【作者】 李泉；

【导师】 邹萍；

【作者基本信息】 华中科技大学 ， 内科学， 2009， 博士

【摘要】 第一部分蛋白酶激活受体-1，2在小鼠急性GVHD模型中的表达目的：急性GVHD的机制仍不明确，但已经公认供者T淋巴细胞的激活和炎症细胞因子的释放发挥了重要的作用。最近的研究提示PAR不仅能降解细胞内蛋白酶，还是炎症和免疫过程中的重要信号分子。本研究的目的是探讨蛋白酶激活受体-1，2(PAR-1，2)在小鼠急性移植物抗宿主病(GVHD)模型中的表达。方法：建立异基因造血干细胞移植(HSCT)后小鼠急性GVHD模型，同基因HSCT小鼠作为对照组。观察小鼠急性GVHD临床表现，组织切片HE染色观察小鼠GVHD的病理改变。实时荧光定量PCR、Western blot和免疫组织化学方法检测PAR-1，2在基因和蛋白水平的表达。结果：异基因HSCT小鼠在+18～+28天出现了典型的急性GVHD征象和病理表现。实时定量PCR结果显示：PAR-1基因在异基因HSCT组小鼠的急性GVHD靶器官——皮肤、肝脏、小肠中表达水平较对照组显著增高(皮肤：0．039±0．013 vs．0．008±0．002，P=0．009；肝脏：0．165±0．006 vs．0．017±0．006，P=0．004；小肠：0．215±0．009 vs．0．016±0．002，P=0．003)；PAR-2基因在异基因HSCT组小鼠肝脏和小肠的表达较对照组增高(肝脏：0．010±0．002 vs．0．003±0．001，P=0．008；小肠：0．006±0．001 vs．0．003±0．001，P=0．024)，两者在其余器官的表达和对照组无统计学差异(P＞0．05)。Western blot检测PAR-1，2在蛋白水平的表达与基因表达的检测结果一致：PAR—1蛋白在异基因HSCT小鼠的皮肤、小肠、肝脏中的表达分别是对照组的6．3±1．4(P=0．022)、3．9±0．8(P=0．035)和6．2±0．6倍(P=0．018)；PAR—2蛋白在aGVHD小鼠的小肠、肝脏中的表达分别是对照组的2．85±0．3倍(P=0．037)和4．0±0．7倍(P=0．012)。免疫组织化学检查显示PAR—1在异基因HSCT小鼠皮肤上皮细胞和腺上皮细胞、肝脏汇管区以及肝细胞、血管内皮细胞，肠道的粘膜上皮和腺上皮表达明显增强；PAR—2在异基因HSCT小鼠小肠的粘膜上皮和腺上皮以及肝脏的肝细胞、血管内皮细胞表达增强，而PAR—1和PAR—2在异基因HSCT小鼠胃、肾、肺组织及对照组小鼠各组织中则无表达或表达很弱。结论：本研究显示PAR-1，2表达增高很有可能参与异基因HSCT后急性GVHD的病理过程，这有助于对急性GVHD病理机制的深入理解，并可能为急性GVHD的防治提供新的作用靶点。第二部分异基因CD8~+T淋巴细胞通过PAR-1途径诱导血管内皮细胞凋亡目的：血管内皮细胞是急性和慢性GVHD中异基因T淋巴细胞的靶组织，内皮细胞损伤也是移植相关血管疾病发生的始动环节。异基因造血干细胞移植后血管内皮损伤的机制仍不明确，本研究的目的是研究异基因CD8~+T淋巴细胞对血管内皮细胞凋亡的影响及其分子机制。方法：磁珠分选正常人外周血CD8~+T淋巴细胞。AnnexinⅤ-FITC试剂盒检测异基因CD8~+T淋巴细胞和蛋白酶激活受体—1(PAR-1)激动剂作用后人脐静脉内皮细胞(HUVEC)和人皮肤微血管内皮细胞(HDMEC)凋亡率，RT—PCR检测PAR-1的mRNA表达，Western blot检测内皮细胞PAR-1、丝裂原活化蛋白激酶(MAPK)和Caspase-3表达。并检测PAR-1抗体、MAPK抑制剂对异基因CD8~+T淋巴细胞和PAR-1激动剂诱导的HUVEC和HDMEC凋亡的影响。结果：HUVEC与HDMEC在基因和蛋白水平均有PAR—1表达。异基因CD8~+T淋巴细胞作用24小时和48小时，HUVEC凋亡率分别为51．7±4．1％和29．4±3．3％，HDMEC凋亡率分别为28．9±2．2％和15．2±1．8％，均显著高于对照组(P＜0．01)；SFLLRN(PAR—1激动剂)诱导两种内皮细胞凋亡的作用与异基因CD8~+T淋巴细胞无统计学差异(P＞0．05)。异基因CD8~+T淋巴细胞作用后，HUVEC与HCMEC中磷酸化p38MAPK表达水平较对照组明显增强(P＜0．05)，30min时分别为对照组的9．8、6．3倍，Caspase-3裂解增加，峰值分别为对照组的5．1、4．3倍；SFLLRN作用后，HUVEC与HCMEC中磷酸化p38MAPK表达水平亦较对照组明显增强(P＜0．05)，30min时分别为对照组的9．8、6．3倍，Caspase-3裂解增加，峰值分别为对照组的5．7、2．9倍。而异基因CD8~+T淋巴细胞和SFLLRN作用后，HUVEC与HCMEC中磷酸化JNK表达水平较对照组无显著改变。PAR—1抗体可显著抑制异基因CD8~+T淋巴细胞诱导的内皮细胞凋亡，PAR—1抗体作用后异基因CD8~+T淋巴细胞诱导的HUVEC与HDMEC凋亡率分别下降了82．8％(t=7．42，P＜0．001)和76．5％(t=4．31，P=0．005)。P38MAPK抑制剂(SB203580)作用后，异基因CD8~+T淋巴细胞诱导的异基因CD8~+T细胞诱导的HUVEC和HDMEC凋亡率分别较未用药组下降76．3％(t=4．53，P=0．003)和81．6％(t=5．96，P＜0．001)。结论：异基因CD8~+T淋巴细胞通过PAR—1途径引起细胞内p38MAPK激活、Caspase-3裂解，从而诱导血管内皮细胞凋亡。这有助于深入理解异基因造血干细胞移植后急性GVHD和移植相关血管疾病的发生机制，并为防治移植并发症提供新的作用靶点。第三部分组织因子在小鼠急性GVHD模型中的表达目的：近年研究发现，血管内皮细胞异基因HSCT后供者T淋巴细胞的靶组织，血管内皮细胞损伤在急性GVHD中发挥了非常重要的作用。由于内皮细胞活化、损伤后TF表达显著增高，而TF不仅是凝血—炎症网络的重要因子，而且还通过其细胞内转导信号介导炎症过程，调节炎症细胞因子的释放。