IFN-γ诱导支气管上皮细胞高表达Galectin-9介导造血干细胞移植后肺免疫保护及机制的研究

发布时间：2018-03-04 03:11

  本文选题：造血干细胞移植　切入点：急性移植物抗宿主病　出处：《华中科技大学》2013年博士论文　论文类型：学位论文

【摘要】：目的：目前肺在急性移植物抗宿主病中是否受到免疫攻击而被赦免尚存在争议。建立一个成熟可靠的小鼠移植后肺免疫保护模型，为后期异基因移植后肺免疫保护的深入研究提供理想的实验模型和依据。 方法：建立小鼠aGVHD模型，分别是C57BL/6J→C57BL/6J，同基因组；C57BL/6J→B6D2F1同种异基因半相合组；C57BL/6J→BALB/C，脾与骨髓细胞为2:1；C57BL/6J→BALB/C，脾与骨髓细胞为4:1；移植成功后观察aGVHD临床评分和生存期，检测外周血白细胞计数和嵌合体，测肺、肝、肠、皮肤组织病理，ELISA检测移植后肺组织中IFN-γ水平。 结果：同种异基因组小鼠42天生存率为20%，在移植后28天小鼠出现GVHD典型临床表现和病理损伤，但肺组织损伤与经典靶器官肝、小肠、皮肤相比明显减轻，在同基因组中移植小鼠42天全部生存，无GVHD临床表现；而MHC完全不相合组出现严重的GVHD表现，移植后12天半数小鼠死亡，移植物脾和骨髓细胞为4:1组还出现严重的间质性肺炎改变；移植濒死状态的小鼠其外周血白细胞计数为（1.69±0.12）×109/L，在移植后第30天和60天可检测到供者型Y染色体；同种异基因组小鼠在移植后第1和2周肺组织中IFN-γ明显升高，其峰值为（188.6±12.7）pg/ml，而完全不相合组（2:1和4:1）在移植后第1和2周IFN-γ有轻度升高，与同种异基因组相比具有显著性差异(P0.05)。 结论：造血干细胞移植后肺存在相对免疫赦免，它与输注的淋巴细胞数、肺内IFN-γ水平相关。 目的：体内探讨IFN-γ依赖性Gal-9在急性移植物抗宿主病中的表达和体内机制。 方法：建立小鼠aGVHD移植模型： C57BL/6J→C57BL/6J，同基因组；C57BL/6J→B6D2F1，野生型组；IFN-γ-/-→B6D2F1，供者IFN-γ-/-组； C57BL/6J→IFN-γR-/-，受者IFN-γR-/-组；观察移植后小鼠的生存期和临床评分，HE染色观察肺、肝、肠、皮肤组织病理，支气管肺泡灌洗术检测灌洗液中总细胞数、总蛋白量及肺湿/干比重；+7天Tunel检测肺中细胞凋亡，+28天流式和ELISA检测T细胞亚群和细胞因子水平；免疫组化、免疫荧光、ELISA和Western blot检测肺、小肠、皮肤、肝中Gal-9的表达；为探讨Gal-9特异性的肺保护，，对供者IFN-γ-/-组和受者IFN-γR-/-组经鼻灌注吸入Gal-9蛋白，观察小鼠生存期和肺病理，ELIAS检测IFN-γ、TNF-a、IL-4和IL-17变化。 结果：同基因移植组小鼠42天全部存活，无GVHD临床表现和病理改变；野生型组小鼠可有aGVHD表现，24天生存率为大于50%，在移植后28天肺组织病理改变比经典靶器官肝、小肠、皮肤明显轻微；供者IFN-γ-/-组和受者IFN-γR-/-组小鼠均有致死性GVHD表现，14天内全部死亡，肺组织出现严重病理改变，BALF中总细胞数和总蛋白量及肺湿/干比重在移植后1周明显增高；Tunel提示异基因组小鼠肺中存在大量凋亡细胞，供者IFN-γ-/-组和受者IFN-γR-/-组凋亡细胞很少见，流式和ELISA检测显示这两组小鼠在移植后第7天肺组织中CD4+和CD8+细胞明显升高；在供者IFN-γ-/-小鼠中IL-4明显升高，受者IFN-γR-/-组中IFN-γ和IL-17升高；免疫组化、免疫荧光、ELISA和Western blot检测均发现在野生型小鼠肺中Gal-9高表达；经鼻灌注吸入Gal-9蛋白可显著改善供者IFN-γ-/-和受者IFN-γR-/-移植小鼠的生存期和肺组织病理，在供者IFN-γ-/-小鼠体内与IL-4和IL-17下降相关，而在受者IFN-γR-/-小鼠体内与IFN-γ和IL-17水平下降相关。 结论：IFN-γ依赖性Gal-9可促进活化T细胞亚群耗竭或/和其分泌的细胞因子降低，在造血干细胞移植后肺损伤的免疫保护中起重要作用。 目的：体外探讨IFN-γ对支气管上皮细胞中Galectin-9的诱导及高表达的Galectin-9对T细胞凋亡的作用和机制。 方法：用纤支镜灌洗刷从正常人的支气管中段分离出支气管上皮细胞（HBE），用CK-18、CK-19和Vimentin鉴定支气管上皮细胞；用浓度为0，0.08，0.4，2，10，50ng/ml的IFN-γ和浓度为10ng/ml IFN-γ刺激支气管上皮细胞0，3，6，12，24，48h，Real-timePCR和Western blot检测Gal-9表达；CD3+免疫磁珠分选人的T细胞，用含10%胎牛血清的RPMI1640培养基，加入ConA和IFN-γ或IL-4或IL-17培养12小时，流式细胞仪检测T细胞亚群、CD69和Tim-3的表达率；将IFN-γ预处理的HBE与T细胞共培养24h，流式检测T细胞凋亡，采用Tim-3-FC阻断和Transwell培养进行阻断；分光光度仪检测T细胞凋亡中Caspase家族的活化，用Caspase-3(z-DEVD-fmk)或Caspase-4抑制剂(Ac-LEVD-cho)对比T细胞凋亡的影响。 