STAT1在G-CSF诱导异基因造血干细胞移植供者T细胞免疫耐受中的作用

发布时间：2018-10-20 10:47

【摘要】： 目的:临床实践表明应用G-CSF作为干细胞动员剂进行的外周血造血干细胞移植,其急性移植物抗宿主反应并不比传统的骨髓移植高。这说明G-CSF存在着诱导免疫耐受的作用。G-CSF可以从T细胞分化、树突状细胞(DCs)、单个核细胞、调节性T细胞等角度对干细胞移植物的免疫功能进行调节,诱导移植物中的T细胞产生免疫耐受。目前的观察表明,G-CSF刺激后,出现Th1型细胞因子(IFN-γ、IL-2、T-bet、IL-12等)明显减少,而Th2型细胞因子(IL-10、IL-4、GATA-3等)显著增多,这一现象在诱导免疫耐受中起着至关重要的作用。但是,目前对T细胞上是否存在G-CSF受体仍存在很大的争议,因此G-CSF能否直接对T细胞产生作用仍不肯定。 JAK-STAT信号转导途径作为机体内非常重要的信号转导途径之一,在机体免疫功能的形成与发挥作用中有重要的作用。这一途径参与体内多种病理生理过程,调节体内众多基因的表达,约有50余种细胞因子通过其进行信号传导。因此,它对机体各种生理功能的维持及对外界刺激做出正确的反应是非常重要的一环。其中,STAT1作为Th1型细胞因子重要的信号转导因子,在IFN-γ、IL-2引起的基因转录过程中居于核心地位。并且很多学者已经证实STAT1是GVHD重要的调控点。但到目前为止,尚没有研究对STAT1在G-CSF诱导的免疫耐受中的变化进行过检测。因此本实验在G-CSF诱导免疫耐受过程中,检测Th1型细胞及Th2型细胞的数量及比值,并检测G-CSF受体、T-bet、GATA-3和STAT1的表达水平的变化,并对其作用进行初步探讨,以进一步加深对G-CSF诱导免疫耐受机制的了解、为对其进行调控打下基础。方法: 1收集标本:分别留取23位骨髓移植供者应用G-CSF动员前及应用G-CSF动员3天后的外周血标本10ml,使用肝素抗凝。 2分组:共分为两组,即供者应用G-CSF动员前的外周血标本和供者应用G-CSF动员后的外周血标本,为配对标本。 3应用免疫磁珠分选法分选CD4+T细胞:从采集的外周血标本分离出单个核细胞后,再应用免疫磁珠分选的方法分离CD4+T细胞并提纯,最后用流式细胞仪检测CD4+T细胞的纯度并计数。 4流式细胞技术检测G-CSF动员前及G-CSF动员后的CD4+T细胞中Th1、Th2的数量及Th1/Th2比值。 5制备cDNA:把分选得来的CD4+T细胞应用Trizol试剂盒提取RNA,并逆转录为cDNA。 6应用实时荧光定量聚合酶链反应(Real-time Quantitative PCR,RQ-PCR)方法分别检测体内应用G-CSF动员前后G-CSFR、T-bet、GATA-3和STAT1表达量的变化。 7将纯化的CD4+T细胞进行细胞培养,并在培养过程中加入不同浓度的G-CSF刺激,观察其刺激T细胞增殖的作用。 8分别将应用G-CSF刺激前后培养的CD4+T细胞收集,提取RNA并逆转录为cDNA. 9 RQ-PCR方法检测体外培养的CD4+T细胞经G-CSF刺激前后的G-CSFR、T-bet、GATAT-3和STAT1表达量变化。结果: 1在流式细胞术检测CD4+T细胞Th1/Th2比值结果显示G-动员后的Th1/Th2比值较动员前的Th1/Th2比值有显著的降低。 2我们对标本进行CD4+T细胞计数,显示G-CSF动员3天后CD4+T细胞的数量和占单个核细胞的比例均增加。 3应用RQ-PCR检测体内应用G-CSF动员后G-CSFR、T-bet、GATA-3及STAT1表达水平的变化。结果显示:虽这些基因的表达均有变化,但经统计学处理,均没有统计学意义。 4体外培养分离纯化的CD4+T淋巴细胞,结果显示:G-CSF可以刺激分离后的CD4+T淋巴细胞增殖,这种作用达峰时间为12-24小时,48小时后开始下降,72小时后作用消失。G-CSF的刺激浓度以200ng/ml为宜,增加浓度,增殖指数未见明显的增加。 5 RQ-PCR检测在体外培养时加入G-CSF刺激前后的CD4+T细胞的G-CSFR、T-bet、GATA-3、STAT1的表达变化。结果显示:在体外加入G-CSF刺激后,CD4+T细胞出现了G-CSFR和GATA-3表达的升高,T-bet和STAT1则出现下降。结论: 1体内应用G-CSF动员后,Th1/Th2比值比动员前显著降低(P0.01)。表明在供者体内T淋巴细胞受到G-CSF刺激后,CD4+T淋巴细胞会向Th2方向分化,这可能诱导了免疫耐受的产生。 2体内应用G-CSF动员后,CD4+T细胞短期内(至少3天内)可以因G-CSF的刺激作用而出现增殖。 3体外培养分离纯化的CD4+T淋巴细胞时加入G-CSF可以刺激分离后的CD4+T淋巴细胞增殖。这种作用12-24小时达到高峰,48小时后开始下降,72小时后消失。G-CSF最适刺激浓度以200ng/ml。 4体内应用G-CSF动员后G-CSFR的表达量未见增高,而在外培养CD4+T细胞并加入G-CSF刺激后会出现G-CSFR表达的增高,这可能与体内、外环境的差异,以及体内G-CSF浓度处于较低水平且浓度不稳定有关。 5体内应用G-CSF动员后GATA-3及T-bet的变化趋势不明显。而在体外培养应用G-CSF后GATA-3出现明显的升高同时T-bet则明显降低,这可能与体内外的环境差异有关。 6 STAT1在体内应用G-CSF动员后并没有减低,但在体外培养加入G-CSF刺激后会出现STAT1的减低,这可能与体内外环境的差异、体内信号转导途径上复杂的网络联系和STAT1在维持Treg细胞的产生及发挥正常作用是必需的有关。目前关于这方面的研究较少,要明白其确切作用及机制,仍需要大量的研究。
