【摘要】： 目的:臨床實(shí)踐表明應(yīng)用G-CSF作為干細(xì)胞動(dòng)員劑進(jìn)行的外周血造血干細(xì)胞移植,其急性移植物抗宿主反應(yīng)并不比傳統(tǒng)的骨髓移植高。這說(shuō)明G-CSF存在著誘導(dǎo)免疫耐受的作用。G-CSF可以從T細(xì)胞分化、樹(shù)突狀細(xì)胞(DCs)、單個(gè)核細(xì)胞、調(diào)節(jié)性T細(xì)胞等角度對(duì)干細(xì)胞移植物的免疫功能進(jìn)行調(diào)節(jié),誘導(dǎo)移植物中的T細(xì)胞產(chǎn)生免疫耐受。目前的觀察表明,G-CSF刺激后,出現(xiàn)Th1型細(xì)胞因子(IFN-γ、IL-2、T-bet、IL-12等)明顯減少,而Th2型細(xì)胞因子(IL-10、IL-4、GATA-3等)顯著增多,這一現(xiàn)象在誘導(dǎo)免疫耐受中起著至關(guān)重要的作用。但是,目前對(duì)T細(xì)胞上是否存在G-CSF受體仍存在很大的爭(zhēng)議,因此G-CSF能否直接對(duì)T細(xì)胞產(chǎn)生作用仍不肯定。 JAK-STAT信號(hào)轉(zhuǎn)導(dǎo)途徑作為機(jī)體內(nèi)非常重要的信號(hào)轉(zhuǎn)導(dǎo)途徑之一,在機(jī)體免疫功能的形成與發(fā)揮作用中有重要的作用。這一途徑參與體內(nèi)多種病理生理過(guò)程,調(diào)節(jié)體內(nèi)眾多基因的表達(dá),約有50余種細(xì)胞因子通過(guò)其進(jìn)行信號(hào)傳導(dǎo)。因此,它對(duì)機(jī)體各種生理功能的維持及對(duì)外界刺激做出正確的反應(yīng)是非常重要的一環(huán)。其中,STAT1作為Th1型細(xì)胞因子重要的信號(hào)轉(zhuǎn)導(dǎo)因子,在IFN-γ、IL-2引起的基因轉(zhuǎn)錄過(guò)程中居于核心地位。并且很多學(xué)者已經(jīng)證實(shí)STAT1是GVHD重要的調(diào)控點(diǎn)。但到目前為止,尚沒(méi)有研究對(duì)STAT1在G-CSF誘導(dǎo)的免疫耐受中的變化進(jìn)行過(guò)檢測(cè)。因此本實(shí)驗(yàn)在G-CSF誘導(dǎo)免疫耐受過(guò)程中,檢測(cè)Th1型細(xì)胞及Th2型細(xì)胞的數(shù)量及比值,并檢測(cè)G-CSF受體、T-bet、GATA-3和STAT1的表達(dá)水平的變化,并對(duì)其作用進(jìn)行初步探討,以進(jìn)一步加深對(duì)G-CSF誘導(dǎo)免疫耐受機(jī)制的了解、為對(duì)其進(jìn)行調(diào)控打下基礎(chǔ)。 方法: 1收集標(biāo)本:分別留取23位骨髓移植供者應(yīng)用G-CSF動(dòng)員前及應(yīng)用G-CSF動(dòng)員3天后的外周血標(biāo)本10ml,使用肝素抗凝。 2分組:共分為兩組,即供者應(yīng)用G-CSF動(dòng)員前的外周血標(biāo)本和供者應(yīng)用G-CSF動(dòng)員后的外周血標(biāo)本,為配對(duì)標(biāo)本。 3應(yīng)用免疫磁珠分選法分選CD4+T細(xì)胞:從采集的外周血標(biāo)本分離出單個(gè)核細(xì)胞后,再應(yīng)用免疫磁珠分選的方法分離CD4+T細(xì)胞并提純,最后用流式細(xì)胞儀檢測(cè)CD4+T細(xì)胞的純度并計(jì)數(shù)。 4流式細(xì)胞技術(shù)檢測(cè)G-CSF動(dòng)員前及G-CSF動(dòng)員后的CD4+T細(xì)胞中Th1、Th2的數(shù)量及Th1/Th2比值。 5制備cDNA:把分選得來(lái)的CD4+T細(xì)胞應(yīng)用Trizol試劑盒提取RNA,并逆轉(zhuǎn)錄為cDNA。 6應(yīng)用實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)(Real-time Quantitative PCR,RQ-PCR)方法分別檢測(cè)體內(nèi)應(yīng)用G-CSF動(dòng)員前后G-CSFR、T-bet、GATA-3和STAT1表達(dá)量的變化。 7將純化的CD4+T細(xì)胞進(jìn)行細(xì)胞培養(yǎng),并在培養(yǎng)過(guò)程中加入不同濃度的G-CSF刺激,觀察其刺激T細(xì)胞增殖的作用。 