结果：培养出的支气管上皮细胞中CK18和CK19大量表达，而vimentin无表达；IFN-γ诱导HBE高表达Gal-9，呈时间和浓度依赖性，其最佳诱导浓度和时间为10ng/ml和24h；免疫磁珠从外周单个核细胞分出T细胞（CD3+）比值为（86.2±5.21）%， ConA刺激T细胞后其CD69表达率为（72.11±3.14）%，加入IFN-γ、IL-4和IL-23其Th1、Th2和Th17细胞表达率分别为(71.3±2.09)%，（62.3±3.51）%，（42.3±6.12）%；将HBE与T细胞进行共培养，未采用IFN-γ预处理组的T细胞凋亡率为（1.9±0.12）%，用IFN-γ预处理组其T细胞凋亡率为（18.5±1.46）%，并可被Tim-3-FC部分阻断（8.1±0.33）%；在transwell共培养中，T细胞凋亡率为（12.6±2.31）%；分光光度计检测发现在IFN-γ预处理的HBE与T细胞共培养中，Caspase-3和Caspase-4活化水平上调，加入Caspase-3抑制剂后T细胞凋亡明显减少，为(13.4±2.13）%，但Caspase-4抑制剂后T细胞凋亡未见明显影响。 结论：IFN-γ诱导HBE高表达Gal-9，后者与活化T细胞上Tim-3相结合，呈caspase-3途径依赖性诱导Th1/Th17细胞凋亡。
[Abstract]:Objective: the lung in acute graft-versus-host disease by immune attack and forgiveness is still controversial. To establish a mature and reliable mice after lung transplantation immune protection model, and based on the experimental model of allogeneic transplantation later in-depth study of lung immune protection provides the ideal.
Methods: to establish a rat model of aGVHD, which is C57BL/6J, C57BL/6J, C57BL/6J, B6D2F1 with the genome; allogeneic haploidentical group; C57BL/6J, BALB/C, spleen and bone marrow cells of 2:1; C57BL/6J, BALB/C, spleen and bone marrow cells were observed for 4:1; aGVHD clinical score and survival after a successful transplant, white blood cell count in peripheral blood the detection and measurement of lung, chimera, liver, intestine, skin tissue, IFN- levels of lung tissue ELISA detection after transplantation.
Results: the allogeneic mouse genome 42 day survival rate was 20% in 28 days after transplantation, mice showed the typical clinical manifestations of GVHD and pathological damage, but lung injury and classic target organ liver, small intestine, skin significantly reduced, in the same genome in transplanted mice all 42 days of survival, no clinical manifestations of GVHD and MHC; mismatched group showed severe GVHD symptoms, 12 and a half the number of death of mice after transplantation, graft spleen and bone marrow cells of plants 4:1 group also appeared serious interstitial pneumonia; mice peripheral blood leukocyte count for transplantation in dying (1.69 + 0.12) * 109/L, in thirtieth days and 60 days after transplantation detection of donor chromosome Y; IFN- gamma allogeneic mice genome at first and second weeks after transplantation in lung tissue was significantly increased, the peak value of (188.6 + 12.7) pg/ml, and completely mismatched group (2:1 and 4:1) in the first and second week after transplantation, IFN- gamma increased slightly, There is a significant difference compared with the allogeneic genomes (P0.05).
