樞mi R-146a的表達(dá)減少了中樞炎性浸潤(rùn)和脫髓鞘的變化。同時(shí)免疫熒光化學(xué)結(jié)果顯示,抑制中樞mi R-146a的表達(dá)顯著減少了中樞神經(jīng)系統(tǒng)白細(xì)胞、單核吞噬細(xì)胞和中性粒細(xì)胞的數(shù)量,也緩解了病灶部位的星形膠質(zhì)細(xì)胞化。(3)檢測(cè)EAE發(fā)病不同階段中樞神經(jīng)系統(tǒng)病灶部位mi R-146a及相關(guān)基因的表達(dá),發(fā)現(xiàn)mi R-146a在病灶部位的表達(dá)與TRAF6及IFN-β的表達(dá)顯著負(fù)相關(guān)。進(jìn)一步利用生物信息學(xué)分析發(fā)現(xiàn),TRAF6基因3’非編碼區(qū)含有兩個(gè)mi R-146a結(jié)合位點(diǎn)(447-468,506-532),體外培養(yǎng)的星形膠質(zhì)細(xì)胞中,轉(zhuǎn)染mi R-146a會(huì)顯著抑制TRAF6的表達(dá)。利用攜帶TRAF6 3’非編碼區(qū)或mi R-146a結(jié)合位點(diǎn)突變的TRAF6 3’非編碼區(qū)雙熒光素酶報(bào)告基因系統(tǒng)轉(zhuǎn)染原代星形膠質(zhì)細(xì)胞發(fā)現(xiàn)TRAF6是mi R-146a的直接作用靶點(diǎn)。進(jìn)一步利用EAE動(dòng)物模型發(fā)現(xiàn),中樞特異性抑制mi R-146a表達(dá)的同時(shí)下調(diào)TRAF6的表達(dá)阻斷了抑制mi R-146a對(duì)EAE的保護(hù)作用,而下調(diào)mi R-146a或過(guò)表達(dá)TRAF6均能緩解EAE的病情,說(shuō)明在EAE的CNS病灶區(qū)域,mi R-146a至少部分的通過(guò)作用于TRAF6調(diào)控中樞內(nèi)源性1型干擾素系統(tǒng)的表達(dá)或分泌參與EAE疾病進(jìn)程。上述結(jié)果表明mi R-146a通過(guò)調(diào)控TRAF6及1型干擾素的表達(dá)及分泌參與EAE疾病進(jìn)程,抑制中樞mi R-146a的表達(dá)會(huì)顯著減輕EAE的病情,為闡明MS/EAE發(fā)病機(jī)制以及疾病治療新靶點(diǎn)的發(fā)現(xiàn)奠定基礎(chǔ)。
[Abstract]:Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS). It is an autoimmune demyelinating disease model of the central nervous system mediated by Th1 and Th17 cells. In the central nervous system, the self activated Th1/Th17 (CD4 +T) cells, CD8+T cells, macrophage / dendritic cells, hypertrophy in the central nervous system. Cells and activated microglia can produce a large number of proinflammatory cytokines IFN- gamma, IL-17, TNF- alpha, GM-CSF and IL-23 to promote cell mediated immune response. Although the pathogenesis is not completely clear, in view of the characteristics of its inflammatory reaction, most of the treatment of multiple sclerosis is mainly based on anti-inflammatory and immunomodulatory treatment of.IFN- beta. The first approved immunomodulatory drugs in the clinic have played an important role in the treatment of MS. However, to date, the source of endogenous IFN- beta and its specific target cells are still not very clear. The inflammatory microenvironment of the central nervous system, the regulation of inflammation, and the response of the central nervous system cells to the inflammatory response It is important for the progress of the MS/EAE disease. Therefore, it is important to explore the response of the central nervous system in the condition of neuroimmunology and inflammation to elucidate the pathogenesis of MS/EAE and to discover new therapeutic targets. In the previous study, the characteristics of multiple targets based on MI RNA and its multitudinous diseases and physiology The important role of the process is to focus on the expression of MI RNA in the lesion site in EAE disease. On this basis, we focus on the role and mechanism of CNS specific mi RNA in MS/EAE inflammatory demyelinating and myelin regeneration from the epigenetic point of view. The results obtained are as follows: (1) the use of 200 mu g MOG35-55 to immunization for 8-12 weeks female C57BL/6 mice were at the peak of onset (20 days after immunization). After anesthesia, PBS was fully perfused in mice. The total RNA was extracted from the spinal cord of the spinal cord of the EAE mice and the same sex mice. The MI RNA PCR array chip of the SABiosciences company was used to detect the difference in the MI RNA in the spinal cord of the spinal cord of the EAE mice. The expression of a, MI R-147, MI R-155, MI R-223, MI R-509-3p significantly up-regulated, MI R-124. The primary astrocytes and microglia were purified and cultured in vitro, and treated with IL-17, TNF- alpha and IL-17 combined with TNF- alpha, the expression of MI R-146a in two cells was significantly increased by inflammatory stimulation, and IL-17 and TNF- alpha had synergistic effects. (2) the construction of MI R-146a sponge was used to inhibit mi. R-146a expressed the lentivirus vector, named it mi R-146a inhibitor, packed the lentivirus and tested the titer. The virus was diluted into 5 x 108IU/ml. with 200 mu g MOG35-55 for 8-12 weeks female C57BL/6 mice, and 20 mu l was given by the lateral ventricle in the early stage (after immunization, 14d) and peak period (after immunization). /mouse lentivirus LV-mi R-146a inhibitor or control lentivirus was used to count the clinical neurological function score of mice. It was found that the expression of central inhibition of MI R-146a in EAE could significantly alleviate the disease. The inhibition of central mi R-146a expression did not affect the proportion of peripheral immune cells, quantity, proliferation, and secretion of inflammatory factors. The expression of MI R-146a in the central nervous system reduced the changes in the central inflammatory infiltration and demyelination. At the same time, the immunofluorescence chemical results showed that the inhibition of the expression of MI R-146a in the central nervous system significantly reduced the number of leukocytes in the central nervous system, the number of monocyte phagocytes and neutrophils, and also the astrocytomization of the site of the disease. (3) detection of the pathogenesis of EAE. The expression of MI R-146a and related genes in the central nervous system of different stages showed that the expression of MI R-146a was negatively correlated with the expression of TRAF6 and IFN- beta. Further use of bioinformatics analysis found that the TRAF6 gene 3 'non coding region contains two mi R-146a junction sites (447-468506-532), and the in vitro culture is star shaped. In glial cells, transfection of MI R-146a significantly inhibits the expression of TRAF6. It is found that TRAF6 is the direct target of MI R-146a using the TRAF6 3 'non coding region double luciferase reporter gene system carrying TRAF6 3' non coding region or MI R-146a binding site mutation, and further using EAE animal model to find the target. The inhibition of the expression of MI R-146a and the expression of TRAF6 inhibited the protective effect of MI R-146a on EAE, and the downregulation of MI R-146a or overexpression of TRAF6 could mitigate the condition of EAE. The results show that MI R-146a is involved in the process of EAE disease through the regulation of the expression and secretion of TRAF6 and type 1 interferon, and the inhibition of the expression of central mi R-146a can significantly reduce the condition of EAE, laying a foundation for the discovery of the pathogenesis of MS/EAE and the discovery of new targets for the treatment of disease.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R744.5

