【作者】 刘景华；

【导师】 于力；

【作者基本信息】 中国人民解放军军医进修学院 ， 血液病学， 2009， 博士

【摘要】 目的:造血干细胞移植后免疫重建及急性移植物抗宿主病（acutegraft-versus-host disease,aGVHD）的研究为本领域研究的热点。NKT细胞是T淋巴细胞中的一新型的调节型细胞,α-GalCer（α-galactosylceramide,α-半乳糖神经酰胺）,能特异性识别并激活NKT细胞,迅速的大量的分泌IFN-γ及IL-4。本研究在小鼠同种异基因骨髓移植模型中探讨移植后受鼠体内α-GalCer的应用是否有促进免疫重建的作用,同时不加重aGVHD,并对其机制进行初步的探讨。方法:1.建立同种异基因清髓性骨髓移植模型。C57BL/6小鼠为供鼠,BALB/c小鼠为受鼠。首先摸索出最小的清髓性辐射剂量,之后以此辐射剂量作为骨髓移植前的预处理,建立两个aGVHD严重程度不同的骨髓移植模型。2.利用aGVHD相对较轻的骨髓移植模型,实验组骨髓移植后立即腹腔注射α-GalCer,对照组骨髓移植后立即腹腔注射溶剂DMSO。移植后同一时间点检测两组小鼠脾脏的单个核细胞计数、CD³⁺、CD4⁺、CD8⁺、B220⁺、CD40⁺、CD86⁺、CD11c⁺、CD80⁺细胞的相对数目;供者细胞嵌合率;胸腺的单个核细胞计数和细胞组份;造血重建。并进行体外造血干细胞集落的培养及体内、外T、B细胞的增殖实验。3.利用aGVHD严重程度不同的两个骨髓移植模型,实验组骨髓移植后立即腹腔注射α-GalCer,对照组骨髓移植后立即腹腔注射溶剂DMSO。在每个移植模型中观察两组小鼠的生存期、aGVHD的临床表现和病理改变、供者细胞嵌合率。并进行移植后6小时血清及移植后d27脾脏单个核细胞培养上清细胞因子的检测;体内、外胸腺、脾脏、外周血、外周淋巴结单个核细胞及T细胞的迁移检测。结果:1.≥7.5Gy TBI为清髓剂量。以7.5Gy TBI为预处理,在输注相同的骨髓细胞数的前提下,输注脾细胞数3×10⁷及1×10⁷,建立两个aGVHD严重程度不同的同种异基因骨髓移植模型。2.在输注脾细胞数1×10⁷骨髓移植模型中,骨髓移植后d70,实验组小鼠脾脏单个核细胞计数、CD3⁺、CD4⁺、CD8⁺、B220⁺、CD40⁺及CD86⁺细胞的比例均明显高于对照组,CD11c⁺、CD80⁺细胞的比例两组间无区别:胸腺单个核细胞计数、CD3⁺、CD4⁺、CD8⁺细胞的比例两组间无区别,且均较正常小鼠明显偏低。移植后早期d7,实验组供者细胞嵌合率高于对照组。移植后d3,实验组外周血白细胞高于对照组。体外造血干细胞集落培养,α-GalCer组较DMSO组CFU数目明显多。体内、外T、B细胞增殖实验,α-GalCer组较DMSO组T、B细胞明显增殖。3.在aGVHD严重程度不同的两个骨髓移植模型中,实验组小鼠较对照组小鼠生存时间明显延长,aGVHD临床表现及病理改变减轻。4.在输注脾细胞数1×10⁷移植模型中,移植后6小时血清中IL-4水平实验组小鼠与腹腔注射α-GalCer的正常小鼠、腹腔注射α-GalCer的单纯照射小鼠无差异,均明显高于腹腔注射DMSO的单纯照射小鼠;血清中IFN-γ水平实验组小鼠与腹腔注射α-GalCer的单纯照射小鼠无差异,稍高于腹腔注射DMSO的单纯照射小鼠,均明显低于腹腔注射α-GalCer的正常小鼠。移植后d27脾脏单个核细胞培养上清中IL-4、IL-10水平实验组高于对照组,而IFN-γ、IL-2水平实验组低于对照组。5.体内、外迁移实验:在α-GalCer的作用下单个核细胞、CD3⁺、CD4⁺、CD8⁺细胞从胸腺、脾脏、外周血中出来,迁移至外周淋巴结。结论:1.BALB/c小鼠接受⁶⁰Coγ射线TBI的致死剂量是≥7.5Gy。在接受7.5Gy TBI的预处理后,输注相同的骨髓细胞数的前提下,输注不同的脾细胞数建立两个aGVHD严重程度不同的骨髓移植模型,随着输注脾细胞数增加,aGVHD的严重程度随之增加。2.移植后α-GalCer的应用促进T细胞、B细胞的免疫重建,同时增加第二刺激信号CD86、CD40的表达。3.α-GalCer加速的免疫重建与α-GalCer促进供者细胞的早期植入、促进造血干细胞的增殖与分化、促进T、B细胞的增殖作用有关,而与改善胸腺的功能无关。4.移植后α-GalCer的应用减轻了aGVHD,而且并未以延迟植入为代价,同时维持植入的持久。减轻aGVHD的机制之一:α-GalCer使受者的NKT细胞分泌大量的IL-4,促使供者的T细胞向Th2分化;减轻aGVHD的机制之二:α-GalCer促使T淋巴细胞从胸腺、脾脏、外周血出来,迁移至外周淋巴结,减少进入至靶器官的淋巴细胞数量,从而减轻淋巴细胞对靶器官的损伤。更多还原

