發(fā)布時(shí)間：2018-04-13 12:24

  本文選題：誘導(dǎo)性多能干細(xì)胞 + 胸腺移植��； 參考：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景加快異基因骨髓移植(allogeneic bone marrow transplantation,allo-BMT)后T細(xì)胞重建能夠降低感染和腫瘤復(fù)發(fā)的幾率。胸腺移植(thymus transplantation,TT)作為一種提高胸腺功能、加快T細(xì)胞重建的方法,已被臨床用于治療DiGeorge綜合征、嚴(yán)重聯(lián)合免疫缺陷病、艾滋病等疾病。前期研究顯示,TT聯(lián)合allo-BMT不但能夠促進(jìn)T細(xì)胞重建,加強(qiáng)抗腫瘤效應(yīng),而且不會(huì)引起嚴(yán)重的移植物抗宿主病(graft versus host disease,GVHD)。然而胸腺供體來(lái)源缺乏制約了 TT 在 allo-BMT 的應(yīng)用。誘導(dǎo)性多能干細(xì)胞(induced pluripotent stem cell,iPS cells)具有和胚胎干細(xì)胞相似的功能,學(xué)者們已成功將iPS細(xì)胞誘導(dǎo)分化為造血祖細(xì)胞、神經(jīng)元、胰島β細(xì)胞、肝細(xì)胞、血管內(nèi)皮細(xì)胞等。iPS細(xì)胞技術(shù)為培養(yǎng)人工胸腺提供了新思路。目前國(guó)內(nèi)外尚未有將iPS細(xì)胞誘導(dǎo)分化為胸腺并運(yùn)用于異基因骨髓移植的研究報(bào)道。研究目的本課題組前期研究將表達(dá)綠色熒光蛋白的C57BL/6小鼠的iPS細(xì)胞,通過(guò)顯微注射到ICR小鼠囊胚腔內(nèi),獲得的嵌合胚胎移植到代孕雌性ICR小鼠子宮內(nèi),最終獲得嵌合子代,用iPS細(xì)胞成功構(gòu)建了嵌合體胸腺。本研究首次將嵌合體胸腺用于allo-BMT時(shí)的TT,探索其對(duì)受體T細(xì)胞重建和GVHD的影響。旨在解決TT的來(lái)源,并尋找一種加快allo-BMT后T細(xì)胞重建的新方法。研究方法1.異基因骨髓內(nèi)骨髓移植、胸腺移植和淋巴細(xì)胞輸注異基因骨髓內(nèi)骨髓移植(intra-bone marrow-bone marrow transplantation,IBM-BMT):受體 BALB/c小鼠移植前24h均接受X射線全身照射。取C57BL/6小鼠的股骨和脛骨的骨髓細(xì)胞(bone marrow cells,BMCs),制成單細(xì)胞懸液。將受體小鼠麻醉,使用微量注射器穿過(guò)關(guān)節(jié)囊將BMC1×107/mouse注入脛骨骨髓腔內(nèi)。TT:取嵌合體小鼠胸腺,分離為大小約2×2×1mm的組織塊,暴露受體左側(cè)腎臟于體腔外,將胸腺組織塊置于腎被膜下,逐層縫合肌肉及皮膚。淋巴細(xì)胞輸注(donor lymphocyte infusion,DLI):取C57BL/6小鼠的脾臟,研磨,計(jì)數(shù),通過(guò)鼠尾靜脈注入5×106/mouse脾臟單細(xì)胞懸液。2.IBM-BMT+TT組的供體胸腺和各組GVHD的觀察實(shí)驗(yàn)分三組:IBM-BMT 組,IBM-BMT+TT 組,IBM-BMT+DLI 組。移植4周后,取移植的供體胸腺行HE染色,觀察組織學(xué)形態(tài)。每周測(cè)量小鼠體重2-3次,同時(shí)觀察GVHD的表現(xiàn)。取受體小鼠的肝臟、小腸、皮膚,10%福爾馬林固定,石蠟包埋,切片,HE染色,觀察其GVHD的病理變化。3.檢測(cè)各組T細(xì)胞亞群在移植4周后,取受體小鼠外周血,流式細(xì)胞儀分析檢查植入狀態(tài)、分析不同實(shí)驗(yàn)組T細(xì)胞亞群比例。取脾臟,流式細(xì)胞儀分析不同組Foxp3+CD4+ T細(xì)胞的比例。研究結(jié)果1.IBM-BMT+TT 組、IBM-BMT+DLI 組、IBM-BMT 組外周血 CD8+T 細(xì)胞組比例分別為(11.10±1.49)%,(8.49±0.82)%,(5.52±0.83)%,兩兩組間差異有統(tǒng)計(jì)學(xué)意義(P0.05)。IBM-BMT+TT組的外周血CD4+T細(xì)胞比例為(9.60±0.69)%,顯著高于 IBM-BMT+DLI 組的(8.07±0.65)%及 IBM-BMT 組(6.42±1.40)%(P0.05)。2.IBM-BMT+TT、IBM-BMT組的脾臟Foxp3+細(xì)胞占CD4+T細(xì)胞比例分別為(1.86±0.36)%、(2.29±0.23)%,顯著高于 IBM-BMT+DLI組(0.07±0.05)%(P0.05)。3.IBM-BMT+TT、IBM-BMT 組的 GVHD 臨床評(píng)分低于 IBM-BMT+DLI 組,病理學(xué)檢查GVHD表現(xiàn)更輕。結(jié)論:1.iPS細(xì)胞來(lái)源的嵌合體胸腺移植,通過(guò)胸腺依賴路徑,能有效加快移植后的CD4+和CD8+ T細(xì)胞數(shù)目的恢復(fù),支持Foxp3+CD4+ T細(xì)胞(即Treg細(xì)胞)的發(fā)育,促進(jìn)T細(xì)胞重建。2.IBM-BMT+TT組GVHD反應(yīng)輕,可能是嵌合體胸腺支持Foxp3+CD4+ T細(xì)胞的產(chǎn)生,抑制GVHD反應(yīng)。3.iPS細(xì)胞來(lái)源的嵌合體胸腺移植能促進(jìn)T細(xì)胞重建,并不引起嚴(yán)重GVHD,為解決TT的來(lái)源和allo-BMT后T細(xì)胞重建遲緩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.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R457.7

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本文編號(hào)：1744478