髓系來(lái)源的抑制性細(xì)胞在多發(fā)性硬化臨床患者及實(shí)驗(yàn)性自身免疫性腦脊髓炎中的變化及機(jī)制
發(fā)布時(shí)間：2018-08-27 05:53
【摘要】：研究背景：多發(fā)性硬化(multiple sclerosis,MS)是一種以中樞神經(jīng)系統(tǒng)(central nervous system, CNS)白質(zhì)炎性脫髓鞘為主要病理特點(diǎn)的自身免疫性疾病。該病的確切病因至今未明,因此,尚無(wú)有效根治該病的措施。糖皮質(zhì)激素是MS急性發(fā)作和復(fù)發(fā)的主要治療藥物。實(shí)驗(yàn)性自身免疫性腦脊髓炎(experimental autoimmune encephalomyelitis,EAE)是人類MS的經(jīng)典動(dòng)物模型,為MS疾病的研究提供充分的實(shí)驗(yàn)依據(jù)。髓系來(lái)源的抑制性細(xì)胞(myeloid-derived suppressor cells,MDSCs)是最初在癌癥患者中被發(fā)現(xiàn)的、在病理情況下異常擴(kuò)增的一群異質(zhì)性髓系前體細(xì)胞。它對(duì)于T細(xì)胞的增殖有著明顯的抑制功能,因此,MDSCs具有免疫調(diào)節(jié)作用。已有研究發(fā)現(xiàn)：MDSCs在EAE發(fā)病期明顯增加,尚沒(méi)有關(guān)于MDSCs在MS臨床患者中的變化及功能的系統(tǒng)性研究。另有一些研究表明：在小鼠創(chuàng)傷及皮膚移植模型中,糖皮質(zhì)激素可促進(jìn)MDSCs勺擴(kuò)增;但尚未有研究討論過(guò)糖皮質(zhì)激素治療對(duì)EAE中MDSCs勺影響。研究目的：闡述MDSCs在MS臨床患者活動(dòng)期以及糖皮質(zhì)激素治療后的功能及變化情況;探討糖皮質(zhì)激素對(duì)EAE中MDSCs的影響;并比較MDSCs的特征及功能在MS臨床研究與EAE模型研究中的異同點(diǎn);為MDSCs能否成為MS新的治療靶點(diǎn)提供實(shí)驗(yàn)依據(jù)。研究方法：(1)通過(guò)流式細(xì)胞術(shù)檢測(cè)MS臨床患者活動(dòng)期及糖皮質(zhì)激素治療后的外周血單個(gè)核細(xì)胞(peripheral blood mononuclear cells,PBMC)中MDSCs及調(diào)節(jié)性T細(xì)胞(regulatory T cells,Treg細(xì)胞)的變化。(2)檢測(cè)活動(dòng)期及糖皮質(zhì)激素治療后MS臨床患者血清中Arg1與iNOS的變化。(3)通過(guò)T細(xì)胞增殖試驗(yàn)檢測(cè)MS臨床患者PBMC中MDSCs的抑制功能。(4)應(yīng)用MOG35-55加完全弗氏佐劑(CFA)混合乳液,皮下注射C57BL/6小鼠,腹腔注射百日咳毒素(pertussis toxin, PTX)建立EAE模型;并通過(guò)尾靜脈注射甲強(qiáng)龍來(lái)建立EAE的糖皮質(zhì)激素治療模型。(5)通過(guò)流式細(xì)胞術(shù)檢測(cè)EAE發(fā)病期及糖皮質(zhì)激素治療后小鼠PBMC,脾臟中的MDSCs及Treg細(xì)胞變化。(6)應(yīng)用qRT-PCR的方法分析小鼠脾臟中Argl與Inos的mRNA表達(dá)情況。(7)通過(guò)T細(xì)胞增殖試驗(yàn)檢測(cè)激素治療組與未治療組EAE小鼠脾臟中MDSCs的抑制功能。研究結(jié)果：(1)在MS患者的活動(dòng)期,PBMC中MDSCs的比例較正常對(duì)照組相比無(wú)顯著增加;但G-MDSCs占PBMC的比例較正常對(duì)照組增加。(2)糖皮質(zhì)激素沖擊治療后,MS患者PBMC中MDSCs的比例明顯增加,與活動(dòng)期相比,有顯著的統(tǒng)計(jì)學(xué)差異(P0.0001)。(3)糖皮質(zhì)激素沖擊治療后,MDSCs的兩個(gè)亞群M-MDSCs與G-MDSCs在PBMC中比例均顯著增加;但是在MDSCs中,僅G-MDSCs所占的細(xì)胞比例較活動(dòng)期明顯增加,差異有統(tǒng)計(jì)學(xué)意義(P=0.0035)。(4)糖皮質(zhì)激素沖擊治療后,MS患者血清中精氨酸酶的活性明顯增加,并且與外周血中G-MDSCs的比例呈明顯相關(guān)性。細(xì)胞內(nèi)染色結(jié)果也證明：與M-MDSCs相比,G-MDSCs分泌了更高水平的精氨酸酶。而血清中iNOS的水平較發(fā)病前無(wú)明顯變化。(5)糖皮質(zhì)激素沖擊治療后的MDSCs能顯著抑制T細(xì)胞增殖,G-MDSCs與M-MDSCs勺抑制功能并沒(méi)有顯著性差異。(6)糖皮質(zhì)激素沖擊治療后,MS患者外周血Treg細(xì)胞占CD4+T細(xì)胞的比例無(wú)明顯變化。(7)在EAE模型中,發(fā)病期給予小鼠糖皮質(zhì)激素治療,可以有效促進(jìn)疾病的緩解。這一實(shí)驗(yàn)過(guò)程模擬了臨床患者的糖皮質(zhì)激素治療過(guò)程。(8)在EAE模型中,發(fā)病期外周血與脾臟中的MDSCs比例即明顯增加,但糖皮質(zhì)激素治療后,外周血中MDSCs的比例較未治療組無(wú)明顯變化細(xì)胞群體中,G-MDSCs亞群在MDSCs中的比例較未治療組相比明顯增加,這一點(diǎn)與臨床患者的研究相一致。而糖皮質(zhì)激素治療后脾臟中的MDSCs及兩個(gè)亞群的比例與未治療組相比均無(wú)明顯變化。(9)在EAE模型中,發(fā)病期小鼠脾臟中Argl與Inos的mRNA表達(dá)量即明顯增加。糖皮質(zhì)激素治療后脾臟中Argl與InoS的mRNA表達(dá)量并沒(méi)有明顯的變化。(10)在EAE模型中,激素治療組與未治療組的MDSCs均能抑制T細(xì)胞增殖,且M-MDSCs的抑制功能較G-MDSCs雖,但兩組間無(wú)顯著差別。(11)在EAE模型中,激素治療組與未治療組小鼠外周血、脾臟的CD4+T細(xì)胞中Treg細(xì)胞的比例無(wú)顯著改變。結(jié)論：(1)在MS臨床患者的活動(dòng)期,外周血PBMC中MDSCs的比例較正常對(duì)照組相比無(wú)明顯增加,糖皮質(zhì)激素沖擊治療可增加PBMC中MDSCs的比例。治療后的MDSCs分泌了高水平的Arg1,并顯著抑制T細(xì)胞增殖。(2)在EAE模型中,發(fā)病期小鼠外周血與脾臟中的MDSCs比例即明顯增加,脾臟中Argl與Inos的mRNA表達(dá)增加。糖皮質(zhì)激素治療可緩解小鼠疾病進(jìn)展但未改變小鼠外周血與脾臟中的MDSCs比例及脾臟中Argl與Inos的nRNA表達(dá)水平。激素治療組與未治療組的MDSCs均能抑制T細(xì)胞增殖,M-MDSCs的抑制功能較G-MDSSCs更強(qiáng),但兩組MDSCs的抑制功能無(wú)明顯差別。(3)雖然EAE模型可以模擬多發(fā)性硬化的臨床過(guò)程,但在關(guān)于MDSCs的動(dòng)力學(xué)變化及功能研究方面并不完全與臨床研究的趨勢(shì)相符。
