【摘要】：目的缺血性卒中是中國致死率最高的疾病,目前尚缺乏安全有效的治療方法。免疫炎癥反應(yīng)在腦缺血的疾病過程中具有重要的影響。免疫系統(tǒng)包括外周和中樞,該過程包括多種免疫細胞的活化,如小膠質(zhì)細胞,巨噬細胞,淋巴細胞,中性粒細胞等。調(diào)節(jié)小膠質(zhì)細胞的活化狀態(tài),抑制T細胞的病理性激活,對MCAO都具有保護作用。本實驗旨在小鼠MCAO模型中探索缺血性卒中的藥物治療方法和機制,包括以下三方面：1、天然白藜蘆醇多聚體MalibatolA (MA)保護缺血性卒中的機制研究；2、重組T細胞受體配體(RTL)保護雌性小鼠的MCAO損傷的行為學研究；3、探索一種以嚙齒類動物為模型的研究卒中后失語的實驗方法。方法利用72小時MCAO腦片的免疫熒光觀察MA對M1/M2型小膠質(zhì)細胞標志物(CD16/32和CD206)在Iba-1+小膠質(zhì)細胞的表達情況；利用PPARγ抑制劑T0070907后,TTC及NSS檢測MA對MCAO梗死體積和神經(jīng)功能的影響,并利用定量PCR檢測對M1/M2型小膠質(zhì)細胞標志物表型的影響；利用凝膠遷移電泳實驗(EMSA)檢測MA對PPARγ的轉(zhuǎn)錄活性的影響；利用染色質(zhì)免疫共沉淀(ChIP)檢測PPAR與YM-1、CD206啟動子序列的結(jié)合情況；利用免疫熒光和蛋白質(zhì)免疫共沉淀檢測PPARγ和sumo的共表達情況。利用兩種結(jié)構(gòu)的RTL(RTL1000, HLA-DRαl-MOG35-55)在雌性DR2轉(zhuǎn)基因小鼠MCAO后3、24、48、72小時分四次給藥,96小時TTC檢測其腦梗死體積；利用為期16天的行為學實驗,包括cylinder實驗、tube turn實驗、corner turn實驗和novel odor recognition檢測RTL治療后的行為學改變。利用超聲檢測和分析技術(shù),觀察小鼠MCAO后的USV變化并探索RTL治療對其的影響,并利用PCR和免疫熒光技術(shù)探討其可能的分子機制。結(jié)果免疫熒光結(jié)果顯示MCAO后小膠質(zhì)細胞激活,MA處理后在Iba-1+細胞中CD16/32表達降低,而CD206增加。T0070907抑制了Malibatol A對MCAO梗死體積的減小作用和對神經(jīng)功能評分的改善作用。T0070907抑制了MCAO梗死側(cè)皮層MA對M1型標志物(CD16,CD32)的降低作用和對M2型標志物(CD206,YM-1)的增加作用。EMSA和ChIP結(jié)果顯示,MA增強了PPAR的轉(zhuǎn)錄活性,并促進了PPARγ與YM-1啟動子序列的結(jié)合,但對CD206無明顯影響。免疫熒光表明,PPARγ與sumo共表達,但是在MCAO后表達降低。免疫共沉淀結(jié)果表明MA能夠促進MCAO后PPARγ與sumo的結(jié)合。RTL能夠降低雌性MCAO梗死體積,其中HLA-DRα1-MOG35-55對梗死體積的影響呈劑量依賴性,其有效劑量高于前期實驗中發(fā)現(xiàn)的對雄性小鼠的有效劑量。Cylinder實驗結(jié)果表明RTL1000能夠特異性的改善MCAO造成的前肢功能損傷。Novel odor recognition結(jié)果在RTL1000治療及未治療未見明顯差別。同時,本實驗建立了一種研究雌性和雄性小鼠MCAO后USV的實驗方法,并發(fā)現(xiàn),MCAO后USV的數(shù)量降低,頻率的復(fù)雜度降低。RTL1000對MCAO后USV的數(shù)量和頻率有影響。MCAO后Foxp2表達降低,可能與USV改變有關(guān)。結(jié)論MA對MCAO的腦保護和炎癥抑制作用與激活PPARγ有關(guān),能夠增強PPARγ轉(zhuǎn)錄活性,進而上調(diào)M2型小膠質(zhì)細胞的表達。MA的抑制炎癥作用可能也與PPARγ的sumo化有關(guān)。RTL1000與HLA-DRα 1-MOG-35-55都能夠顯著的降低雌性小鼠MCAO梗死體積；RTL1000能夠特異性的改善MCAO后小鼠運動功能,并對MCAO后USV的數(shù)量和頻率都有影響。
[Abstract]:Objective Ischemic stroke is the most serious disease in China, and there is still a lack of safe and effective methods for the treatment of ischemic stroke. The immune inflammatory reaction plays an important role in the course of the disease of cerebral ischemia. The immune system includes an outer periphery and a hub, the process comprising activation of a variety of immune cells, such as microglia, macrophages, lymphocytes, neutrophils, and the like. regulating the activation state of the microglia, inhibiting the pathological activation of the T cells, and protecting the MCAO. The purpose of this study is to explore the methods and mechanisms for the treatment of ischemic stroke in the MCAO model of the mouse, including the following three aspects: 1. the study of the mechanism of the protection of ischemic stroke by a natural white-and-white aloe-polymer Maliatroa (MA); 2. A study on the behavior of recombinant T-cell receptor ligand (RTL) in the protection of MCAO injury in female mice. Methods The expression of M1/ M2 microglial cell markers (CD16/ 32 and CD206) in the Iba-1 + microglia was observed by the immunofluorescence of 72-hour MCAO brain slices, and the effects of MA on the volume of MCAO and the neurological function were detected by TTC and NSS after using the PPAR inhibitor T0070907. The effect of MA on the phenotype of M1/ M2 microglial