本文選題：造血干細胞移植　切入點：急性移植物抗宿主病　出處：《華中科技大學》2013年博士論文　論文類型：學位論文

【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擊而被赦免尚存在爭議。建立一個成熟可靠的小鼠移植后肺免疫保護模型，為后期異基因移植后肺免疫保護的深入研究提供理想的實驗?zāi)Ｐ秃鸵罁?jù)。 方法：建立小鼠aGVHD模型，分別是C57BL/6J→C57BL/6J，同基因組；C57BL/6J→B6D2F1同種異基因半相合組；C57BL/6J→BALB/C，脾與骨髓細胞為2:1；C57BL/6J→BALB/C，脾與骨髓細胞為4:1；移植成功后觀察aGVHD臨床評分和生存期，檢測外周血白細胞計數(shù)和嵌合體，測肺、肝、腸、皮膚組織病理，ELISA檢測移植后肺組織中IFN-γ水平。 結(jié)果：同種異基因組小鼠42天生存率為20%，在移植后28天小鼠出現(xiàn)GVHD典型臨床表現(xiàn)和病理損傷，但肺組織損傷與經(jīng)典靶器官肝、小腸、皮膚相比明顯減輕，在同基因組中移植小鼠42天全部生存，無GVHD臨床表現(xiàn)；而MHC完全不相合組出現(xiàn)嚴重的GVHD表現(xiàn)，移植后12天半數(shù)小鼠死亡，移植物脾和骨髓細胞為4:1組還出現(xiàn)嚴重的間質(zhì)性肺炎改變；移植瀕死狀態(tài)的小鼠其外周血白細胞計數(shù)為（1.69±0.12）×109/L，在移植后第30天和60天可檢測到供者型Y染色體；同種異基因組小鼠在移植后第1和2周肺組織中IFN-γ明顯升高，其峰值為（188.6±12.7）pg/ml，而完全不相合組（2:1和4:1）在移植后第1和2周IFN-γ有輕度升高，與同種異基因組相比具有顯著性差異(P0.05)。 結(jié)論：造血干細胞移植后肺存在相對免疫赦免，它與輸注的淋巴細胞數(shù)、肺內(nèi)IFN-γ水平相關(guān)。 目的：體內(nèi)探討IFN-γ依賴性Gal-9在急性移植物抗宿主病中的表達和體內(nèi)機制。 方法：建立小鼠aGVHD移植模型： C57BL/6J→C57BL/6J，同基因組；C57BL/6J→B6D2F1，野生型組；IFN-γ-/-→B6D2F1，供者IFN-γ-/-組； C57BL/6J→IFN-γR-/-，受者IFN-γR-/-組；觀察移植后小鼠的生存期和臨床評分，HE染色觀察肺、肝、腸、皮膚組織病理，支氣管肺泡灌洗術(shù)檢測灌洗液中總細胞數(shù)、總蛋白量及肺濕/干比重；+7天Tunel檢測肺中細胞凋亡，+28天流式和ELISA檢測T細胞亞群和細胞因子水平；免疫組化、免疫熒光、ELISA和Western blot檢測肺、小腸、皮膚、肝中Gal-9的表達；為探討Gal-9特異性的肺保護，，對供者IFN-γ-/-組和受者IFN-γR-/-組經(jīng)鼻灌注吸入Gal-9蛋白，觀察小鼠生存期和肺病理，ELIAS檢測IFN-γ、TNF-a、IL-4和IL-17變化。 結(jié)果：同基因移植組小鼠42天全部存活，無GVHD臨床表現(xiàn)和病理改變；野生型組小鼠可有aGVHD表現(xiàn)，24天生存率為大于50%，在移植后28天肺組織病理改變比經(jīng)典靶器官肝、小腸、皮膚明顯輕微；供者IFN-γ-/-組和受者IFN-γR-/-組小鼠均有致死性GVHD表現(xiàn)，14天內(nèi)全部死亡，肺組織出現(xiàn)嚴重病理改變，BALF中總細胞數(shù)和總蛋白量及肺濕/干比重在移植后1周明顯增高；Tunel提示異基因組小鼠肺中存在大量凋亡細胞，供者IFN-γ-/-組和受者IFN-γR-/-組凋亡細胞很少見，流式和ELISA檢測顯示這兩組小鼠在移植后第7天肺組織中CD4+和CD8+細胞明顯升高；在供者IFN-γ-/-小鼠中IL-4明顯升高，受者IFN-γR-/-組中IFN-γ和IL-17升高；免疫組化、免疫熒光、ELISA和Western blot檢測均發(fā)現(xiàn)在野生型小鼠肺中Gal-9高表達；經(jīng)鼻灌注吸入Gal-9蛋白可顯著改善供者IFN-γ-/-和受者IFN-γR-/-移植小鼠的生存期和肺組織病理，在供者IFN-γ-/-小鼠體內(nèi)與IL-4和IL-17下降相關(guān)，而在受者IFN-γR-/-小鼠體內(nèi)與IFN-γ和IL-17水平下降相關(guān)。 