脐带血造血干细胞逃逸NK细胞杀伤机制的实验研究

发布时间：2018-05-12 21:29

本文选题：脐带血造血干细胞 + 免疫逃逸　；参考：《复旦大学》2009年硕士论文

【摘要】： 目的:近年来有研究发现异基因脐带血造血干细胞(UCB-HSCs)能够在免疫功能正常的受者体内长期存活。但是UCB-HSCs免疫逃逸能力的机制尚未阐明。而自然杀伤细胞(NK细胞)在杀伤异基因造血干细胞中发挥了主要作用,因此,我们研究UCB-HSCs逃避NK细胞的杀伤的能力,并且探索这种免疫逃逸能力与其细胞表面表达NK细胞活化性和抑制性两类受体的关系,以期找到UCB-HSCs免疫逃逸能力的机制。方法:(1)利用免疫磁珠法分别从成人脐带血、外周血及骨髓中分离UCB-HSCs、外周血造血干细胞(PB-HSCs)及骨髓造血干细胞(BM-HSCs);利用免疫磁珠法及流式细胞分选法从成人外周血中分离NK细胞;(2)以NK细胞和NK-92细胞为效应细胞,并分别以UCB-HSCs为靶细胞,以PB-HSCs、BM-HSCs以及去除了造血干细胞的外周血单个核细胞(PB-MNCs)为对照靶细胞,用乳酸脱氢酶(LDH)释放法分析效应细胞对靶细胞的杀伤率;以NK细胞为效应细胞,并分别以UCB-HSCs、PB-HSCs、BM-HSCs为靶细胞,通过克隆增殖实验分析效应细胞对靶细胞增殖能力的抑制率;(3)利用流式细胞技术分析UCB-HSCs、PB-HSCs、BM-HSCs、PB-MNCs表面NK细胞活化性受体NKp30、NKp44、NKp46的配体以及NK细胞抑制性受体的配体HLA-G分子的表达情况;(4)利用融合蛋白及抗体阻断NKp30、NKp44、NKp46以及HLA-G,观察NK细胞对UCB-HSCs、PB-HSCs、BM-HSCs、PB-MNCs杀伤作用的变化。结果:(1)UCB-HSCs具有逃避NK细胞杀伤的能力,与对照组细胞相比差别有统计学意义;(2)UCB-HSCs、PB-HSCs、BM-HSCs表面低表达NK细胞活化性受体NKp30、NKp44、NKp46的配体,与PB-MNCs相比,差别有统计学意义;(3)UCB-HSCs表面高表达HLA-G分子,与PB-HSCs、BM-HSCs、PB-MNCs相比,差别有统计学意义。结论:(1)UCB-HSCs具有逃避NK细胞杀伤的能力;(2)UCB-HSCs表面低表达NK细胞活化性受体NKp30、NKp44、NKp46的配体可能是其逃逸NK细胞杀伤的机制之一;(3))UCB-HSCs表面高表达HLA-G分子是其逃逸NK细胞杀伤的主要机制。
[Abstract]:Objective: in recent years, it has been found that UCB-HSCsof allogeneic cord blood hematopoietic stem cells can survive for a long time in recipients with normal immune function. However, the mechanism of immune escape ability of UCB-HSCs has not been clarified. Natural killer cells (NK cells) play a major role in killing allogeneic hematopoietic stem cells. Therefore, we study the ability of UCB-HSCs to escape the killing of NK cells. In order to find out the mechanism of immune escape ability of UCB-HSCs, the relationship between the ability of immune escape and the expression of NK cell activative and inhibitory receptors on the cell surface was explored in order to find out the mechanism of immune escape ability of UCB-HSCs. Methods 1) using immunomagnetic bead method to separate umbilical cord blood from adults, UCB-HSCs, PB-HSCsand BM-HSCs2 were isolated from peripheral blood and bone marrow. NK cells were separated from adult peripheral blood by immunomagnetic bead method and flow cytometry.) NK cells and NK-92 cells were used as effector cells. Using UCB-HSCs as target cells, PB-HSCsBM-HSCs and peripheral blood mononuclear cells (PBMC) without hematopoietic stem cells as control, the cytotoxicity of effector cells to target cells was analyzed by lactate dehydrogenase release assay, and NK cells were used as effector cells. UCB-HSCsPB-HSCsPM-HSCs were used as target cells. The inhibitory rate of effector cells on target cell proliferation was analyzed by clone proliferation assay. Flow cytometry was used to analyze the ligands of NK cell activating receptor NKp30 and NKp44NKp46 on the surface of PB-MNCs and the HLA-G molecule of NK cell inhibitory receptor. In order to observe the cytotoxicity of NK cells to UCB-HSCsPM-HSCsBM-HSCsPB-MNCs, NKp30 nkp44 and HLA-G4 were blocked by fusion protein and antibody against UCB-HSCsPM-HSCsPB-MNCs. Results the UCB-HSCs had the ability to evade NK cell killing. There was a significant difference between UCB-HSCsPB-HSCsPB-HM-HSCs and the low expression of NK cell activation receptor NKp30NKp44 / NKp46 on the surface of UCB-HSCs. Compared with PB-MNCs, the UCB-HSCs expressed high expression of HLA-G molecules on the surface of UCB-HSCs. The difference was statistically significant compared with that of PB-HSCsBM-HSCsN PB-MNCs. Conclusion UCB-HSCs have the ability to evade NK cell killing. The low expression of NK cell activating receptor NKp30 / NKp4nkp46 on the surface of UCB-HSCs may be one of the mechanisms of escaping NK cell killing by overexpression of HLA-G molecules on the surface of UCB-HSCs.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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