【作者】 梁晨；

【导师】 韩明哲； 冯四洲；

【作者基本信息】 中国协和医科大学 ， 内科学， 2010， 博士

【摘要】 第一部分：干扰素-γ与骨髓间充质干细胞免疫调节协同作用机制研究[目的]1探讨骨髓间充质干细胞(MSC)在体外对T淋巴细胞增殖的抑制作用,研究其主要作用机制。2探讨干扰素—γ(IFN—γ)作用下MSC免疫活性变化情况,阐明IFN—γ与MSC在免疫调节中的协同作用。[方法]1采用密度梯度离心法或直接贴壁法从正常成人骨髓中分离扩增MSC,取培养后3-5代细胞用于实验研究。2酶联免疫吸附试验(ELISA)分别检测MSC单独培养、MSC与IFN-γ共培养后培养上清液中前列腺素E2(PGE2)、肝细胞生长因子(HGF)和转化生长因子—β1(TGF-β1)表达水平,比较IFN-γ作用下MSC分泌可溶性因子水平变化情况；采用抗CD3单克隆抗体(mAb)和抗CD28mAb刺激T淋巴细胞增殖,在培养体系中加入HGF或TGF-β1等细胞因子,5-溴脱氧尿嘧啶核苷(BrdU)ELISA试剂盒检测上述细胞因子对T细胞增殖的影响。3提取MSC细胞RNA,采用逆转录—聚合酶链反应(RT—PCR)方法检测MSC细胞内吲哚胺2,3—双加氧酶(IDO)表达水平；将MSC与IFN-γ共培养48h后,RT—PCR方法再次检测IDO表达水平,与MSC单独培养时进行比较；采用不同浓度IFN-γ与MSC共培养,检测MSC细胞内IDO表达水平变化情况；在T细胞培养体系中加入犬尿酸(100μM),BrdU试剂盒检测T细胞增殖变化,探讨IDO的免疫抑制作用。4将MSC加入T细胞培养体系中,BrdU试剂盒检测T细胞增殖水平,验证MSC在体外对T淋巴细胞增殖的抑制作用；在反应体系中分别加入IFN—γ及鼠抗人IFN—γmAb,检测T细胞增殖水平变化情况。[结果]1采用密度梯度离心法和直接贴壁法培养获得的MSC在形态和增殖速度方面没有明显差异。原代细胞接种于塑料培养瓶后呈集落样生长,经过2-3周左右细胞贴壁生长融合达80～90%,消化后平均每个底面积25 cm2的培养瓶可获得3～4×105细胞,之后按照1：2或1：3比例进行传代。传至第3-4代时,可获得约3～5×106细胞,供实验使用或液氮冻存。2将3份MSC标本按1×105细胞／孔接种于24孔板,每24h留取细胞培养上清液。在培养24-48h内即可检测到PGE2、HGF和TGF—β1三种细胞因子表达。并且随着培养时间延长,上清液中上述细胞因子浓度逐渐升高,至培养后144h(6d),三种细胞因子表达水平均趋于稳定。设置实验组与对照组,实验组中MSC与IFN—γ(100ng／m1)共培养,对照组中MSC单独培养。144h后留取两组细胞培养上清液,实验组上清液中PGE2浓度为1715.5167±628.5726pg／ml,而对照组PGE2浓度为1344.5163±709.3583pg／ml,较实验组明显降低,差异具有显著统计学意义(P=0.001)。实验组上清液中HGF浓度为4031.7733±1496.8027pg／ml,对照组HGF浓度为2452.4267±1375.3291 pg／ml,较实验组明显降低(P=0.011)。实验组上清液中TGF—β1浓度为1753.5363±413.8059 pg／ml,对照组TGF—β1浓度为1026.6080±450.5418pg／ml,亦较实验组明显降低(P<0.001)。外周血T细胞在抗CD3mAb和抗CD28mAb的刺激下显著增殖,加入HGF浓度分别为10ng／ml、20ng／ml和40ng／ml时,T细胞增殖的抑制率分别为(17.4750±9.8577)%、(27.7183±8.6342)%和(36.6233±11.8959)%,随着HGF浓度升高,对T细胞增殖的抑制作用也越强。HGF浓度为40ng／ml时,T细胞增殖抑制率明显高于HGF浓度10ng／ml组(P=0.005)。加入TGF—β1浓度分别为500pg／ml、1000pg／ml和2000pg／ml时,T细胞增殖的抑制率分别为(13.2163±6.0858)%、(20.6529±7.3306)%和(32.5013±11.2595)%。TGF—β1浓度为2000pg／ml时,T细胞增殖抑制率明显高于TGF—β1浓度500pg／ml组(P<0.001)和1000pg／ml组(P=0.015)。3在T淋巴细胞培养体系中加入外源性犬尿酸(100μM)后,T细胞增殖明显受抑,较阳性对照组明显减低(P=0.004)。MSC单独培养后提取细胞总RNA,半定量RT—PCR方法未检测到明显IDOmRNA表达。将MSC与IFN—γ(100ng／ml)共培养后,IDOmRNA明显高表达。此外,在极低浓度IFN—γ(1ng/ml)作用下,即可检测到IDOmRNA表达。4 MSC显著抑制抗CD3mAb和抗CD28mAb刺激下的T细胞增殖,并且随着MSC细胞密度增高,对T细胞增殖的抑制作用也越强。将MSC(2×104细胞／孔)与不同供者来源T细胞(1×105细胞／孔)共培养,T细胞增殖均显著受抑(P<0.001)。在上述MSC与T细胞混合培养体系中加入IFN—γ(100ng/ml),不会影响MSC对T细胞增殖的抑制作用(P=0.272)；相反,在反应体系中加入鼠抗人IFN—γmAb(5μg/ml)以拮抗IFN—γ的作用,MSC的免疫抑制作用即显著减弱(P=0.002),T细胞增殖部分恢复。[结论]1 MSC组成性表达PGE2、HGF及TGF-β1等可溶性因子,其表达水平在体外实验中均可抑制T淋巴细胞增殖,此为MSC免疫抑制作用的主要机制之一。在一定水平IFN-γ作用下,MSC分泌上述三种免疫抑制性细胞因子水平上调,证实IFN-γ与MSC在免疫抑制中的协同作用。2 IDO通过降解色氨酸为犬尿酸抑制T淋巴细胞增殖。MSC单独培养时不表达IDO活性,但是在IFN-γ诱导作用下,IDO表达水平显著增高,此亦为MSC发挥免疫抑制作用的主要机制之一3骨髓MSC在体外显著抑制异基因T淋巴细胞增殖,并且此抑制作用没有MHC限制性。外源性IFN-γ不仅不会削弱MSC的免疫抑制活性,而且同样具有抑制T细胞增殖的作用,加入IFN-γmAb后可以逆转MSC对T淋巴细胞增殖的抑制作用。第二部分：造血干细胞移植供受者乙型肝炎病毒感染的临床随访研究[目的]分析移植前乙型肝炎病毒(HBV)感染的供、受者在异基因造血干细胞移植(allo—HSCT)后,受者HBV血清学标记变化及对临床结果的影响。[方法]对我院2002年9月至2008年11月间allo—HSCT治疗前供、受者合并HBV感染的79例患者进行随访研究,对其疾病预后与转归、移植后肝功能及HBV血清学标记变化等临床资料进行回顾性分析。[结果]1移植前供、受者HBV感染对受者预后无明显影响。2 HBsAgI阳性组患者20例,13例(65.0%)出现HBV激活,时间为移植后1(0.5-10)月,9例(45.0%)并发乙肝相关肝炎。3 HBsAg阴性组患者35例,4例(11.4%)移植后HBsAg转为阳性,即出现乙肝血清学转换,其中1例伴随严重慢性移植物抗宿主病(cGVHD).4 HBsAg阳性组患者移植后出现HBV激活及+100d内肝功能损害的比例均明显高于HBsAg阴性组患者(P<0.05)。52例(10.0%)HBsAg阳性患者在移植后清除体内HBV,其供者乙肝血清学标志均为HBsAb阳性。[结论]1供、受者HBV感染不是allo—HSCT禁忌证。2 HBsAg阳性是移植后发生HBV激活的高危因素,对于这类患者应进行规范性拉米夫定预防性治疗。3 HBcAb／HBeAb阳性患者移植后可能出现乙肝血清学转换,在免疫抑制剂减量过程中应密切监测其乙肝血清学标记变化情况。4 allo—HSCT可以通过过继免疫治疗清除患者体内HBV。更多还原