我们推测TF在急性GVHD中发挥了重要作用。本课题的目的是研究对组织因子(TF)在小鼠急性GVHD模型中的表达。方法：建立异基因HSCT后小鼠急性GVHD模型，同基因HSCT小鼠作为对照组。观察小鼠急性GVHD表现。实时定量PCR和Western blot方法检测同基因和异基因HSCT小鼠各组织中TF在基因和蛋白水平的表达。结果：异基因HSCT组小鼠在+18～+28d出现典型的急性GVHD临床和病理表现，并于移植后28d全部死亡。同基因HSCT组小鼠未见明显的急性GVHD临床和病理表现，均存活。实时定量PCR检测发现，异基因HSCT组小鼠的急性GVHD靶组织——皮肤、肝脏、胃、小肠中TFmRNA表达水平较对照组显著增强，分别为对照组的15．1±2．1，5．5±1．4，9．7±2．3，14．3±2．9倍(P＜0．01)；Western blot检测发现，在异基因HSCT组小鼠的皮肤、肝脏、胃和小肠中TF蛋白表达水平分别为对照组的13．5±2．7，6．2±0．9，7．9±1．6，15．3±3．2倍，亦较对照组明显增高(P＜0．01)。结论：TF表达增高与急性GVHD靶组织损伤相关。TF很可能参与了异基因HSCT后急性GVHD的病理过程。第四部分组织因子在急性GVHD中的作用机制研究目的：研究显示血管内皮细胞也是急性和慢性移植物抗宿主病中异基因T淋巴细胞的靶组织。异基因T淋巴细胞可诱导血管内皮细胞TF的表达。大量的研究显示，内皮细胞活化、损伤后可组织因子表达显著增高，其不仅是凝血—炎症网络的重要因子，而且还通过其细胞内转导信号介导炎症过程，调节炎症细胞因子的释放。本研究的目的是探讨组织因子TF在急性GVHD血管内皮活化、损伤中的作用及其机制。方法：磁珠阳性分选正常人外周血CD4~+和CD8~+T淋巴细胞。流式细胞术检测异基因CD4~+或CD8~+T淋巴细胞作用后脐静脉内皮细胞(HUVEC)表面TF、血管细胞黏附分子-1(VCAM-1)阳性表达率；实时定量PCR方法检测HUVEC中肿瘤坏死因子-α(TNF-α)、干扰素-γ(IFN-γ)、白介素-6(IL-6)在基因水平的表达；Western blot方法检测HUVEC中丝裂原活化蛋白激酶(MAPK)的表达。实时定量PCR和流式细胞术检测抗TF抗体、p38MAPK阻滞剂或JNK阻滞剂对异基因T淋巴细胞诱导的血管内皮细胞中炎症因子表达的影响。结果：①异基因CD4~+和CD8~+T细胞可显著诱导HUVEC的TF、VCAM-1、TNF-α、IFN-γ和IL-6表达。②异基因CD4~+和CD8~+T细胞作用后，HUVEC内的p38MAPK和JNK磷酸化增强，而磷酸化ERK无变化。SB203580(p38MAPK阻滞剂)作用后，异基因CD4~+T淋巴细胞和CD8~+T淋巴细胞诱导6h的HUVEC膜表面TF表达分别下降39．9±4．3％(n=6，P＜0．05)和64．4±6．1％(n=6，P＜0．01)；SP600125(JNK阻滞剂)作用后，异基因CD4~+T细胞和CD8~+T细胞诱导6h的HUVEC表面TF表达分别下降66．1±5．2％(P＜0．01)和56．8±3．4％(P＜0．01)。③抗TF抗体、p38MAPK和JNK阻滞剂均可显著下调异基因T淋巴细胞诱导HUVEC中的VCAM-1、TNF-α、IFN-γ和IL-6表达。结论：TF是血管内皮细胞参与急性GVHD免疫损伤的关键因子。异基因T淋巴细胞诱导血管内皮细胞TF表达和其他炎症因子表达可能是急性GVHD导致组织损伤的重要机制。阻断血管内皮细胞TF的表达可减少内皮细胞中引起急性GVHD的炎症细胞因子的产生，从而减轻急性GVHD，而不影响对异基因HSCT受者有利的移植物抗肿瘤作用。更多还原

【Abstract】 PartⅠGene and Protein Expression of Proteinase-activatedReceptor-1, 2 in a Murine Model of Acute Graft-versus-host DiseaseObjective: The mechanism of acute GVHD was known without certainty, but theactivation of donor T cells and release of the proinflammatory cytokines such as TNF-α,IFN-γand IL-1 may be involved.Recent findings have demonstrated PARs are not onlydegradative enzymes, but also important signaling molecules involved in inflammation andimmunology.The aim of this study was to explore the expression of proteinase-activatedreceptor-1, 2(PAR-1, 2) in a murine model of acute GVHD.Method: A murine model of acute GVHD after allogeneic HSCT was established, andsyngeneic HSCT mice were used as the controls.Quantitative real-time PCR, Western blotand immunohistochemical test were done to detect the gene and protein expression ofPAR-1, 2 in multiple organs of allogeneic HSCT mice and the controls.Result: Allogeneic HSCT mice showed classical symptoms and histological changesof acute GVHD.PAR-1 mRNA