非血缘脐血移植后发生植入前综合征效应细胞的探索

发布时间：2018-04-21 15:24

本文选题：脐血移植 + 植入前综合征　；参考：《安徽医科大学》2017年硕士论文

【摘要】：目的植入前综合征(PES)是一种高发于非血缘脐血移植(UCBT)后在中性粒细胞植入之前,以发热、皮疹、腹泻等为主要临床表现的一种免疫反应。发生重症PES的患者,移植相关死亡率明显增高。本研究通过对UCBT后发生PES患者外周血进行供受者基因嵌合状态检测,了解发生PES时细胞的来源;根据血清细胞因子检测结果发现可溶性ST2(sST2)在PES发生时明显升高,选择新鲜脐血和外周血干细胞,采用IL-33和sST2进行体外刺激,探索发生PES的效应细胞,为PES发病机制的研究奠定基础。方法本研究分为两部分。第一部分:UCBT后发生PES的细胞来源的研究。根据本中心先前的研究发现UCBT后PES发生的中位时间为UCBT后第7天(+7d),选择移植患者+7d的外周血标本,采用短串联重复序列聚合酶链式反应(STR-PCR)技术检测发生PES组和未发生PES组患者供受者嵌合状态,分析发生PES的细胞来源。第二部分:UCBT后发生PES的效应细胞的探讨。一、UCBT后PES发生时各种免疫细胞及其亚群和相关细胞因子的检测。检测PES发生时的T、B、NK和单核细胞及其亚群的变化;检测患者移植前、移植当天、PES发生时不同时间点的外周血的白细胞数,血浆C-反应蛋白和血清中相关细胞因子的变化。二、不同来源的移植物体外刺激实验:sST2在PES发生时显著增高,而ST2是IL-33的孤儿受体,拟从IL-33/ST2信号通路来推证PES的效应细胞。应用IL-33,sST2刺激新鲜脐血、外周血干细胞(动员采集的同胞供者),每种类型的细胞分4组:实验组:A.IL-33+sST2组;对照组:B.sST2组;C.IL-33组;D.空白组。经体外培养0、12、24和72小时后,细胞计数板计算刺激前后细胞总数,流式细胞术检测刺激前后细胞亚群及ST2受体表达情况。结果第一部分UCBT后发生PES的细胞来源的研究STR-PCR检测脐血及受者移植前后的标本比对供受者的嵌合状态,结果显示:UCBT+7d供者的嵌合度发生PES组明显高于未发生PES组,分别为 70.58±22.86%与37.18±18.96%,两组之间有显著的统计学差异,P0.0001。第二部分UCBT后发生PES的效应细胞的探讨一、UCBT后发生PES时各种免疫细胞及其亚群和相关细胞因子的检测1.白细胞的变化PES组与无PES组在移植前、移植当天、PES发生时的白细胞计数组间无统计学差异,P0.05。2.PES发生时T、B、NK和单核细胞的细胞亚群检测结果:CD45+CD3+T淋巴细胞、CD45+CD3-CD19+B淋巴细胞、CD45+CD3-CD56+NK细胞、CD45+CD3-CD14+单核细胞分别占90%,0.3%,2.1%和0.2%。PES组与无PES组的T、B、NK和单核细胞的各细胞亚群比例无明显统计学差异,P0.05。3.C-反应蛋白的变化在移植前、移植当天、PES发生时,PES组和无PES组两组之间无显著统计学差异,P0.05。4.血清细胞因子的变化发生PES时细胞因子IL-6、MCP-1和sST2在PES组明显高于无PES组(P0.05);PES组自身比较患者发生PES时也显著高于移植前、移植当天的水平(P0.05)。PES患者治疗有效者细胞因子IL-6和MCP-1明显下降并恢复至发生前水平(P0.01),而sST2与PES发生时相比虽有所下降但无统计学差异(P0.05)。发生PES时IFN-γ、IL-2、IL-1Rα、IL-4、IL-8、IL-17A、IL-33、MIP-1α、MIP-1β和TNF-α表达水平两组相近无统计学差异(P0.05),同时PES组患者在不同的时间点,自身对比其表达水平相近无统计学差异(P0.05)。二、不同来源的移植物体外刺激实验1.细胞数量的变化:新鲜脐血和外周血干细胞经IL-33、sST2刺激培养后,各组刺激前后细胞数量无明显增加(P0.05),两种来源不同的细胞刺激培养12h后均出现多形性变化,此后随培养时间延长形态变化不显著。2.T、B、NK和单核细胞各亚群的变化:新鲜脐血经Ficoll提取单个核细胞后,脐血提取的单个核细胞比例分别为T淋巴细胞30-50%、B淋巴细胞1-3%,NK细胞5-8%,单核细胞5-13%。外周血干细胞中T淋巴细胞20-35%、B淋巴细胞2-5%,NK细胞3-9%,单核细胞18-32%。经刺激培养12、24和72h后,四组的T、B、NK和单核细胞亚群比例无明显差异(P0.05),随着培养时间的延长, 有少量细胞凋亡,实验各组细胞不同培养时间点的细胞亚群比例也无明显统计学差异(P0.05)。3.T、B、NK和单核细胞ST2受体表达情况:新鲜脐血和外周血干细胞中ST2受体表达相似,单核细胞上ST2受体表达量高达53-90%,其次是B淋巴细胞亚群约为30-60%,T淋巴细胞及NK细胞亚群表达量低,分别为2-5%和3-10%。而四组的各细胞亚群ST2受体表达无明显变化(P0.05),实验各组细胞不同培养时间点的各细胞亚群ST2表达也无显著变化(P0.05)。结论1.UCBT后PES是供者开始植入时引发多种细胞因子参与的免疫反应,其效应细胞推测来源于供者,供者的早期嵌合是PES发生的高危因素。2.血清中单核细胞相关的趋化因子(MCP-1)在PES发生时明显升高,而淋巴细胞相关的较特异的细胞因子(IL-2、IL-4、IL-8、IL-17A)在PES发生时无显著变化,PES的临床表现主要在皮肤、肠道等组织中,推断组织中的单核巨噬细胞可能是PES的主要效应细胞。3.加入IL-33和sST2进行单纯的体外培养,未见到免疫细胞的增殖和ST2受体表达的增高,但不能确定细胞因子水平是否发生变化,需进一步检测细胞培养液中细胞因子的变化,观察IL-33/ST2信号通路及sST2是否对下游细胞信号转导存在影响,从而引起细胞因子的分泌。同时本实验也需要加测各免疫细胞活化的指标,如HLA-DR,CD27,CCR等等,为揭示PES的发生机制提供依据。
[Abstract]:Objective preimplantation syndrome (PES) is an immune response to the main clinical manifestations of fever, rash, and diarrhoea before neutrophils implantation after high incidence of non related umbilical cord blood transplantation (UCBT). Patients with severe PES have a significant increase in transplant related mortality. This study was conducted through the study of peripheral blood in patients with PES after UCBT. Detection of gene chimerism to understand the source of cells when PES occurs. According to the results of serum cytokine detection, it is found that soluble ST2 (sST2) is obviously elevated when PES occurs, select fresh umbilical blood and peripheral blood stem cells, use IL-33 and sST2 to stimulate in vitro, explore the effect cells of