【相似文獻(xiàn)】

相關(guān)期刊論文 前8條

1 薛鴻林;;阿片類(lèi)藥物聯(lián)合使用致中樞抑制1例[J];藥物流行病學(xué)雜志;2012年11期

2 H.H.Jasper ,張德星 ,孫以安;中樞抑制的現(xiàn)代概念[J];生理科學(xué)進(jìn)展;1965年01期

3 卞春甫,段世明;東莨菪堿中樞抗腎上腺素能作用的研究[J];徐州醫(yī)學(xué)院學(xué)報(bào);1980年02期

4 甘思德;王家惠;;千里光B鹼的中樞抗膽鹼能作用和抗毒效應(yīng)[J];軍事醫(yī)學(xué)科學(xué)院院刊;1982年01期

5 戴立波;何海梅;方平飛;李煥德;;中樞抑制藥物與P-糖蛋白相互作用的研究進(jìn)展[J];中國(guó)臨床藥理學(xué)雜志;2011年10期

6 高洪利,王津津;頸段硬膜外除痛治療出現(xiàn)中樞抑制1例報(bào)告[J];張家口醫(yī)學(xué)院學(xué)報(bào);1994年04期

7 李鳳林;;中樞α-受體在降壓藥作用方式中的作用[J];黑龍江醫(yī)藥;1980年05期

8 ;[J];;年期

相關(guān)重要報(bào)紙文章 前1條

1 東南大學(xué)附屬中大醫(yī)院神經(jīng)內(nèi)科 牟曉東 程守勤 整理;安眠藥:用還是不用[N];健康報(bào);2009年

相關(guān)博士學(xué)位論文 前1條

1 王戩萌;miR-146a通過(guò)TRAF6調(diào)控中樞內(nèi)源性IFN-β參與實(shí)驗(yàn)性自身免疫性腦脊髓炎病程發(fā)生發(fā)展的機(jī)制研究[D];吉林大學(xué);2016年

相關(guān)碩士學(xué)位論文 前2條

1 岳明;一次力竭運(yùn)動(dòng)與肌酸補(bǔ)充后小鼠中樞糖、乳酸、神經(jīng)遞質(zhì)代謝的特點(diǎn)[D];北京體育大學(xué);2013年

2 欒天宇;中樞NMU信號(hào)途徑及NMU2R小分子激動(dòng)劑的初步研究[D];華東師范大學(xué);2008年

，

本文編號(hào)：2024749