8分別將應(yīng)用G-CSF刺激前后培養(yǎng)的CD4+T細(xì)胞收集,提取RNA并逆轉(zhuǎn)錄為cDNA. 9 RQ-PCR方法檢測(cè)體外培養(yǎng)的CD4+T細(xì)胞經(jīng)G-CSF刺激前后的G-CSFR、T-bet、GATAT-3和STAT1表達(dá)量變化。 結(jié)果: 1在流式細(xì)胞術(shù)檢測(cè)CD4+T細(xì)胞Th1/Th2比值結(jié)果顯示G-動(dòng)員后的Th1/Th2比值較動(dòng)員前的Th1/Th2比值有顯著的降低。 2我們對(duì)標(biāo)本進(jìn)行CD4+T細(xì)胞計(jì)數(shù),顯示G-CSF動(dòng)員3天后CD4+T細(xì)胞的數(shù)量和占單個(gè)核細(xì)胞的比例均增加。 3應(yīng)用RQ-PCR檢測(cè)體內(nèi)應(yīng)用G-CSF動(dòng)員后G-CSFR、T-bet、GATA-3及STAT1表達(dá)水平的變化。結(jié)果顯示:雖這些基因的表達(dá)均有變化,但經(jīng)統(tǒng)計(jì)學(xué)處理,均沒(méi)有統(tǒng)計(jì)學(xué)意義。 4體外培養(yǎng)分離純化的CD4+T淋巴細(xì)胞,結(jié)果顯示:G-CSF可以刺激分離后的CD4+T淋巴細(xì)胞增殖,這種作用達(dá)峰時(shí)間為12-24小時(shí),48小時(shí)后開(kāi)始下降,72小時(shí)后作用消失。G-CSF的刺激濃度以200ng/ml為宜,增加濃度,增殖指數(shù)未見(jiàn)明顯的增加。 5 RQ-PCR檢測(cè)在體外培養(yǎng)時(shí)加入G-CSF刺激前后的CD4+T細(xì)胞的G-CSFR、T-bet、GATA-3、STAT1的表達(dá)變化。結(jié)果顯示:在體外加入G-CSF刺激后,CD4+T細(xì)胞出現(xiàn)了G-CSFR和GATA-3表達(dá)的升高,T-bet和STAT1則出現(xiàn)下降。 結(jié)論: 1體內(nèi)應(yīng)用G-CSF動(dòng)員后,Th1/Th2比值比動(dòng)員前顯著降低(P0.01)。表明在供者體內(nèi)T淋巴細(xì)胞受到G-CSF刺激后,CD4+T淋巴細(xì)胞會(huì)向Th2方向分化,這可能誘導(dǎo)了免疫耐受的產(chǎn)生。 2體內(nèi)應(yīng)用G-CSF動(dòng)員后,CD4+T細(xì)胞短期內(nèi)(至少3天內(nèi))可以因G-CSF的刺激作用而出現(xiàn)增殖。 3體外培養(yǎng)分離純化的CD4+T淋巴細(xì)胞時(shí)加入G-CSF可以刺激分離后的CD4+T淋巴細(xì)胞增殖。這種作用12-24小時(shí)達(dá)到高峰,48小時(shí)后開(kāi)始下降,72小時(shí)后消失。G-CSF最適刺激濃度以200ng/ml。 4體內(nèi)應(yīng)用G-CSF動(dòng)員后G-CSFR的表達(dá)量未見(jiàn)增高,而在外培養(yǎng)CD4+T細(xì)胞并加入G-CSF刺激后會(huì)出現(xiàn)G-CSFR表達(dá)的增高,這可能與體內(nèi)、外環(huán)境的差異,以及體內(nèi)G-CSF濃度處于較低水平且濃度不穩(wěn)定有關(guān)。 5體內(nèi)應(yīng)用G-CSF動(dòng)員后GATA-3及T-bet的變化趨勢(shì)不明顯。而在體外培養(yǎng)應(yīng)用G-CSF后GATA-3出現(xiàn)明顯的升高同時(shí)T-bet則明顯降低,這可能與體內(nèi)外的環(huán)境差異有關(guān)。 6 STAT1在體內(nèi)應(yīng)用G-CSF動(dòng)員后并沒(méi)有減低,但在體外培養(yǎng)加入G-CSF刺激后會(huì)出現(xiàn)STAT1的減低,這可能與體內(nèi)外環(huán)境的差異、體內(nèi)信號(hào)轉(zhuǎn)導(dǎo)途徑上復(fù)雜的網(wǎng)絡(luò)聯(lián)系和STAT1在維持Treg細(xì)胞的產(chǎn)生及發(fā)揮正常作用是必需的有關(guān)。目前關(guān)于這方面的研究較少,要明白其確切作用及機(jī)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
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