[Abstract]:Objective: Clinical practice shows that G-CSF is used as a stem cell mobilization agent for peripheral blood stem cell transplantation, and its acute graft-versus-host response is not higher than that of conventional bone marrow transplantation. This suggests that G-CSF has the effect of inducing immune tolerance. G-CSF can regulate the immune function of stem cell grafts from T cell differentiation, dendritic cells (DCs), mononuclear cells, regulatory T cells and other angles, and induce T cells in the graft to generate immune tolerance. At present, there is a significant decrease in Th1 type cytokines (IFN-, IL-2, T-bet, IL-12, etc.) after G-CSF stimulation, while Th2 cytokines (IL-10, IL-4, GATA-3, etc.) have increased significantly, which plays an important role in inducing immune tolerance. However, there is still a great controversy on whether G-CSF receptor is present on T cells, and whether G-CSF can directly play a role in T cells is still uncertain. JAK-STAT signal transduction pathway is one of the most important signal transduction pathways in organism. It is necessary to take part in many pathophysiological processes in the body, regulate the expression of many genes in the body, and make about 50 kinds of cytokines through it Therefore, it is very heavy for the maintenance of various physiological functions of the body and the correct response to external stimuli STAT1, as an important signal transduction factor of Th1 type cytokines, resides in the transcription of the genes induced by IFN-jun and IL-2. Core status. And many scholars have confirmed STAT1 is very important However, we have not studied the changes of STAT1 in immune tolerance induced by G-CSF. Therefore, in the process of G-CSF induced immune tolerance, the number and ratio of Th1 cells and Th2 cells were detected, and the expression levels of G-CSF receptor, T-bet, GATA-3 and STAT1 were detected, and the effects of G-CSF receptor, T-bet, GATA-3 and STAT1 were investigated. The knowledge of the system is to regulate it. to lay a foundation Methods: 1 Collection of specimens: Twenty-three bone marrow transplantation donors were taken to apply G-CSF mobilization before and after mobilization of G-CSF for 3 days. Blood samples 10ml, heparin anti-coagulation. 2 groups: two groups, namely, donor application G-CSF mobilization, peripheral blood sample and donor application G-CSF mobilised peripheral blood samples, paired specimens. CD4 + T cells were sorted by immunomagnetic bead sorting: After isolated mononuclear cells were isolated from the collected peripheral blood samples, CD4 + T cells were isolated and purified by immunomagnetic beads sorting. and finally, detecting the purity of CD4 + T cells by flow cytometry and counting. CD4 + T cells in CD4 + T cells and Th1/ Th2 ratio in CD4 + T cells CD4 + T cells from CD4 + T cells were used to extract RNA and reverse transcribed into cDNA. Real-time quantitative polymerase chain reaction (RQ-PCR) was used to detect G in vivo. Changes of G-1R, T-bet, GATA-3 and STAT1 expression levels before and after CSF mobilization. 7 Purified CD4 + T Cells were cultured, and G-CSF stimulated by different concentrations were added during culture to observe the effect of stimulating T cell proliferation. Using. 