結(jié)論：IFN-γ依賴性Gal-9可促進活化T細胞亞群耗竭或/和其分泌的細胞因子降低，在造血干細胞移植后肺損傷的免疫保護中起重要作用。 目的：體外探討IFN-γ對支氣管上皮細胞中Galectin-9的誘導及高表達的Galectin-9對T細胞凋亡的作用和機制。 方法：用纖支鏡灌洗刷從正常人的支氣管中段分離出支氣管上皮細胞（HBE），用CK-18、CK-19和Vimentin鑒定支氣管上皮細胞；用濃度為0，0.08，0.4，2，10，50ng/ml的IFN-γ和濃度為10ng/ml IFN-γ刺激支氣管上皮細胞0，3，6，12，24，48h，Real-timePCR和Western blot檢測Gal-9表達；CD3+免疫磁珠分選人的T細胞，用含10%胎牛血清的RPMI1640培養(yǎng)基，加入ConA和IFN-γ或IL-4或IL-17培養(yǎng)12小時，流式細胞儀檢測T細胞亞群、CD69和Tim-3的表達率；將IFN-γ預處理的HBE與T細胞共培養(yǎng)24h，流式檢測T細胞凋亡，采用Tim-3-FC阻斷和Transwell培養(yǎng)進行阻斷；分光光度儀檢測T細胞凋亡中Caspase家族的活化，用Caspase-3(z-DEVD-fmk)或Caspase-4抑制劑(Ac-LEVD-cho)對比T細胞凋亡的影響。 結(jié)果：培養(yǎng)出的支氣管上皮細胞中CK18和CK19大量表達，而vimentin無表達；IFN-γ誘導HBE高表達Gal-9，呈時間和濃度依賴性，其最佳誘導濃度和時間為10ng/ml和24h；免疫磁珠從外周單個核細胞分出T細胞（CD3+）比值為（86.2±5.21）%， ConA刺激T細胞后其CD69表達率為（72.11±3.14）%，加入IFN-γ、IL-4和IL-23其Th1、Th2和Th17細胞表達率分別為(71.3±2.09)%，（62.3±3.51）%，（42.3±6.12）%；將HBE與T細胞進行共培養(yǎng)，未采用IFN-γ預處理組的T細胞凋亡率為（1.9±0.12）%，用IFN-γ預處理組其T細胞凋亡率為（18.5±1.46）%，并可被Tim-3-FC部分阻斷（8.1±0.33）%；在transwell共培養(yǎng)中，T細胞凋亡率為（12.6±2.31）%；分光光度計檢測發(fā)現(xiàn)在IFN-γ預處理的HBE與T細胞共培養(yǎng)中，Caspase-3和Caspase-4活化水平上調(diào)，加入Caspase-3抑制劑后T細胞凋亡明顯減少，為(13.4±2.13）%，但Caspase-4抑制劑后T細胞凋亡未見明顯影響。 結(jié)論：IFN-γ誘導HBE高表達Gal-9，后者與活化T細胞上Tim-3相結(jié)合，呈caspase-3途徑依賴性誘導Th1/Th17細胞凋亡。
[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.
緇撴灉錛氬煿鍏誨嚭鐨勬敮姘旂涓婄毊緇嗚優(yōu)涓瑿K18鍜孋K19澶ч噺琛ㄨ揪,鑰寁imentin鏃犺〃杈撅紱IFN-緯璇卞HBE楂樿〃杈綠al-9,鍛堟椂闂村拰嫻撳害渚濊禆鎬

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