【Abstract】 Objective1 To investigate the immunosuppressive effect of bone marrow derived mesenchymal stem cells (MSCs) on T cell proliferation in vitro. To study the main mechanisms of MSC-mediated immunomodulation.2 To investigate the changes in MSC immunomodulatory activity in the presence of Interferon-gamma (IFN-y). To explore the synergistic effects of IFN-y in the immunomodulatory role of MSC.Methods1 Human MSCs were generated from bone marrow aspirates of healthy donors, recruited after informed consent. MSCs were obtained with density-gradient centrifugation or direct plating and cultured in 25cm2 flasks. MSCs at passage 3 to 5 were cryopreserved and used for futrher study.2 MSCs were cultured in the presence or absence of IFN-y (100ng/ml), the supernatants were collected for measurement of PGE2、HGF and TGF-β1 by ELISA kits. T lymphocytes proliferated under the stimulation of anti-human CD3 monoclonal antibody (mAb) and anti-human CD28 mAb.HGF and TGF-β1 were added respectively into the culture system, their effects on T cell proliferation were examined by BrdU ELISA kit.3 MSCs were cultured in the presence or absence of IFN-y (100ng/ml) for 48h and total RNA was extracted from cells and reverse-transcribed to cDNA. The cDNA was analysed for the expression of human indoleamine 2,3-dioxygenase (IDO) mRNA by semiquantitative RT-PCR. The expression levels were compared in these two conditions. Kynurenine (100μM) was added into the T lymphocytes culture system, its suppressive effects on T cell proliferation was examined by BrdU ELISA kit.4 Mononuclear cells (MNC) were extracted from peripheral blood from different healthy donors. MSC were cultured for 24h prior to MNC addition. After 96h of incubation, BrdU labeling solution was added to each well and incubated for a further 24h. The cell proliferation was tested by BrdU ELISA kit. Furthermore, recombinant human IFN-γ(100ng/ml) and anti-IFN-γmAb (5μg/ml) were added into the culture system. The T cell proliferation was tested in these different conditions.Results1 MSCs could be isolated from normal human bone marrow. MSC cells obtained with density-gradient centrifugation or direct plating had similar characteristics in cell morphology and proliferation kinetics. After the MNCs were inoculated in the plastic culture flask, the cells developed to an adherent fibroblast-like population. Two to there weeks later, the cells could be digested with trpysin and expanded. By the third to fourth passage,3～5×106 MSC per donor could be retrieved for further experiments.2 MSCs from three different donos were inoculated in flat-bottomed 24-well plates (1×105/well). The supernatant of the culture media was collected every 24h. The immunosuppressive cytokines PGE2、HGF and TGF—β1 coule be detected within 24～48h. Concentrarions of these cytokines continually increased until 144h after inoculation.Furthmore, MSCs were divided into two groups, in experimental group, MSC was cocultured with IFN—γ(100ng/ml) and in control group, MSC was cultured alone. After 144h, the supernatant was collected for examination. In the experimental group, the concentrations of PGE2、HGF and TGF-β1 were 1715.5167±628.5726 pg/ml,4031.7733±1496.8027pg/ml and 1753.5363±413.8059 pg/ml respectively. In the control group, the concentrations of PGE2、HGF and TGF-β1 were 1344.5163±709.3583 pg/ml,2452.4267±1375.3291 pg/ml and 1026.6080±450.5418 pg/ml respectively. Therefore, PGE2、HGF and TGF-β1 expressions of MSC were significantly up-regulated by IFN—γ. For these three cytokines, the concentrations between experimental group and control group had statistical significance, the P value was 0.001,0.011 and less than 0.001 respectively.The MNCs obtained from peripheral blood proliferated in the presence of anti-human CD3 mAb and anti-human CD28 mAb. When the concentrations of HGF were10ng/ml,20ng/ml and 40ng/ml, the inhibitory rates of T cell proliferation were (17.4750±9.8577)%、(27.7183±8.6342)% and (36.6233±11.8959)% respectively. With the increased concentration of HGF, the inhibitory rate also increased.When HGF concentration was 40ng/ml, the inhibitory rate of T cell proliferation was significantly higher than the 10ng/ml HGF group (P=0.005).When the concentrations of TGF-β1 were 500pg/ml, 1000pg/ml and 2000pg/ml, the inhibitory rates of T cell proliferation were (13.2163±6.0858)%、(20.6529±7.3306)% and (32.5013±11.2595)% respectively. With the increased concentration of TGF-β1, the inhibitory rate also increased.When TGF-β1 concentration was 2000pg/ml, the inhibitory rate of T cell proliferation was significantly higher than the 500pg/ml group (P<0.001) and the 1000pg/ml group (P=0.015).3 T lymphocytes proliferated under the stimulation of anti-human CD3 mAb and anti-human CD28 mAb. When exogenous kynurenine (100Mm) was added into the culture system, the rate of T cell proliferation was significantly restrained (P=0.004).The expression of IDO mRNA was unable to be detected when MSC was cultured alone. In contrast, The IDO mRNA expression was remarkably enhanced when MSCs cultured in the presence of IFN-γ. Even the concentration of IFN-y was lng/ml, the IDO mRNA expression still could be detected.4 Bone marrow-derived MSC remarkably suppressed allogeneic T cell proliferation in vitro. With the increased concentration of MSC, the inhibitory rate was also increased. The suppressive role of MSC had no major histocompatibility complex (MHC) restriction.IFN-y did not break, but promoted the immunosuppressive capacity of human MSC. Addition of exogenous IFN-y (100ng/ml) into the T cell culture system