PES, and lay the foundation for the study of pathogenesis of PES. Methods this study was divided into two parts. Part one: a study of the cell origin of PES after UCBT. According to previous studies in this center, the median time of PES after UCBT was found to be seventh days after UCBT (+7d), and the peripheral blood samples of the transplanted +7d were selected and the PES group was detected by the short tandem repeat polymerase chain reaction (STR-PCR) technique. PES group of donor chimerism, analysis of PES cell origin. Second part: the effect cells of PES after UCBT. 1, detection of various immune cells and their subsets and related cytokines at the occurrence of UCBT PES. The changes in T, B, NK and mononuclear cells and its subgroups at the occurrence of PES; detection of patients before transplantation, On the day of transplantation, the number of white blood cells in peripheral blood at different time points, plasma C- reactive protein and serum related cytokines at different time points. Two, external stimulation experiments of different sources of transplantation: sST2 increased significantly at PES, and ST2 is an orphan receptor of IL-33, which is intended to promote PES effector cells from IL-33/ST2 signaling pathway. I L-33, sST2 stimulates fresh cord blood and peripheral blood stem cells (mobilized compatriots donor). Each type of cell is divided into 4 groups: experimental group: group A.IL-33+sST2; control group: B.sST2 group; C.IL-33 group; D. blank group. Cell count board is used to count the total number of cells before and after stimulation in vitro and after 72 hours in vitro. Flow cytometry is used to detect the cell subcell before and after stimulation. Results of group and ST2 receptor expression. Results the cell origin of PES after part one UCBT was studied by STR-PCR to detect the chimerism of umbilical cord blood and recipients before and after transplantation. The results showed that the chimerism of UCBT+7d donor in PES group was significantly higher than that of the non PES group, which was 70.58 + 22.86% and 37.18 + 18.96%. The two groups were among the two groups. Significant statistical differences, P0.0001. second part UCBT after the occurrence of PES effect cells, UCBT after PES, the detection of various immune cells and its subsets and related cytokines 1. leukocytes changes in the PES group and no PES group before the transplantation, the day of transplantation, the leukocyte count groups at the time of PES occurred, no statistical difference, P0.05.2.PES The cell subsets of T, B, NK and mononuclear cells were detected at the time of occurrence: CD45+CD3+T lymphocyte, CD45+CD3-CD19+B lymphocyte, CD45+CD3-CD56+NK cell, CD45+CD3-CD14+ mononuclear cells accounted for 90%, 0.3%, 2.1% and 0.2%.PES and 0.2%.PES without PES group T, B, NK and mononuclear cell subgroups There was no significant difference between the two groups in the PES group and the non PES group before the transplant, the day of the transplant, and the two groups without PES. The changes of serum cytokines in P0.05.4. were IL-6, MCP-1 and sST2 were significantly higher in the PES group than in the non PES group (P0.05). The cytokines IL-6 and MCP-1 of the patients with P0.05.PES were significantly decreased and recovered to the pre occurrence level (P0.01), while sST2 and PES were decreased, but there was