髓系来源的抑制性细胞在多发性硬化临床患者及实验性自身免疫性脑脊髓炎中的变化及机制
发布时间：2018-08-27 05:53
【摘要】：研究背景：多发性硬化(multiple sclerosis,MS)是一种以中枢神经系统(central nervous system, CNS)白质炎性脱髓鞘为主要病理特点的自身免疫性疾病。该病的确切病因至今未明,因此,尚无有效根治该病的措施。糖皮质激素是MS急性发作和复发的主要治疗药物。实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)是人类MS的经典动物模型,为MS疾病的研究提供充分的实验依据。髓系来源的抑制性细胞(myeloid-derived suppressor cells,MDSCs)是最初在癌症患者中被发现的、在病理情况下异常扩增的一群异质性髓系前体细胞。它对于T细胞的增殖有着明显的抑制功能,因此,MDSCs具有免疫调节作用。已有研究发现：MDSCs在EAE发病期明显增加,尚没有关于MDSCs在MS临床患者中的变化及功能的系统性研究。另有一些研究表明：在小鼠创伤及皮肤移植模型中,糖皮质激素可促进MDSCs勺扩增;但尚未有研究讨论过糖皮质激素治疗对EAE中MDSCs勺影响。研究目的：阐述MDSCs在MS临床患者活动期以及糖皮质激素治疗后的功能及变化情况;探讨糖皮质激素对EAE中MDSCs的影响;并比较MDSCs的特征及功能在MS临床研究与EAE模型研究中的异同点;为MDSCs能否成为MS新的治疗靶点提供实验依据。研究方法：(1)通过流式细胞术检测MS临床患者活动期及糖皮质激素治疗后的外周血单个核细胞(peripheral blood mononuclear cells,PBMC)中MDSCs及调节性T细胞(regulatory T cells,Treg细胞)的变化。(2)检测活动期及糖皮质激素治疗后MS临床患者血清中Arg1与iNOS的变化。(3)通过T细胞增殖试验检测MS临床患者PBMC中MDSCs的抑制功能。(4)应用MOG35-55加完全弗氏佐剂(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;并通过尾静脉注射甲强龙来建立EAE的糖皮质激素治疗模型。(5)通过流式细胞术检测EAE发病期及糖皮质激素治疗后小鼠PBMC,脾脏中的MDSCs及Treg细胞变化。(6)应用qRT-PCR的方法分析小鼠脾脏中Argl与Inos的mRNA表达情况。(7)通过T细胞增殖试验检测激素治疗组与未治疗组EAE小鼠脾脏中MDSCs的抑制功能。研究结果：(1)在MS患者的活动期,PBMC中MDSCs的比例较正常对照组相比无显著增加;但G-MDSCs占PBMC的比例较正常对照组增加。(2)糖皮质激素冲击治疗后,MS患者PBMC中MDSCs的比例明显增加,与活动期相比,有显著的统计学差异(P0.0001)。(3)糖皮质激素冲击治疗后,MDSCs的两个亚群M-MDSCs与G-MDSCs在PBMC中比例均显著增加;但是在MDSCs中,仅G-MDSCs所占的细胞比例较活动期明显增加,差异有统计学意义(P=0.0035)。(4)糖皮质激素冲击治疗后,MS患者血清中精氨酸酶的活性明显增加,并且与外周血中G-MDSCs的比例呈明显相关性。细胞内染色结果也证明：与M-MDSCs相比,G-MDSCs分泌了更高水平的精氨酸酶。而血清中iNOS的水平较发病前无明显变化。(5)糖皮质激素冲击治疗后的MDSCs能显著抑制T细胞增殖,G-MDSCs与M-MDSCs勺抑制功能并没有显著性差异。(6)糖皮质激素冲击治疗后,MS患者外周血Treg细胞占CD4+T细胞的比例无明显变化。(7)在EAE模型中,发病期给予小鼠糖皮质激素治疗,可以有效促进疾病的缓解。这一实验过程模拟了临床患者的糖皮质激素治疗过程。(8)在EAE模型中,发病期外周血与脾脏中的MDSCs比例即明显增加,但糖皮质激素治疗后,外周血中MDSCs的比例较未治疗组无明显变化细胞群体中,G-MDSCs亚群在MDSCs中的比例较未治疗组相比明显增加,这一点与临床患者的研究相一致。而糖皮质激素治疗后脾脏中的MDSCs及两个亚群的比例与未治疗组相比均无明显变化。(9)在EAE模型中,发病期小鼠脾脏中Argl与Inos的mRNA表达量即明显增加。糖皮质激素治疗后脾脏中Argl与InoS的mRNA表达量并没有明显的变化。(10)在EAE模型中,激素治疗组与未治疗组的MDSCs均能抑制T细胞增殖,且M-MDSCs的抑制功能较G-MDSCs虽,但两组间无显著差别。(11)在EAE模型中,激素治疗组与未治疗组小鼠外周血、脾脏的CD4+T细胞中Treg细胞的比例无显著改变。结论：(1)在MS临床患者的活动期,外周血PBMC中MDSCs的比例较正常对照组相比无明显增加,糖皮质激素冲击治疗可增加PBMC中MDSCs的比例。治疗后的MDSCs分泌了高水平的Arg1,并显著抑制T细胞增殖。(2)在EAE模型中,发病期小鼠外周血与脾脏中的MDSCs比例即明显增加,脾脏中Argl与Inos的mRNA表达增加。糖皮质激素治疗可缓解小鼠疾病进展但未改变小鼠外周血与脾脏中的MDSCs比例及脾脏中Argl与Inos的nRNA表达水平。激素治疗组与未治疗组的MDSCs均能抑制T细胞增殖,M-MDSCs的抑制功能较G-MDSSCs更强,但两组MDSCs的抑制功能无明显差别。(3)虽然EAE模型可以模拟多发性硬化的临床过程,但在关于MDSCs的动力学变化及功能研究方面并不完全与临床研究的趋势相符。
[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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R744.5
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