骨髓增生异常综合征患者巨噬细胞亚群的研究

发布时间：2018-06-03 08:15

本文选题：骨髓增生异常综合征 + 巨噬细胞　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的:通过检测健康对照组及不同危险分组骨髓增生异常综合征患者骨髓巨噬细胞占单个核细胞的比例,骨髓Ⅰ型巨噬细胞和Ⅱ型巨噬细胞比值,及Ⅰ型和Ⅱ型巨噬细胞表面抗原的表达,并且通过体外培养刺激Ⅰ型巨噬细胞,并检测IL-1β及TNF-αm RNA表达,比较正常对照组及实验组的不同,探究巨噬细胞在骨髓增生异常综合征发病中的作用。背景:MDS是一组起源于造血干细胞的髓系克隆性疾病。该病的主要特点为全血细胞减少和高风险向急性白血病转化。普遍的发病高龄化(平均发病年龄在70-75岁)使得对MDS患者的管理更为复杂,与此同时还有伴随的非血液系统的并发症,以及老年患者对于某些高强度治疗的耐受力较差。除此之外,对于进展为AML的患者,其标准治疗的疗效要低于初治AML患者。虽然近年来对MDS来的治疗取得了一些进展,来那度胺、地西他滨、阿扎胞苷以及造血干细胞移植等治疗方法延长了病人的生存期,但是MDS患者预后差、生存期短、缺乏有效治疗手段的局面并没有从根本上改变,贫血、出血、感染等并发症明显降低了患者的生存质量,并可直接导致患者死亡。微环境的改变和免疫下调导致了这种分化异常。近年来,肿瘤微环境是肿瘤发病机制与治疗的研究热点之一。肿瘤微环境主要由细胞、信号分子、细胞外基质、细胞因子等组成。目前国内外学者认为,单独的肿瘤细胞不会致病,而是通过“招募”正常的细胞,通过细胞间的相互作用,分泌抑制性细胞因子,从而抑制机体免疫细胞的杀伤功能等机制,形成有利于肿瘤细胞生长的微环境,以此来促进肿瘤的进展和转移。近年来有研究发现,巨噬细胞也是造血微环境的成分细胞,影响造血干细胞的迁移与定居。而在MDS中也发现有异常的单核细胞积累的现象,影响细胞的迁移。因此MDS骨髓巨噬细胞的异常,可能对MDS的致病起到一定的作用。巨噬细胞在极化为M2后,可通过多种途径对抗肿瘤免疫产生抑制。研究认为TAMs为M2巨噬细胞。另一方面,研究发现TAMs能够抑制NK细胞及CTL细胞的杀伤功能。TAMs表达HLA-C、HLA-E和HLA-G等膜结合分子能够抑制NK细胞的激活。巨噬细胞同样表达PD-1及CTAL-4的配体。PD-1及CTAL-4通常在活化的免疫效应细胞(如B细胞、T细胞及NK细胞)高表达,激活后抑制T细胞的细胞毒作用,从而抑制机体的免疫。材料与方法:首先,我们应用流式细胞术(Flow Cytometry,FCM)检测38名MDS患者(LR-MDS患者15名,HR-MDS患者23名)和21名正常对照(normal control,NC)骨髓中以CD14标记的骨髓单核细胞比例及CD14的平均荧强度(MFI),进而以CD14+、CD68+标记巨噬细胞,检测巨噬细胞占骨髓单核细胞及骨髓单个核细胞的比例;以CD64、CD40标记Ⅰ型巨噬细胞(M1),CD206、CD163标记为Ⅱ型巨噬细胞(M2),比较MDS组与正常对照组M1/M2的不同,以及巨噬细胞表面CD64、CD40、CD206、CD163表达的不同。然后,通过体外培养刺激MDS及正常对照组M1巨噬细胞,提取两组M1巨噬细胞的m RNA,比较两组巨噬细胞IL-1β及TNF-αm RNA表达的不同。结果:1、MDS组及正常对照组骨髓单核细胞差异1.1骨髓单核细胞比例正常对照组骨髓单核细胞比例为(2.11±0.93)%,MDS-低危组骨髓单核细胞比例为(1.96±1.53)%,MDS-高危组骨髓单核细胞比例为(3.66±3.38)%。正常对照组与低危组相比骨髓单核细胞比例差异无统计学意义;较高危组相比骨髓单核细胞比例降低,差异有统计学意义(p0.05)。高危组较低危组相比比例升高,差异有统计学意义(p0.05)。1.2 CD14平均荧光强度正常对照组骨髓单核细胞CD14平均荧光强度为639.05±359.78,MDS-低危组骨髓单核细胞CD14平均荧光强度为501.43±374.44,MDS-高危组骨髓单核细胞CD14平均荧光强度为458.26±306.72。正常对照组与低危组相比骨髓单核细胞CD14平均荧光强度差异无统计学意义;较高危组相比骨髓单核细胞CD14平均荧光强度升高,差异有统计学意义(p0.05)。高危组较低危组相比骨髓单核细胞CD14平均荧光强度差异无统计学意义。2、MDS组及正常对照组骨髓巨噬细胞细胞差异2.1骨髓M1、M2巨噬细胞占单核细胞比例正常对照组M1巨噬细胞占单核细胞比例为(6.41±7.09)%,MDS-低危组M1巨噬细胞占单核细胞比例为(8.08±10.31)%,MDS-高危组M2巨噬细胞占单核细胞比例为(7.80±9.41)%,三组之间相比差异均无统计学意义正常对照组M2巨噬细胞占单核细胞比例为(1.82±2.47)%,MDS-低危组M2巨噬细胞占单核细胞比例为(3.18±3.79)%,MDS-高危组M2巨噬细胞占单核细胞比例为(3.93±3.81)%,MDS-高危组较正常对照组相比M2巨噬细胞占单核细胞比例明显升高,且差异有统计学意义(p0.05)2.2骨髓M1巨噬细胞/M2巨噬细胞比值正常对照组骨髓M1/M2为3.50±3.22,MDS-低危组骨髓M1/M2为1.68±0.78,MDS-高危组骨髓M1/M2为1.80±0.88,正常组骨髓M1/M2与MDS-低危组及MDS-高危组骨髓M1/M2相比明显降低,且差异有统计学意义(p0.05)。MDS-低危组与MDS-高危组骨髓M1/M2相比差异无统计学意义。2.3其他MDS组骨髓巨噬细胞表面CD64、CD40、CD206、CD163表达与正常对照组骨髓巨噬细胞相比差异均无统计学意义。3、MDS组及正常对照组M1巨噬细胞IL-1β及TNF-αmRNA表达差异3.1 MDS组及正常对照组M1巨噬细胞IL-1βmRNA表达正常对照组IL-1βmRNA表达为2.07±1.66,MDS-低危组为0.5±0.6,MDS高危组为0.98±0.72,正常对照组与MDS-低危组及MDS-高危组相比IL-1βm RNA表达均升高,且差异有统计学意义(p0.05),MDS-低危组与MDS-高危组相比差异无统计学意义。3.2 MDS组及正常对照组M1巨噬细胞TNF-αm RNA表达正常对照组TNF-αmRNA表达为1.20±0.75,MDS组TNF-αmRNA表达为0.55±0.62,正常对照组与MDS组相比IL-1βm RNA表达升高,且差异有统计学意义(p0.05)。结论:(1)MDS患者BM中存在异常单核细胞的蓄积,可能与疾病的进展有关;(2)MDS患者与正常的对照组相比,M2巨噬细胞占单核细胞的比例明显升高,随着疾病的进展,MDS患者骨髓M2占单核细胞比例进一步升高;(3)MDS患者骨髓M1/M2与正常对照组相比有明显差异,提示MDS患者存在巨噬细胞极化的异常;(4)MDS患者骨髓巨噬细胞极化的异常,可能与MDS疾病的发生与进展有关,但具体机制仍待进一步研究。
