iPS细胞来源的嵌合体胸腺移植对异基因骨髓移植小鼠的T细胞重建和GVHD的影响

发布时间：2018-04-13 12:24

本文选题：诱导性多能干细胞 + 胸腺移植　；参考：《南方医科大学》2017年硕士论文

【摘要】：背景加快异基因骨髓移植(allogeneic bone marrow transplantation,allo-BMT)后T细胞重建能够降低感染和肿瘤复发的几率。胸腺移植(thymus transplantation,TT)作为一种提高胸腺功能、加快T细胞重建的方法,已被临床用于治疗DiGeorge综合征、严重联合免疫缺陷病、艾滋病等疾病。前期研究显示,TT联合allo-BMT不但能够促进T细胞重建,加强抗肿瘤效应,而且不会引起严重的移植物抗宿主病(graft versus host disease,GVHD)。然而胸腺供体来源缺乏制约了 TT 在 allo-BMT 的应用。诱导性多能干细胞(induced pluripotent stem cell,iPS cells)具有和胚胎干细胞相似的功能,学者们已成功将iPS细胞诱导分化为造血祖细胞、神经元、胰岛β细胞、肝细胞、血管内皮细胞等。iPS细胞技术为培养人工胸腺提供了新思路。目前国内外尚未有将iPS细胞诱导分化为胸腺并运用于异基因骨髓移植的研究报道。研究目的本课题组前期研究将表达绿色荧光蛋白的C57BL/6小鼠的iPS细胞,通过显微注射到ICR小鼠囊胚腔内,获得的嵌合胚胎移植到代孕雌性ICR小鼠子宫内,最终获得嵌合子代,用iPS细胞成功构建了嵌合体胸腺。本研究首次将嵌合体胸腺用于allo-BMT时的TT,探索其对受体T细胞重建和GVHD的影响。旨在解决TT的来源,并寻找一种加快allo-BMT后T细胞重建的新方法。研究方法1.异基因骨髓内骨髓移植、胸腺移植和淋巴细胞输注异基因骨髓内骨髓移植(intra-bone marrow-bone marrow transplantation,IBM-BMT):受体 BALB/c小鼠移植前24h均接受X射线全身照射。取C57BL/6小鼠的股骨和胫骨的骨髓细胞(bone marrow cells,BMCs),制成单细胞悬液。将受体小鼠麻醉,使用微量注射器穿过关节囊将BMC1×107/mouse注入胫骨骨髓腔内。TT:取嵌合体小鼠胸腺,分离为大小约2×2×1mm的组织块,暴露受体左侧肾脏于体腔外,将胸腺组织块置于肾被膜下,逐层缝合肌肉及皮肤。淋巴细胞输注(donor lymphocyte infusion,DLI):取C57BL/6小鼠的脾脏,研磨,计数,通过鼠尾静脉注入5×106/mouse脾脏单细胞悬液。2.IBM-BMT+TT组的供体胸腺和各组GVHD的观察实验分三组:IBM-BMT 组,IBM-BMT+TT 组,IBM-BMT+DLI 组。移植4周后,取移植的供体胸腺行HE染色,观察组织学形态。每周测量小鼠体重2-3次,同时观察GVHD的表现。取受体小鼠的肝脏、小肠、皮肤,10%福尔马林固定,石蜡包埋,切片,HE染色,观察其GVHD的病理变化。3.检测各组T细胞亚群在移植4周后,取受体小鼠外周血,流式细胞仪分析检查植入状态、分析不同实验组T细胞亚群比例。取脾脏,流式细胞仪分析不同组Foxp3+CD4+ T细胞的比例。研究结果1.IBM-BMT+TT 组、IBM-BMT+DLI 组、IBM-BMT 组外周血 CD8+T 细胞组比例分别为(11.10±1.49)%,(8.49±0.82)%,(5.52±0.83)%,两两组间差异有统计学意义(P0.05)。IBM-BMT+TT组的外周血CD4+T细胞比例为(9.60±0.69)%,显著高于 IBM-BMT+DLI 组的(8.07±0.65)%及 IBM-BMT 组(6.42±1.40)%(P0.05)。2.IBM-BMT+TT、IBM-BMT组的脾脏Foxp3+细胞占CD4+T细胞比例分别为(1.86±0.36)%、(2.29±0.23)%,显著高于 IBM-BMT+DLI组(0.07±0.05)%(P0.05)。3.IBM-BMT+TT、IBM-BMT 组的 GVHD 临床评分低于 IBM-BMT+DLI 组,病理学检查GVHD表现更轻。结论:1.iPS细胞来源的嵌合体胸腺移植,通过胸腺依赖路径,能有效加快移植后的CD4+和CD8+ T细胞数目的恢复,支持Foxp3+CD4+ T细胞(即Treg细胞)的发育,促进T细胞重建。2.IBM-BMT+TT组GVHD反应轻,可能是嵌合体胸腺支持Foxp3+CD4+ T细胞的产生,抑制GVHD反应。3.iPS细胞来源的嵌合体胸腺移植能促进T细胞重建,并不引起严重GVHD,为解决TT的来源和allo-BMT后T细胞重建迟缓和免疫排斥的提供了新方法。
[Abstract]:To speed up the background of allogeneic bone marrow transplantation (allogeneic bone marrow transplantation, allo-BMT) reconstruction of T cells can reduce the chance of infection and tumor recurrence. Thymus transplantation (thymus transplantation TT) as a way to improve the function of thymus, accelerate T cell reconstruction, has been clinically used for the treatment of DiGeorge syndrome, severe combined immunodeficiency disease, aids other diseases. Previous studies have shown that TT and allo-BMT not only can promote T cell reconstitution, enhance the anti-tumor effect, and does not cause severe graft-versus-host disease (graft versus host disease, GVHD). However, the thymus donor shortage restricts the TT application in allo-BMT. Induced pluripotent stem cells (induced pluripotent stem cell iPS, cells) and embryonic stem cells have similar function, the researchers have successfully induced iPS cell differentiation into hematopoietic progenitor cells, neurons, beta Cells, liver cells, vascular endothelial cells and.IPS cell technology provides a new idea for the cultivation of artificial thymus. At home and abroad have been the induction of differentiation of iPS cells in thymus and applied research reports of allogeneic bone marrow transplantation. The study group will research the expression of green fluorescent protein in C57BL/6 mice by iPS cells. Microinjection into ICR mouse blastocyst cavity, the chimeric embryos transplanted into a surrogate female ICR in mouse uterus, obtain chimeric, iPS cells