BMSCs联合骨髓移植建立嵌合体并诱导免疫耐受形成的机制研究

发布时间：2018-05-15 18:00

本文选题：间充质干细胞 + 免疫耐受　；参考：《第四军医大学》2012年博士论文

【摘要】：研究意义异体器官移植已成为脏器功能衰竭治疗中的一种常规治疗方案。在器官移植领域，目前主要使用免疫抑制药物来避免、减轻和逆转排斥反应，但是使用这些药物的同时也带来了一系列问题，如感染几率增加、恶性肿瘤发病率提高、费用昂贵等。尽管如此，这些方法仍不能避免慢性移植物功能衰竭，因此，大量研究致力于寻求一种更好的方法建立免疫耐受。经骨髓移植建立混合嵌合体被证实是建立异体移植免疫耐受的理想方法之一。然而，在诱导混合嵌合体的过程中，受体必须经过辐射、放疗及化疗等预处理方案，以提高造血干细胞移植成活率，这点很大程度上限制了该技术的推广应用。到目前为止，仍没有一种切实可靠、适用于临床的预处理方案。 BMSC在体外易于培养扩增，BMSCs的低免疫原性与免疫调节两方面特性使得它在治疗移植排斥方面具有潜在优势。目前，人们已经利用BMSCs的免疫调节特性对自身免疫性疾病、全身炎症反应综合症及GVHD等多种疾病展开治疗，并取得良好的效果。基于目前通过骨髓移植建立嵌合体尚无低毒性预处理方案，本研究拟尝试用BMSCs对受体进行预处理，然后进行骨髓移植，观察是否可以成功建立混合嵌合体并形成免疫耐受。基于Th17/Treg细胞在免疫排斥与耐受中的重要地位，本研究进一步通过体外实验研究BMSCs对Th17/Treg细胞分化的影响，并对其机制进行探索。材料与方法 1.将供体（Balb/c）小鼠来源BMSCs进行体外培养扩展，通过尾静脉向受体输注，然后将供体骨髓向受体移植，观察在不使用免疫毒性药物的情况下是否可以移植成活形成嵌合体；在此基础上，将供体及第三方（ICR）小鼠来源皮片向受体移植，观察其存活情况，分析是否形成免疫耐受； 2.体外培养受体CD4~+T细胞作为反应细胞，获取供体脾细胞为刺激细胞，二者进行单向混合淋巴细胞反应，观察在异体抗原刺激下初始T细胞向Th17/Treg细胞分化情况；进一步在该体系中加入BMSCs进行混合培养，观察BMSCs对Th17/Treg细胞分化的影响，通过对细胞因子等进行检测，初步探讨其可能机制； 3.通过1-D-MT抑制BMSCs细胞内IDO功能，观察BMSCs对Th17/Treg细胞分化的影响，分析IDO在BMSCs干预Th17/Treg细胞分化中的作用。结果 1.BMSCs对受体预处理后进行骨髓移植，受体外周血中可持续检测到供体来源细胞，在移植4周后，供体细胞占外周血总细胞约20-25%；在该模型上进行皮肤移植，发现供体来源皮肤可长期存活，值得关注的是，供体来源皮肤的毛发在移植后可持续正常生长，提示未发生移植物慢性功能丧失。病理切片显示，供体来源皮片结构完整，，仅有少量单个核细胞浸润，而第三方来源皮片则出现表皮脱落，基底层大量单个核细胞浸润等；通过取脾细胞进行单项混合淋巴细胞反应，发现成功建立嵌合体的小鼠脾细胞对于供体抗原具有低反应性，而对第三方抗原反应性正常； 2.通过单项混合淋巴细胞反应结果发现，异体抗原刺激初始T细胞，可促使其向Th17/Treg细胞分化；BMSCs干预后，反应性T细胞增殖明显受抑制，同时Th17细胞比例减少，而Treg细胞增多，进一步分析发现，BMSCs可降低反应体系中IL-1β、IL-2、IL-6及IL-17，同时提高IL-10、TGFβ1及IDO； 3.通过1-D-MT预孵育可抑制BMSCs细胞内IDO功能，该BMSCs对T细胞增殖及Th17/Treg细胞分化的影响作用明显降低，但培养体系内介导TGFβ1及IL-6水平与正常BMSCs组比较未见明显差异。结论在不使用任何免疫毒性药物的情况下，通过BMSCs联合骨髓移植可成功建立混合嵌合体并形成供体特异性免疫耐受，在此模型上进行供体皮肤移植可长期存活；BMSCs可促使异体抗原刺激引起的Th17/Treg细胞分化向Treg细胞方向偏移，IDO在其中可能具有关键性作用。
[Abstract]:research meaning
Allogeneic organ transplantation has become a routine treatment in the treatment of organ failure. In the field of organ transplantation, the main use of immunosuppressive drugs is to avoid, reduce and reverse rejection, but the use of these drugs also brings a series of problems, such as increased risk of infection, higher incidence of malignant tumor, and high cost. In spite of this, these methods still can not avoid chronic graft failure, so a great deal of research has been made to seek a better way to establish immune tolerance. The establishment of mixed chimerism through bone marrow transplantation is one of the ideal methods for the establishment of allograft immune tolerance. However, in the process of inducing mixed chimerism, the receptor is a receptor. Radiation, radiotherapy and chemotherapy are required to improve the survival rate of hematopoietic stem cell transplantation, which greatly restricts the application of this technique. So far, there is still no reliable and reliable clinical preconditioning scheme.
BMSC is easy to expand in vitro. The two characteristics of BMSCs's low immunogenicity and immunomodulation make it potential advantages in the treatment of transplant rejection. At present, people have used the immune regulation of BMSCs to treat autoimmune diseases, systemic inflammatory response syndrome and GVHD and other diseases, and have achieved good results. Fruit. Based on the lack of low toxicity preconditioning for the establishment of chimerism through bone marrow transplantation, this study intends to attempt to pretreat the receptor with BMSCs and then carry out a bone marrow transplant to observe whether a mixed chimerism can be successfully established and the immune tolerance is formed. Based on the importance of Th17/Treg cells in immune rejection and tolerance, this study has been studied. One step was to study the effect of BMSCs on differentiation of Th17/Treg cells in vitro and explore its mechanism.