[Abstract]:Objective: to detect the ratio of bone marrow macrophages to mononuclear cells, the ratio of bone marrow macrophages to type I macrophages and the expression of surface antigen of type I and type II macrophages in the healthy control group and the patients with different risk group myelodysplastic syndrome, and to stimulate type I macrophages and detect IL- in vitro. 1 beta and TNF- alpha m RNA expression, compare the difference between the normal control group and the experimental group, and explore the role of macrophage in the pathogenesis of myelodysplastic syndrome. Background: MDS is a group of myeloid cloned diseases originating from hematopoietic stem cells. The main feature of this disease is the transformation of whole blood cell and high risk to acute leukemia. The general incidence of disease is high. Aging (average age of onset at 70-75 years) makes the management of MDS more complex, accompanied by complications associated with non blood systems, and poor tolerance for some elderly patients with high intensity treatment. Besides, for patients with AML, the therapeutic effect of the standard treatment is lower than that of the initial AML patients. Some progress has been made in the treatment of MDS. The treatment methods such as amido, diazibin, arazarin, and hematopoietic stem cell transplantation have extended the patient's survival time, but the prognosis of the patients with MDS is poor, the survival time is short, and the lack of effective treatment is not fundamentally changed, and the complications such as anemia, bleeding and infection have been significantly reduced. The quality of life of the patient can lead to the death of the patient directly. The change of microenvironment and the downregulation of the immune system lead to this differentiation. In recent years, the tumor microenvironment is one of the hot spots in the pathogenesis and treatment of tumor. The tumor microenvironment is mainly composed of cells, signal molecules, extracellular matrix, cytokine and so on. In addition, the individual tumor cells do not cause disease, but by "recruiting" normal cells, secreting inhibitory cytokine through intercellular interaction, inhibiting the killing function of immune cells and forming microenvironment conducive to the growth of tumor cells, in order to promote the progression and metastasis of tumor. Macrophages are also components of the hematopoietic microenvironment, which affect the migration and settlement of hematopoietic stem cells. In MDS, abnormal mononuclear cell accumulation is found to affect cell migration. Therefore, abnormal MDS bone marrow macrophages may play a role in the pathogenesis of MDS. Macrophages can pass through the polarization of M2. The research suggests that TAMs is M2 macrophage. On the other hand, the study found that TAMs can inhibit the expression of HLA-C in NK and CTL cells, and the membrane binding molecules such as HLA-E and HLA-G can inhibit the activation of NK cells. Activated immune effector cells (such as B, T and NK cells) were