no statistical difference (P0.05). There was no statistical difference between the two groups of IFN- gamma, IL-2, IL-1R alpha. (P0.05), at the same time, there was no significant difference in the expression level of the patients in group PES at different time points (P0.05). Two, the number of 1. cells in the external stimulation experiment of different sources: the number of fresh cord blood and peripheral blood stem cells by IL-33, sST2 after stimulation, the number of cells before and after stimulation was not significantly increased (P0.05), two kinds of cells. The cells with different sources had polymorphic changes after the stimulation of 12h. After that, the changes in the morphological changes were not significant.2.T, B, NK and monocyte subgroups. After the fresh cord blood was extracted by Ficoll, the proportion of mononuclear cells extracted from umbilical blood was T lymphocyte 30-50%, B lymphocyte 1-3%, NK cell 5-8%, The T lymphocyte 20-35%, B lymphocyte 2-5% and NK cells 3-9% in the monocyte 5-13%. peripheral blood stem cells. After the monocyte 18-32%. was stimulated to cultivate 12,24 and 72h, there was no significant difference in the proportion of T, B, and monocyte subsets in the four groups. With the prolongation of culture time, there were a small amount of cell apoptosis. There was no significant difference in the proportion of cell subgroups (P0.05).3.T, B, NK and the expression of ST2 receptor in monocyte: the expression of ST2 receptor in fresh umbilical blood and peripheral blood stem cells was similar, the expression of ST2 receptor on mononuclear cells was as high as 53-90%, followed by the B lymphocyte subgroup of about 30-60%, T lymphocyte and NK cell subgroup low, respectively. There was no significant change in the expression of ST2 receptor in each cell subgroup of the four groups (P0.05), and there was no significant change in the ST2 expression of each cell subgroup of the cells at different time points in the experimental group (P0.05). Conclusion 1.UCBT PES was a immunization reaction caused by a variety of cytokines when the donor began to be implanted, and its effector cells were derived from donors and donors. Early chimerism is a high risk factor for the occurrence of PES..2. serum monocyte related chemokine (MCP-1) increases significantly at the time of PES, while lymphocyte related specific cytokines (IL-2, IL-4, IL-8, IL-17A) have no significant changes in the occurrence of PES. The clinical manifestations of PES are mainly in the skin, intestinal and other tissues, and infer the single tissue. The nucleus macrophage may be the main effect cell of PES,.3. added to IL-33 and sST2 for simple culture in vitro, not the proliferation of immune cells and the increase of ST2 receptor expression, but it can not determine whether the level of cytokine changes. It is necessary to further detect the change of cytokine in the cell culture fluid and observe the IL-33/ST2 signaling pathway and sST2 Whether there is an effect on the signal transduction of the downstream cells to cause the secretion of cytokines, we also need to measure the activation of various immune cells, such as HLA-DR, CD27, CCR and so on, to provide the basis for revealing the mechanism of PES.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R457.7

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