[Abstract]:Background: Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory demyelination of the white matter in the central nervous system (CNS). The exact etiology of the disease is still unknown. Therefore, there is no effective radical treatment for MS. Glucocorticoids are acute onset and recurrence of MS. Experimental autoimmune encephalomyelitis (EAE) is a classic animal model of human MS, which provides sufficient experimental evidence for the study of MS diseases. A group of heterogeneous myeloid precursors abnormally amplified under physiological conditions. It has a significant inhibitory effect on the proliferation of T cells. Therefore, MDSCs have an immunomodulatory effect. It has been shown that glucocorticoids can promote the amplification of MDSCs in traumatic and skin transplantation models in mice, but no studies have discussed the effect of glucocorticoids on MDSCs in EAE. Methods: (1) Flow cytometry was used to detect peripheral blood mononuclear cells (PBMCs) in patients with MS at active stage and after glucocorticoid therapy. Changes of MDSCs and regulatory T cells (Treg cells) in mononuclear cells (PBMC). (2) Changes of serum Arg1 and iNOS in patients with MS during active phase and after glucocorticoid treatment. (3) Inhibitory function of MDSCs in PBMC of MS patients was detected by T cell proliferation test. (4) Application of MOG35-55 plus CFA. Mixed emulsion, subcutaneous injection of C57BL/6 mice, establishment of EAE model by intraperitoneal injection of pertussis toxin (PTX), and establishment of EAE glucocorticoid treatment model by tail vein injection of methylprednisolone. (5) the changes of MDSCs and Treg cells in PBMC and spleen of mice after EAE treatment and glucocorticoid treatment were detected by flow cytometry. 6) The expression of Argl and Inos mRNA in spleen of mice was analyzed by qRT-PCR. (7) The inhibition of MDSCs in spleen of EAE mice treated with hormone and untreated with hormone was detected by T cell proliferation test. The results showed that: (1) The proportion of MDSCs in PBMC was not significantly increased compared with that in normal control group during the active phase of MS patients; The proportion of MDSCs in PBMC of MS patients was significantly higher than that in normal control group. (2) The proportion of MDSCs in PBMC of MS patients was significantly higher than that in active phase (P 0.0001). (3) After glucocorticoid shock therapy, the proportion of M-MDSCs and G-MDSCs in PBMC of two subgroups of