highly expressed and activated to inhibit the cytotoxicity of T cells and thus inhibit the body's immunity. Materials and methods: first, we used flow cytometry (FCM) to detect 38 MDS patients (15 LR-MDS patients, 23 HR-MDS patients) and 21 normal controls (normal control). NC) the proportion of bone marrow mononuclear cells labeled with CD14 and the average fluorescence intensity of CD14 (MFI) in bone marrow, and then CD14+, CD68+ labeled macrophages, and the ratio of macrophages to mononuclear cells and bone marrow mononuclear cells in bone marrow; and CD64, CD40 labeled macrophages (M1), CD206, CD163 labeled as type II macrophages (M2). The expression of CD64, CD40, CD206 and CD163 on the surface of macrophage was different in the normal control group. Then, the m RNA of the two groups of M1 macrophages was extracted by stimulating MDS and the normal control group of M1 macrophages in vitro, and the difference between the IL-1 beta and TNF- alpha of the two groups was compared. Results: 1, the bone marrow mononuclear cells of the 1 group and the normal control group were fine. The proportion of 1.1 bone marrow mononuclear cells in the normal control group was (2.11 + 0.93)%, the ratio of bone marrow mononuclear cells in MDS- low risk group was (1.96 + 1.53)%, and the proportion of bone marrow mononuclear cells in high risk group of MDS- was (3.66 + 3.38)%. The ratio of bone marrow mononuclear cells in normal control group was not statistically significant compared with that of low risk group; the higher risk group was not significant. The ratio of bone marrow mononuclear cells decreased, the difference was statistically significant (P0.05). The ratio of high risk group was higher than that of low risk group, the difference was statistically significant (P0.05), the average fluorescence intensity of CD14 in bone marrow mononuclear cells of normal control group was 639.05 + 359.78, and the average fluorescence intensity of bone marrow mononuclear cells in MDS- low risk group was 501.43, and the average fluorescence intensity was 501.43 in MDS- low risk group. The average fluorescence intensity of CD14 in bone marrow mononuclear cells of MDS- high risk group was 458.26 + 306.72., and the average fluorescence intensity of CD14 in bone marrow mononuclear cells was not statistically significant compared with that of low risk group, and the average fluorescence intensity of CD14 in bone marrow mononuclear cells in high risk group was higher than that in bone marrow mononuclear cells (P0.05). The high risk group was lower than the low risk group. There was no significant difference in the average fluorescence intensity of CD14 compared with bone marrow mononuclear cells. The difference of bone marrow macrophages in MDS group and normal control group was 2.1 bone marrow M1, M2 macrophage accounted for mononuclear cells in normal control group, M1 macrophages accounted for (6.41 + 7.09)% of mononuclear cells. The proportion of M1 macrophages in MDS- low risk group was (8.08 + 10.) (8.08 + 10.). 