cell marker was detected by quantitative PCR. The effect of MA on the transcription activity of PPAR was detected by gel-transfer electrophoresis (EMSA), and the binding of PPAR with YM-1 and CD206 promoter was detected by means of chromatin immunoprecipitation (ChIP). The co-expression of PPAR antigen and sumo was detected by immunofluorescence and protein immune co-precipitation. Two structures of RTL (RTL1000, HLA-DR-l-MOG35-55) were administered four times at 3, 24, 48, 72 hours after MCAO in female DR2 transgenic mice, and the volume of cerebral infarction was detected by TTC at 96 h; and the behavior change after RTL treatment was detected using a 16-day behavioral experiment, including a cyclinder experiment, a tube return experiment, a corner return experiment, and a novoor recovery test. The changes of the USV and the effect of RTL on the mouse MCAO were observed by means of ultrasonic detection and analysis, and the possible molecular mechanism was discussed by means of PCR and immunofluorescence. Results The results showed that the expression of CD16/ 32 in Iba-1 + cells decreased with the increase of CD206 after MA treatment. T0070907 inhibited the effect of Malibucol A on the volume of MCAO infarction and the improvement of the neurological score. T0070907 inhibited the decrease of the MCAO infarct-side cortical MA on the M1-type marker (CD16, CD32) and the increase in the M2-type marker (CD206, YM-1). The results of EMSA and ChIP show that MA enhances the transcriptional activity of PPAR and promotes the combination of PPAR and YM-1 promoter sequences, but has no significant effect on CD206. Immunofluorescence showed that PPAR was co-expressed with sumo, but decreased after MCAO. The results show that MA can promote the combination of PPAR and sumo after MCAO. The effect of HLA-DR-1-MOG35-55 on the infarct volume was dose-dependent, and the effective dose of HLA-DR-1-MOG35-55 was higher than that in the male mice. The Cyclinder test results show that the RTL1000 can specifically improve the functional damage of the forelimb caused by the MCAO. No significant difference was seen in the treatment and non-treatment of the RL1000. At the same time, this experiment set up a method to study the USV after MCAO in female and male mice, and found that the number of USV after MCAO is reduced and the complexity of frequency is reduced. The RTL1000 has an impact on the number and frequency of the USV after MCAO. The decrease of Foxp2 expression after MCAO may be related to the change of USV. Conclusion The inhibitory effect of MA on the brain protection and inflammation of MCAO is related to the activation of PPAR, which can enhance the transcription activity of PPAR, and then up-regulate the expression of M2-type microglia. The inhibitory effect of MA may also be related to the sumo-ization of PPAR. Both RTL1000 and HLA-DR-1-MOG-35-55 can significantly reduce the MCAO infarct volume of female mice; the RTL1000 can specifically improve the mouse motion function after MCAO, and has an effect on the number and frequency of the USV after MCAO.
【學位授予單位】：南京大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R743.3

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