Materials and methods
1. the donor (Balb/c) mouse derived BMSCs was cultured and expanded in vitro, transfused through the tail vein to the receptor, and then transplanted the donor bone marrow to the receptor to observe whether it could be transplanted into the chimeras without the use of immuno toxic drugs. On this basis, the donor and the three party (ICR) mouse skin slices were transplanted to the receptor. Observe their survival and analyze whether immune tolerance is formed.
2. in vitro culture receptor CD4~+T cells were used as reactive cells to obtain donor spleen cells as stimulating cells, and the two ones were subjected to unidirectional mixed lymphocyte reaction. The differentiation of initial T cells to Th17/Treg cells was observed under the stimulation of allogenic antigen, and BMSCs was added to the system to observe the differentiation of BMSCs to Th17/Treg cells. Through the detection of cytokines, the possible mechanism is preliminarily discussed.
3. inhibit IDO function in BMSCs cells by 1-D-MT, observe the effect of BMSCs on Th17/Treg cell differentiation, and analyze the role of IDO in BMSCs intervening Th17/Treg cell differentiation.
Result
1.BMSCs was pretreated with bone marrow transplantation, and the donor cells were continuously detected in the peripheral blood. After 4 weeks of transplantation, the donor cells accounted for about 20-25% of the total peripheral blood cells, and the skin transplantation was carried out on the model to find that the donor skin could survive for a long time. It is worth paying attention to the hair source of the donor's skin after transplantation. The continuous normal growth showed no chronic loss of function of the graft. Pathological sections showed that the skin slices of the donor source were intact, only a small amount of mononuclear cells were infiltrated, while the epidermis of the third source skin appeared to fall off, and the basal layer was infiltrated with mononuclear cells, and a single mixed lymphocyte reaction was found by taking the spleen cells. Mouse spleen cells with chimeric chimerism have low reactivity to donor antigens and normal reactivity to third party antigens.
2. through the results of single mixed lymphocyte reaction, it was found that the allogenic antigen stimulated the initial T cells to differentiate into Th17/Treg cells, and the proliferation of reactive T cells was obviously inhibited by the BMSCs stem, and the proportion of Th17 cells decreased, while Treg cells were increased and further analyzed, and BMSCs could reduce IL-1 beta, IL-2, IL-6 and IL-17 in the reaction system. At the same time, IL-10, TGF beta 1 and IDO were improved.
3. the pre incubation of 1-D-MT could inhibit the IDO function in BMSCs cells, and the effect of BMSCs on T cell proliferation and Th17/Treg cell differentiation was significantly reduced, but the level of TGF beta 1 and IL-6 in the culture system was not significantly different from that in the normal BMSCs group.
conclusion
In the absence of any immuno toxic drugs, BMSCs combined bone marrow transplantation can successfully establish mixed chimeras and form donor specific immune tolerance. The donor skin graft can survive for a long time on this model; BMSCs can induce Th17/ Treg cell differentiation caused by allogenic stimulation to the direction of Treg cells, IDO in it There may be a key role in it.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

【参考文献】