Conclusion: there is a relative immune pardon in the lung after hematopoietic stem cell transplantation, which is related to the number of transfused lymphocytes and the level of IFN- gamma in the lung.
Objective: To investigate the expression and mechanism of IFN- gamma dependent Gal-9 in acute graft versus host disease (graft-versus-host disease) in vivo.
Methods: mice aGVHD transplantation model: C57BL/6J, C57BL/6J, C57BL/6J, B6D2F1, the same genome; wild type group; IFN- gamma - - B6D2F1, IFN- - gamma donor group; C57BL/6J, IFN- y R-/-, recipient IFN- gamma R-/- group; mice survival after transplantation were observed and clinical observation of lung, HE score. Staining of liver, intestine, skin biopsy, total cell detection of lavage fluid in the bronchoalveolar lavage number, total protein and lung wet / dry weight; +7 days Tunel detected lung cell apoptosis in +28 days, flow cytometry and ELISA detection of T cell subsets and cytokines; immunohistochemistry, immunofluorescence, ELISA Western and blot detection of lung, small intestine, skin, liver Gal-9 expression in the lung protection; to investigate the specificity of Gal-9, IFN- group of donor and recipient IFN- gamma - gamma R-/- group inhaled Gal-9 protein nasal perfusion, observe the survival of mice and lung pathology, ELIAS detection of IFN- TNF-a and IL-17 IL-4, gamma. Change.
Results: isotransplantation mice survived for 42 days, without changing the GVHD clinical manifestation and pathology; wild type mice with aGVHD, the 24 day survival rate was greater than 50% in 28 days after transplantation, the pathological changes of the lung tissue than the classical target organ liver, small intestine, skin was slight; donor IFN- gamma - group and by IFN- gamma R-/- mice had fatal GVHD, all died within 14 days, lung tissue appeared serious pathological changes, total cell number and total amount of protein in BALF and lung wet / dry weight in 1 after transplantation increased Zhou Mingxian; Tunel indicates the presence of large amount of apoptotic cells in the lung of mice allogeneic donor. IFN- gamma - group and recipient IFN- gamma R-/- group of apoptotic cells is rare, flow cytometry and ELISA assay showed that CD4+ and CD8+ cells increased significantly in the two groups of mice at seventh days after transplantation in donor lung tissue; IL-4 IFN- gamma - / - mice increased significantly, by IFN- IFN- R-/- in the group of gamma gamma And IL-17 increased; immunohistochemistry, immunofluorescence, ELISA and Western blot were now in wild-type mice in the lung Gal-9 expression; nasal inhalation of Gal-9 protein can significantly improve perfusion of donor IFN- and recipient IFN- gamma - gamma R-/- mice transplanted lung tissue pathology and survival period, related decline in donor IFN- gamma - / - mice in vivo and IL-4 and IL-17, and in IFN- infected R-/- mice with IFN- gamma gamma and IL-17 levels decreased.
Conclusion: IFN- gamma dependent Gal-9 promotes the depletion and / or secretion of cytokines in activated T cell subsets, and plays an important role in the immune protection of lung injury after hematopoietic stem cell transplantation.
Objective: To investigate the effect of IFN- gamma on the induction of Galectin-9 in bronchial epithelial cells and the effect and mechanism of high expression of Galectin-9 on apoptosis of T cells in vitro.
Methods: using fiberbronchoscope isolated from normal human bronchial brush middle of bronchial epithelial cells (HBE), CK-18, CK-19 and Vimentin identification of bronchial epithelial cells; with the concentration of IFN- and concentration of 0,0.08,0.4,2,10,50ng/ml 10ng/ml gamma gamma IFN- stimulate the bronchial epithelial cells with 0,3,6,12,24,48h, the expression of Real-timePCR and Western blot Gal-9 CD3+ immune detection; magnetic beads selection T cell culture medium containing 10% fetal bovine serum RPMI1640, ConA and IFN- with gamma or IL-4 or IL-17 for 12 hours, the detection of T cell subsets by flow cytometry, the expression of CD69 and Tim-3 HBE and T cell rate; IFN- gamma pretreatment of co cultured 24h, flow cytometry detection of T cell apoptosis, Tim-3-FC and Transwell were cultured by blocking the activation of Caspase block; UV spectrophotometer to detect the apoptosis of T cells in the family, with Caspase-3 (z-DEVD-fmk) or Caspase-4 inhibitor (Ac-LEVD-cho) on The effect of T cell apoptosis.
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