expression was significantly increased in the skin, liver,small intestine of allogeneic HSCT mice (skin: 0.039±0.013 vs.controls: 0.008±0.002,P=0.009; liver: 0.165±0.006, vs.controls: 0.017±0.006, P=0.004; small intestine:0.215±0.009 vs.controls: 0.016±0.002, P=0.003) but not in the stomach, lung and kidneyof allogeneic HSCT mice (P>0.05).PAR-2 mRNA expression in the liver and smallintestine of allogeneic HSCT mice (liver: 0.010±0.002 vs.controls: 0.003±0.001, P=0.008;small intestine: 0.006±0.001 vs.controls: 0.003±0.001, P=0.024) but not in the other organs(P>0.05) was found to be significantly elevated.The protein expression of PAR-1, 2 werein accordance with the mRNA expression as shown by western blot.The levels of PAR-1protein expression in the skin, small intestine, liver of allogeneic HSCT mice wereincreased by 5.3, 2.9 and 5.2-fold compared with the controls respectively (P<0.01).Thelevels of PAR-2 protein expression in the small intestine and liver of allogeneic HSCT micewere increased by 2.85 and 4.0-fold compared with the controls respectively (P<0.01). Immunohistochemical test showed there was strong PAR-1 and PAR-2 immunoreactivity inthe hepatocyte, vascular endothelial cell of the liver, epithelium mucosae, glandularepithelium of small intestine of allogeneic HSCT mice.Enhanced immunoreactivity ofPAR-1 was also found in epithelial cell and glandular epithelium of the skin.But there wasabsent or weak PAR-1 and PAR-2 immunoreactivity in the organs of controls and the lung,kidney, stomach of allogeneic HSCT mice.Conclusion: Increased expression of PAR-1, 2 in the target organs of acute GVHDsuggests PAR-1, 2 may contribute to pathogenesis of acute GVHD after allogeneic HSCT.It may lead to a better understanding of acute GVHD and may provide a promising methodto alleviate acute GVHDPartⅡProteinase-Activated Receptor-1 Mediates Allogeneic CD8~+ TCell-Induced Apoptosis of Vascular Endothelial CellsObjective:Vascular endothelial-cells injury plays a pivotal role in the pathogenesis ofgraft-versus-host disease and transplant-associated endothelial injury syndrome.In thiscontext, allogeneic reactive cytotoxic T cell may contribute to the apoptosis of vascularendothelial cells.The aim of this study was to investigate the molecular mechanism ofallogeneic CD8~+T cell-induced apoptosis of vascular endothelial cells.Method: Allogeneic CD8~+T cells were isolated from PBMC by positive selectionusing magnetic beads coated with anti-CD8 antibody.Apoptosis of human umbilical veinendothelial cells (HUVECs) and human dermal microvascular endothelial cells (HDMECs)were detected by AnnexinⅤ-FITC labeling.Gene and protein expression ofproteinase-activated receptor-1(PAR-1) in vascular endothelial cells were tested byRT-PCR and Western blotting.Western blotting was also used to detect the change ofMAPK and Caspase-3 expression in the vascular endothelial cells.The influence ofSFLLRN (PAR-1 agonist), ATAP2 (PAR-1 antibody), SB203580 (inhibitor of p38MAPK),SP600125 (inhibitor of JNK) on apoptosis were also examined. Result: After co-culturing with allogeneic CD8~+T cells for 24 h and 48 h, theapoptosis rates of HUVECs were 51.7±4.1% and 29.4±3.3% respectively (P<0.01, vsuntreated HUVECs), the apoptosis rates of HDMECs were 28.9±2.2% and 15.2±1.8%respectively (P<0.01, vs untreated HDMECs).The effect of PAR-1 agonist on apoptosis ofHUVECs and HDMECs was similar to the effect of allogeneic CD8~+T cells.These effectswere largely prevented by ATAP2 and SB203580 (p<0.05).ATAP2 reduced allogeneicCD8~+T cells-induced apoptosis of HUVECs and HDMECs by 82.8%, 76.5% comparedwith the untreated cells respectively (P<0.05).SB203580 reduced allogeneic CD8~+Tcells-induced apoptosis of HUVECs and HDMECs by 76.3%, 81.6% compared with theuntreated cells respectively (P<0.05).Allogeneic CD8~+T cells and PAR-1 agonist enhancedcleavage of Caspase-3 and led to p38MAPK phosphorylation.The levels of Caspase-3protein expression in HUVECs and HDMECs were increased by 4.7, 1.9-fold comparedwith the controls respectively at 12-h point (P<0.01).The levels of phosphorylatedp38MAPK in HUVECs and HDMECs were increased by 8.8, 5.3-fold compared with thecontrols respectively (P<0.05).Conclusion: Allogeneic CD8~+T cells induced apoptosis of vascular endothelial cellsthrough PAR-1 dependent modulation of intrinsic apoptotic pathway via cleavage ofCaspase-3 and phosphorylation of p38MAPK.It may lead to a better understanding ofacute GVHD and transplant-associated vascular disease.Measures to block PAR-1 mayalleviate acute GVHD and transplant-associated endothelial injury syndrome.PartⅢExpression of Tissue Factor in a Murine Model of AcuteGraft-versus-host DiseaseObjective: The aim of this study was to explore the expression of tissue factor (TF) ina murine model of acute graft-versus-host disease (GVHD).Method: A murine model of acute GVHD after allogeneic HSCT was established, and syngeneic HSCT mice were used as the controls.Quantitative real-time PCR and Westernblot test were done to detect the gene and protein expression of TF in multiple organs ofallogeneic HSCT mice and the controls.Result: Allogeneic HSCT mice showed classical symptoms