8, the CD4 + T cells cultured before and after stimulation with G-CSF were collected, RNA was extracted and reverse transcribed into cDNA. in vitro culture The expression of G-1R, T-bet, GATAT-3 and STAT1 in CD4 + T cells after G-CSF stimulation was changed. 1. The Th1/ Th2 ratio of CD4 + T cells was detected by flow cytometry. The Th1/ Th2 ratio after G-mobilization was higher than that before mobilization. The ratio of h2 was significantly lower. We counted the CD4 + T cell count in the specimen to show the number of CD4 + T cells after 3 days after G-CSF mobilization. and the proportion of the individual nuclear cells increased. 3, the application of the RQ-PCR detection in vivo application G The changes of G-, R, T-bet, GATA-3 and STAT1 levels after CSF mobilization were observed. The purified CD4 + T lymphocytes showed that G-CSF could stimulate CD4 + T lymphocytes after separation. The concentration of G-CSF decreased after 48 hours, and disappeared after 72 hours. The stimulation concentration of G-CSF was 200ng/ ml. 5 RQ-PCR was used to detect the G-1R, T-be of CD4 + T cells before and after G-CSF stimulation. t,G The expression of ATA-3 and STAT1 showed that after G-CSF stimulation was added in vitro, CD4 T-bet and STAT1 showed a decrease in the expression of G-1R and GATA-3 in + T cells. After the mobilization of G-CSF in vivo, the ratio of Th1/ Th2 was significantly lower than before mobilization (P0.01). After the stimulation of G-CSF, CD4 + T lymphocytes differentiate into Th2, which may induce the production of immune tolerance. After mobilization of G-CSF in vivo, CD4 + T cells are short-term (within at least 3 days) Proliferation can occur due to stimulation of G-CSF. 3. In vitro culture and isolation of purified CD4 + T lymphocytes stimulated the proliferation of CD4 + T lymphocytes after separation. This effect reached the peak at 12-24 hours, began to decrease after 48 hours, and disappeared after 72 hours. The optimum concentration of G-CSF was 200ng/ ml. The expression level of G-CSF R after G-CSF mobilization was not increased in 4 patients. In addition to the addition of G-CSF stimulation, the expression of G-CSF R may be increased after the addition of G-CSF, which may be related to the difference in vivo and external environment, as well as in vivo G. The CSF concentration was at a low level and the concentration was not stable. The trend of GATA-3 and T-bet after G-CSF mobilization was not obvious in 5 patients. There was a significant increase in GATA-3 after G-CSF, while T-bet decreased significantly, which could be related to environmental differences outside the body.. 6 STAT1 was not reduced after G-CSF mobilization in vivo, but in the body
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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