successfully constructed chimeric thymus. This study will be the first time for the allo-BMT chimeric thymus TT, explore their effects on T cell receptor and GVHD. To solve the reconstruction the source of TT, and to find a new method to speed up the allo-BMT of T cells after reconstruction. Methods 1. allogeneic bone marrow transplantation and bone marrow transplantation, thymus lymphocyte infusion of allogeneic bone marrow Bone marrow transplant (intra-bone marrow-bone marrow transplantation, IBM-BMT): BALB/c receptor mice before transplantation 24h underwent whole body X irradiation. C57BL/6 mice femur and tibia bone marrow cells (bone, marrow, cells, BMCs) into single cell suspension. The recipient mice anesthesia, using micro syringe BMC1 * 107/mouse tibial bone marrow injection.TT: chimeric mice thymus cavity block through the joint capsule, were about the size of 2 * 2 * 1mm tissue exposed to external cavity receptors in the left kidney, thymus tissue in the renal capsule, sutured muscle and skin. Lymphocyte infusion (donor lymphocyte, infusion, DLI): C57BL/6 mice spleen, grinding, count 5 * 106/mouse spleen cell suspension group.2.IBM-BMT+TT donor thymus and GVHD were observed through the experiments were divided into three groups after intravenous injection of IBM-BMT group, IBM-BMT+TT group, IBM-BMT+DLI Group. 4 weeks after transplantation, the donor thymus HE staining after transplantation, observe the histological morphology. Body weight was measured weekly in 2-3 times, while observing the performance of GVHD. The receptor in liver, intestine, skin, 10% formalin fixed, paraffin embedded sections, HE staining, observe the pathological changes of.3. GVHD to detect the T cell subsets in 4 weeks after transplantation, the receptor in peripheral blood of mice, examined the engraftment state of flow cytometric analysis of the percentage of T cells in different experimental groups. Subsets of spleen, analysis of different group of Foxp3+CD4+ T cells by flow cytometry. Results the proportion of IBM-BMT+DLI group, 1.IBM-BMT+TT group, IBM-BMT group of peripheral blood cells in CD8+T group respectively (11.10 + 1.49)% and (8.49 + 0.82)% and (5.52 + 0.83)%, the difference was statistically significant between the 22 groups (P0.05) of peripheral blood CD4+T cell ratio in the.IBM-BMT+TT group (9.60 + 0.69)%, significantly higher than that of group IBM-BMT+DLI (8.07 % + 0.65) and IBM-BMT group (6.42 + 1.40)% (P0.05).2.IBM-BMT+TT, IBM-BMT group of spleen Foxp3+ cells accounted for the proportion of CD4+T cells were (1.86 + 0.36)% and (2.29 + 0.23)%, significantly higher than that of group IBM-BMT+DLI (0.07 + 0.05)% (P0.05).3.IBM-BMT+TT, GVHD group IBM-BMT clinical score the lower than the IBM-BMT+DLI group, pathological examination showed GVHD lighter. Conclusion: the Thymus Transplantation Chimera derived from 1.iPS cells, through the path dependence of the thymus, can effectively accelerate the CD4+ transplantation and CD8+ T cell number recovery, support Foxp3+CD4+ T cells (Treg cells) development, promote the reconstitution of T cell group.2.IBM-BMT+TT GVHD reaction light may be chimeric thymus support Foxp3+CD4+ of T cells, inhibition of GVHD reaction of.3.iPS cells derived chimeric thymus transplantation can promote T cell reconstitution, does not cause serious GVHD, T cells and allo-BMT TT source reconstruction solution after slow and free A new method of repulsion is provided.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R457.7

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