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4 李德冠;P38 MAPK抑制劑聯(lián)合G-CSF對(duì)全身γ射線照射小鼠輻射損傷的實(shí)驗(yàn)治療研究[D];北京協(xié)和醫(yī)學(xué)院;2012年

5 周曉旭;普伐他汀干預(yù)p38 MAPK、IFN-gamma/STAT1、IL-6/STAT3信號(hào)傳導(dǎo)通路預(yù)防動(dòng)脈粥樣硬化的實(shí)驗(yàn)研究[D];上海交通大學(xué);2008年

6 鄧昊;STAT1和Survivin及相關(guān)蛋白在胃癌中相關(guān)性及其臨床病理學(xué)意義的研究[D];華中科技大學(xué);2006年

7 郭曉玲;G-CSF誘導(dǎo)T淋巴細(xì)胞向TH2分化的機(jī)制研究[D];中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院;2008年

8 姚志峰;G-CSF對(duì)壓力超負(fù)荷下小鼠心室重構(gòu)和心力衰竭的影響[D];復(fù)旦大學(xué);2008年

9 阮曄;應(yīng)用基因芯片技術(shù)對(duì)Graves病、甲狀腺乳頭狀癌相關(guān)基因表達(dá)的研究[D];第二軍醫(yī)大學(xué);2004年

10 鄧昊;STAT1和Survivin及相關(guān)蛋白在胃癌中相關(guān)性的研究及其臨床意義[D];華中科技大學(xué);2006年

相關(guān)碩士學(xué)位論文 前10條

1 蔡圣鑫;STAT1在G-CSF誘導(dǎo)異基因造血干細(xì)胞移植供者T細(xì)胞免疫耐受中的作用[D];河北醫(yī)科大學(xué);2009年

2 劉春鳳;STAT1基因沉默對(duì)哮喘小鼠IFN-γ、IL-5、ICAM-1表達(dá)的影響[D];瀘州醫(yī)學(xué)院;2012年

3 盧年芳;不同亞型干擾素-α抗乙型肝炎病毒活性的實(shí)驗(yàn)研究[D];重慶醫(yī)科大學(xué);2004年

4 劉濟(jì);非霍奇金淋巴瘤血漿GM-CSF、G-CSF表達(dá)的臨床意義及其與中醫(yī)辨證分型的關(guān)系[D];福建中醫(yī)藥大學(xué);2010年

5 楊華;G-CSF聯(lián)合辛伐他汀動(dòng)員血管內(nèi)皮祖細(xì)胞的實(shí)驗(yàn)研究[D];昆明醫(yī)學(xué)院;2011年

6 周江朝;應(yīng)用G-CSF動(dòng)員骨髓干細(xì)胞治療重型顱腦損傷的研究[D];大連醫(yī)科大學(xué);2010年

7 王巍;電離輻射對(duì)食管癌細(xì)胞中H2AX、STAT1表達(dá)的影響[D];河北醫(yī)科大學(xué);2011年

8 宋磊;G-CSF對(duì)局灶腦缺血再灌注大鼠脂質(zhì)過(guò)氧化及神經(jīng)細(xì)胞凋亡的影響[D];吉林大學(xué);2010年

9 王鵬;IFNγ-STAT1信號(hào)通路對(duì)打破內(nèi)毒素耐受作用機(jī)制的研究[D];山東大學(xué);2012年

10 謝曉強(qiáng);G-CSF動(dòng)員骨髓干細(xì)胞向缺血再灌注損傷腎臟歸巢并促進(jìn)腎臟修復(fù)的研究[D];天津醫(yī)科大學(xué);2011年

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