and histological changesof acute GVHD.The levels of TF mRNA expression in the skin, stomach, small intestine,liver of allogeneic HSCT mice were increased by 15.1±2.1, 5.5±1.4, 9.7±2.3, 14.3±2.9-foldcompared with the controls respectively (P<0.01).The levels of TF protein in the skin,stomach, small intestine, liver of allogeneic HSCT mice were increased by 13.5±2.7,6.2±0.9, 7.9±1.6, 15.3±3.2-fold compared with the controls respectively (P<0.01).ButmRNA and protein expression of TF in the kidney and lung of allogeneic HSCT mice wasnot elevated compared with the controls.Conclusion: Increased expression of TF in the target organs of acute GVHD suggestsTF may contribute to pathogenesis of acute GVHD after allogeneic HSCT.It may lead to abetter understanding of acute GVHD and may provide a promising method to alleviateacute GVHDPartⅣPathogenetic Role of Tissue Factor in acuteGraft-versus-host DiseaseObjective: Recent findings suggest that tissue factor (TF) play an important role intransplant immunology.The aim of this study was to study the pathogenetic role ofallogeneic T lymphocytes and tissue factor (TF) in GVHD.Method: TF mRNA and protein expression in organs of allogeneic HSCT mice andthe controls were determined by real-time PCR and Western blot.The effect of allogeneic Tcells on TF, VCAM-1, TNF-α, IFN-γand IL-6 expression and activation of MAPKs inHUVECs was detected by flowcytometry, real-time PCR or Western blot.The influence ofTF antibody, SB203580 and SP600125 on allogeneic T cells-induced proinflammatory cytokines expression was also investigated.Result: 1.Allogeneic CD4~+T cell and CD8~+T cell enhanced TF, VCAM-1, TNF-α,IFN-γand IL-6 expression in TNF-αprestimulated HUVECs compared to the controls.2.Allogeneic CD4~+T cell and CD8~+T cell enhanced p38MAPK and JNK phosphorylation inHUVECs compared to the controls, but ERK phosphorylation was not affected.SB203580and SP600125 reduced allogeneic CD4~+T cell and CD8~+T cells -induced TF expression inHUVECs compared to non-treated HUVECs.3.TF antibody, SB203580 and SP600125down-regulated allogeneic T cells-induced VCAM-1, TNF-α, IFN-γ, IL-6 expression inHUVECs compared to non-treated HUVECs.Conclusion: TF is the key factor in immune-mediated endothelial-injury in acuteGVHD.Allogeneic T cells-induced expression of TF and other proinflammatory cytokinesin vascular endothelial cells may contribute to the pathogenesis of GVHD.Measures whichblock TF expression of vascular endothelial cells may inhibit production ofproinflammatory cytokines contributing to acute GVHD and alleviate acute GVHD.更多还原