MDSCs was significantly higher than that in normal control group. (4) After glucocorticoid shock therapy, the activity of arginase in serum of MS patients was significantly increased, and the ratio of G-MDSCs in peripheral blood was significantly correlated. The results of intracellular staining also showed that G-MDSCs secreted more water than M-MDSCs. (5) MDSCs significantly inhibited the proliferation of T cells, but there was no significant difference between G-MDSCs and M-MDSCs spoon inhibitory function. (6) After glucocorticoid shock therapy, the percentage of Treg cells in peripheral blood of MS patients in CD4 + T cells did not change significantly. (7) In the EAE model, the treatment of mice with glucocorticoids during the onset of the disease can effectively promote the remission of the disease. This experimental process simulates the treatment process of clinical patients with glucocorticoids. (8) In the EAE model, the proportion of MDSCs in peripheral blood and spleen during the onset of the disease was significantly increased, but the proportion of MDSCs in peripheral blood was significantly increased after glucocorticoids treatment. The proportion of G-MDSCs subsets in MDSCs was significantly higher in the non-treated group than in the untreated group, which was consistent with the clinical study. The expression of Argl and Inos mRNA in spleen was significantly increased after glucocorticoid treatment. There was no significant change in the expression of Argl and InoS mRNA in spleen after glucocorticoid treatment. (10) In EAE model, MDSCs in hormone treatment group and untreated group could inhibit T cell proliferation, and the inhibitory function of M-MDSCs was better than that of G-MDSCs, but there was no significant difference between the two groups. Conclusion: (1) In the active phase of MS patients, the proportion of MDSCs in peripheral blood PBMC was not significantly increased compared with the normal control group, and the proportion of MDSCs in PBMC was increased after treatment with glucocorticoid. In EAE model, the ratio of MDSCs in peripheral blood and spleen increased significantly, and the expression of Argl and Inos mRNA in spleen increased. Glucocorticoid therapy could alleviate the progression of disease in mice, but did not change the ratio of MDSCs in peripheral blood and spleen and Argl in spleen. MDSCs in hormone treatment group and untreated group inhibited the proliferation of T cells, and the inhibitory function of M-MDSCs was stronger than that of G-MDSSCs, but there was no significant difference in the inhibitory function of MDSCs between the two groups. (3) Although EAE model could simulate the clinical process of multiple sclerosis, there was no difference in the study of the dynamics and function of MDSCs. This is consistent with the trend of clinical research.