31)%, the proportion of M2 macrophages in MDS- high risk group was (7.80 + 9.41)%. There was no statistically significant difference between the three groups. The proportion of M2 macrophages in the normal control group was (1.82 + 2.47)%, and the proportion of M2 macrophages in the low risk group of MDS- was (3.18 + 3.79)%, and the proportion of M2 macrophages in the high-risk group of MDS- was (the proportion of mononuclear cells). 3.93 + 3.81)%, MDS- high risk group compared with normal control group, M2 macrophage accounted for a significant increase in mononuclear cells, and the difference was statistically significant (P0.05) 2.2 bone marrow M1 macrophage /M2 macrophage ratio normal control group bone marrow M1/M2 is 3.50 + 3.22, MDS- low risk group bone marrow M1/M2 is 1.68 + 0.78, MDS- high-risk group bone marrow M1/M2 is 1.80 + 0.88, normal Group bone marrow M1/M2 was significantly lower than that of MDS- low risk group and MDS- high risk group, and the difference was statistically significant (P0.05) in.MDS- low risk group and MDS- high-risk group, there was no significant difference in bone marrow M1/M2 between the high-risk group of MDS- and.2.3 other MDS groups. There was no statistical significance.3, MDS group and normal control group M1 macrophage IL-1 beta and TNF- alpha mRNA expression difference 3.1 MDS group and normal control group M1 macrophage IL-1 beta mRNA expression of IL-1 beta mRNA expression was 2.07 + 1.66, low risk group was 0.5 + 0.6, high-risk group was 0.98 + 0.72, normal control group compared with the low risk group and high-risk group The expression of -1 beta m RNA increased, and the difference was statistically significant (P0.05). There was no statistically significant difference between the MDS- low risk group and the MDS- high risk group. The M1 macrophage TNF- alpha m RNA was 1.20 + 0.75 in the normal control group and 0.55 + 0.62 in the normal control group, and the normal control group was compared with the normal control group. The expression of NA was increased and the difference was statistically significant (P0.05). Conclusion: (1) the accumulation of abnormal mononuclear cells in BM of MDS patients may be related to the progress of the disease. (2) the proportion of M2 macrophages in mononuclear cells increased significantly compared with the normal control group, and with the progression of the disease, the proportion of bone marrow M2 in the MDS patients was further more than that of mononuclear cells. (3) the bone marrow M1/M2 of MDS patients was significantly different from that of the normal control group, suggesting that there was an abnormal polarization of macrophage in MDS patients; (4) the abnormal polarization of bone marrow macrophages in MDS patients may be related to the occurrence and progress of MDS disease, but the specific mechanism still need to be studied one step.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R551.3

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