【Abstract】 Objective:research on immune reconstitution and acute graft-versus-host disease(aGVHD) after hemopoietic stem cell transplantation(HSCT) is the hot in area.NKT cells are a distinct subset of T lymphocytes,as one kind of new regulatory cells.α-GalCer(α-galactosylceramide) can recognize and activate NKT cells,so induces NKT cells rapidly secrete large amounts of IFN-γ「and IL-4.We investigate whetherα-GalCer could enhance immune reconstitution without aggravating aGVHD in an experimental model of HSCT,and futher explore the mechanism.Methods:1.C57BL/6 mice were as donors,and BALB/c mice as recipients,two HSCT model with different severity of aGVHD were estabilished. 2.In HSCT model with lighter aGVHD,mice of experimental group were injected intraperitoneally withα-GalCer immediately after HSCT,while mice of control group received the diluent only.Splenocyte counts,CD3~+,CD4~+,CD8~+,B220~+, CD40~+,CD86~+,CD11c~+ and CD80~+ cell relative counts,donor chimerism, thymocyte counts,thymocyte constitute and hemological reconstitution were compared at the same time post transplantation between two groups,and we performed peripheral blood mobilized stem cell colony forming unit(CFU) assays in methylcellulose in vitro and lymphocyte proliferation assay in vitro and in vivo.3.In two HSCT model,Survival,clinical and pathological aGVHD,and donor chimerism were evaluated between two groups of each HSCT model,and serum 6 hours post transplantation and splenocyte cultural supernant day 27 post transplantation were examined by ELISA for cytokines of IL-4,IL-2,1L-10 and IFN-γ,and migration assays spleen,thymus,periphral blood and LN monocyte were performed in vitro and in vivo.Results:1.Two HSCT models with different severity of aGVHD were estabilished.2.In HSCT model of 1×10~7 splenocyte,on day 70 post transplantation,splenocyte counts,CD3~+,CD4~+,CD8~+,B220~+,CD40~+ and CD86~+ cell relative counts were obviously higher in experimental group than in control group.At the same time thymocyte counts and thymocyte constitute were no different between two groups,and both were obviously lower than normal mice.On day 7,donor chimerism was higher in experimental group than in control group.On day 3,hemological reconstitution was faster in experimental group than in control group,and CFU number per 10~3 CD34~+ cell was higher inα-GalCer culture group than in DMSO culture group.Lymphocyte proliferation assay in vitro and in vivo both showed thatα-GalCer promoted T cells and B cells proliferation.3.In two HSCT model,mice in experimental group both survived longer than in control group,and clinical and pathological aGVHD were lighter.4. In HSCT model of 1×10~7 splenocyte,IL-4 level of serum 6 hours post transplantation were no different among experimental group mice,normal mice injected withα-GalCer and mice irradiated and injected withα-GalCer,and all were higher than mice irradiated and injected with DMSO;IFN-γlevel were no different between experimental group mice and normal mice injected withα-GalCer,both were a litter higher than mice irradiated and injected with DMSO and obviously lower than normal mice injected withα-GalCer.On day 27 post transplantation,IL-4 and IL-10 level of splenocyte cultural supernant in experimental group were higher than in control group,while IFN-γand IL-2 level were lower.5.Migration assays in vitro and in vivo showed thatα-GalCer caused moncyte,CD3~+,CD4~+ and CD8~+ cell egress from thymus,spleen and peripheral blood to LN.Conclusions:1.Two HSCT models with different severity of aGVHD are established.2.α-GalCer accelerates immune reconstitution of T cells and B cells,and simultaneously enhances expression of the second stimulation signal CD86 and CD40.3.Acceleration of immune reconstitution byα-GalCer is correlated with enhancing early engraftment,hemopoietic stem cell proliferation and differation and proliferation of T cells and B cells byα-GalCer,which is independent of improving thymus.4.α-GalCer reduces aGVHD,which does not at the cost of preventing or impairing engraftment.One mechanism is that stimulation of host NKT cells through administration ofα-GalCer regulates acute aGVHD by inducing Th2 polarization of donor T cells.The other mechanism is thatα-GalCer causes T cells egress from thymus,spleen and peripheral blood to LN,which reduces T cells in circulating blood and prevents T cells from infiltrating into targets organ.更多还原