【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
【分類號(hào)】：R744.5
[Abstract]:Background: Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory demyelination of the white matter in the central nervous system (CNS). The exact etiology of the disease is still unknown. Therefore, there is no effective radical treatment for MS. Glucocorticoids are acute onset and recurrence of MS. Experimental autoimmune encephalomyelitis (EAE) is a classic animal model of human MS, which provides sufficient experimental evidence for the study of MS diseases. A group of heterogeneous myeloid precursors abnormally amplified under physiological conditions. It has a significant inhibitory effect on the proliferation of T cells. Therefore, MDSCs have an immunomodulatory effect. It has been shown that glucocorticoids can promote the amplification of MDSCs in traumatic and skin transplantation models in mice, but no studies have discussed the effect of glucocorticoids on MDSCs in EAE. Methods: (1) Flow cytometry was used to detect peripheral blood mononuclear cells (PBMCs) in patients with MS at active stage and after glucocorticoid therapy. Changes of MDSCs and regulatory T cells (Treg cells) in mononuclear cells (PBMC). (2) Changes of serum Arg1 and iNOS in patients with MS during active phase and after glucocorticoid treatment. (3) Inhibitory function of MDSCs in PBMC of MS patients was detected by T cell proliferation test. (4) Application of MOG35-55 plus CFA. Mixed emulsion, subcutaneous injection of C57BL/6 mice, establishment of EAE model by intraperitoneal injection of pertussis toxin (PTX), and establishment of EAE glucocorticoid treatment model by tail vein injection of methylprednisolone. (5) the changes of MDSCs and Treg cells in PBMC and spleen of mice after EAE treatment and glucocorticoid treatment were detected by flow cytometry. 6) The expression of Argl and Inos mRNA in spleen of mice was analyzed by qRT-PCR. (7) The inhibition of MDSCs in spleen of EAE mice treated with hormone and untreated with hormone was detected by T cell proliferation test. The results showed that: (1) The proportion of MDSCs in PBMC was not significantly increased compared with that in normal control group during the active phase of MS patients; The proportion of MDSCs in PBMC of MS patients was significantly higher than that in normal control group. (2) The proportion of MDSCs in PBMC of MS patients was significantly higher than that in active phase (P 0.0001). (3) After glucocorticoid shock therapy, the proportion of M-MDSCs and G-MDSCs in PBMC of two subgroups of MDSCs was significantly higher than that in normal control group. (4) After glucocorticoid shock therapy, the activity of arginase in serum of MS patients was significantly increased, and the ratio of G-MDSCs in peripheral blood was significantly correlated. The results of intracellular staining also showed that G-MDSCs secreted more water than M-MDSCs. (5) MDSCs significantly inhibited the proliferation of T cells, but there was no significant difference between G-MDSCs and M-MDSCs spoon inhibitory function. (6) After glucocorticoid shock therapy, the percentage of Treg cells in peripheral blood of MS patients in CD4 + T cells did not change significantly. (7) In the EAE model, the treatment of mice with glucocorticoids during the onset of the disease can effectively promote the remission of the disease. This experimental process simulates the treatment process of clinical patients with glucocorticoids. (8) In the EAE model, the proportion of MDSCs in peripheral blood and spleen during the onset of the disease was significantly increased, but the proportion of MDSCs in peripheral blood was significantly increased after glucocorticoids treatment. The proportion of G-MDSCs subsets in MDSCs was significantly higher in the non-treated group than in the untreated group, which was consistent with the clinical study. The expression of Argl and Inos mRNA in spleen was significantly increased after glucocorticoid treatment. There was no significant change in the expression of Argl and InoS mRNA in spleen after glucocorticoid treatment. (10) In EAE model, MDSCs in hormone treatment group and untreated group could inhibit T cell proliferation, and the inhibitory function of M-MDSCs was better than that of G-MDSCs, but there was no significant difference between the two groups. Conclusion: (1) In the active phase of MS patients, the proportion of MDSCs in peripheral blood PBMC was not significantly increased compared with the normal control group, and the proportion of MDSCs in PBMC was increased after treatment with glucocorticoid. In EAE model, the ratio of MDSCs in peripheral blood and spleen increased significantly, and the expression of Argl and Inos mRNA in spleen increased. Glucocorticoid therapy could alleviate the progression of disease in mice, but did not change the ratio of MDSCs in peripheral blood and spleen and Argl in spleen. MDSCs in hormone treatment group and untreated group inhibited the proliferation of T cells, and the inhibitory function of M-MDSCs was stronger than that of G-MDSSCs, but there was no significant difference in the inhibitory function of MDSCs between the two groups. (3) Although EAE model could simulate the clinical process of multiple sclerosis, there was no difference in the study of the dynamics and function of MDSCs. This is consistent with the trend of clinical research.
【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
【分類號(hào)】：R744.5
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