had no significant effect on the inhibitory capacity of MSC (P=0.272).By contrast,5μg/ml IFN-y mAb was added into the culture system, the inhibitory rate of T cell proliferation was significantly decreased (P=0.002). Therefore, T cell proliferation was partially restored.Conclusion1 Human MSC constitutively expressed PGE2-. HGF and TGF-β1 at immunosuppressive concentrations. It was maybe one of the main mechanisms in MSC immunosuppressive activity. The pro inflammatory cytokine IFN-y did not ablate MSC inhibition of T cell proliferation but up-regulated PGE2、HGF and TGF-β1, confirming the synergistic effects of IFN-y with MSC in immunosuppression.2 IFN-y induced MSC expression of IDO, involved in tryptophan catabolism. This activity also contributed to MSC immunosuppressive effect.3 Human bone marrow-derived MSC notably suppressed allogeneic T cell proliferation in vitro. The suppressive role of MSC had no MHC restriction. Exogenous IFN-γdid not break, but promoted the immunosuppressive capacity of human MSC. Addition of IFN-y mAb into the culture system partially restored the T cell proliferation. ObjectiveTo investigate the prognosis and changes in hepatitis B serologic markers in patients whose donors or themselves were infected with HBV before allogeneic hematopoietic stem cell transplantation (allo-HSCT).MethodsWe analyzed retrospectively the clinical outcomes of 79 patients receiving allo-HSCT in our hospital between September 1992 and November 2008, including 55 patients with HBV infection and 24 patients with HBV infected donors.Results1 HBV infection did not interfere with the clinical outcomes of allo-HSCT.2 In HBsAg+group,13 (65.0%) patients developed HBV reactivation between 0.5 and 10 months after transplantation,9 (45.0%) of them developed HBV-related hepatitis.3 For the 35 HBsAg- patients with HBcAb/HBeAb positivity,4 (11.4%) of them were observed for HBV seroconversion (HBsAg became positive after immunosuppressive therapy),1 patient was concomitant with severe chronic graft-versus-host disease (cGVHD).4 There was a significant difference in HBV recativation rate between the HBsAg+and HBsAg- group (P<0.01). The incidence of hepatitis occurred within 100 days after HSCT was also higher in HBsAg+patients (P<0.05)5 2 HBsAg+patients were observed for clearance of HBV through adoptive immunity transfer, both donors were positve for HBsAb.Conclusion1 Donors or recipients infected with HBV was not considered an absolute contraindication to HSCT.2 HBsAg positivity was a high risk factor for HBV reactivation. Prophylactic lamivudine treatment could be helpful.3 For the patients with HBcAb/HBeAb positivity, HBV seroconversion could be observed, especially in patients following immunosuppressant withdrawal. Therefore, it was critical to pay close attention to the changes of HBV serologic markers in them.4 Adoptive immunity transfer was effective in clearing HBV infection in patients undergoing allo-HSCT更多还原