复合组织移植中淋巴细胞克隆间的相互抑制作用及其机制

发布时间：2018-07-10 15:58

本文选题：异基因复合组织移植 + 免疫耐受　；参考：《第三军医大学》2012年博士论文

【摘要】：目的及背景：烧伤、创伤和肿瘤等造成大范围的肢体组织缺损很难通过传统的自体组织移植获得良好的形态学与功能学重建。利用异基因皮肤、肌肉、骨骼、神经、肌腱、血管等不同发育来源的多种组织构成的复合植物，即异基因复合组织移植（Composite TissueAllotransplantation, CTA）来替代缺损的肢体可能成为解决这一难题的有效手段之一。尽管目前临床已成功进行了多例CTA移植，但医学界对于权衡长期应用免疫抑制剂造成的感染、肿瘤的风险以及复合组织移植带来的益处上尚存在争议。如何诱导机体对于CTA的特异性免疫耐受可能成为推动CTA临床应用的重要问题。但目前医学界对于机体对CTA免疫反应的动力学特点以及其机制的认识尚不完全。因此，深入探讨CTA的免疫机理，可能为诱导CTA的长期特异性免疫耐受提供新的思路和理论依据。从动物模型及临床病例的研究中发现，CTA引起的免疫排斥反应往往低于单一组织或器官移植，但这种低免疫应答强度的原因尚不清楚。其中，免疫细胞的抗原竞争（antigen competition）可能是CTA低免疫原性的机制之一。抗原竞争理论最早源于移植实验中所发现的一种次要组织相容性（mH）抗原刺激可抑制机体对另一种mH抗原的免疫反应的现象。之后，在模式抗原以及病毒抗原肽免疫反应的研究中已经证实，不同克隆的淋巴细胞免疫反应的抗原识别过程中可通过竞争性结合抗原、向APC及其他淋巴细胞递呈抑制性信号、分泌免疫抑制性细胞因子的方式抑制其他克隆淋巴细胞的活化。然而，这一现象仅在一些识别相同抗原的TCR转基因的T细胞克隆的模型中具有显著效应，而针对不同抗原的T细胞间的相互抑制作用则少有报道。 CTA移植物由于其具有多种组织抗原的特点使其成为研究淋巴细胞克隆间相互作用的良好模型。CTA的低免疫原性也提示在其免疫反应中存在针对不同组织抗原的淋巴细胞克隆间相互抑制的可能性。然而淋巴细胞的竞争性抑制在CTA移植免疫耐受中的作用亦主要源于现象的推测，而缺乏直接的证据支持。多种组织抗原刺激是否能够抑制淋巴细胞对异基因移植物的免疫排斥反应，延长移植物的存活时间亦未见报道。因此，我们在本研究中设计了“差异化”的多种组织抗原刺激的CTA移植模型，以及多种MHC抗原刺激的体外与在体模型，以探讨在移植，特别是CTA移植的免疫反应中淋巴细胞克隆间相互抑制作用及其可能机制。从而为进一步了解CTA的免疫机理，诱导CTA的免疫耐受提供新的理论依据。方法： 1.将BN大鼠来源的含不同组织成分的的“差异化”CTA移植物移植至F344大鼠，构建CTA模型。分组为：S组（腹股沟皮片），SL组（腹股沟皮瓣），SLM组（腹股沟肌皮瓣）。观察不同组织成分移植物的存活时间。 2.在CTA术后第7天，于受体大鼠内眦静脉取血后，分离外周血淋巴细胞，与供体来源的淋巴细胞进行混合淋巴细胞反应，利用WST-8比色法检测淋巴细胞增殖率。 3.分离BalB/c（H-2d）、C3H（H-2k）、C57BL/6（H-2b）Qa-1WT及C57BL/6（H-2b）Qa-1KO三种不同MHC表型的近交系小鼠脾脏淋巴细胞。分别以单一MHC表型的淋巴细胞，或1：1混合的两种MHC表型的淋巴细胞作为刺激细胞，在体外进行单向混合淋巴细胞反应。利用WST-8比色法检测淋巴细胞增殖率。 4.收集混合培养第72小时的淋巴细胞培养基上清液，，用ELISA法检测TGF-β1、TNF-α与IL-10细胞因子的分泌。 5.收集混合培养第72小时的淋巴细胞，提取总RNA，利用RT-qPCR检测其IL-10、Foxp3基因mRNA的表达水平。 6.将单一BalB/c（H-2d）来源、单一C3H（H-2k）来源或同时将BalB/c与C3H来源的全层皮片移植至C57BL/6（H-2b）小鼠，观察皮片存活时间及皮片基底部淋巴细胞浸润情况。 7.免疫组织化学染色检测Foxp3+细胞在各组皮肤移植物基底部的分布情况。结果： 1.不同组织成分CTA的存活时间分别为：S组6.25±0.25天，SL组为9±0.707天，SLM组为10.5±0.645天，SL及SLM组移植物存活时间较S组存活时间显著延长（p=0.006），而SLM组存活时间较SL组有延长趋势，但无显著统计学差异（p=0.136）。 2.不同组织成分CTA受体对供体来源淋巴细胞的增殖率分别为：S组1.632±0.103，SL组1.588±0.294，SLM组1.508±0.232，组间无显著差异。 3.两种刺激细胞以1:1混合后同时刺激引起的T淋巴细胞增殖反应弱于相同数量的单一刺激细胞引起的细胞增殖。 4.两种MHC抗原刺激组的IL-10浓度显著高于单一MHC抗原刺激组（p0.001）。而TNF-α、TGF-β1浓度在各MLR反应组间无显著差异。 5.两种MHC抗原刺激组淋巴细胞IL-10mRNA表达显著高于单一MHC抗原刺激组（p0.001），而调节性T细胞标志Foxp3基因表达在各反应组及无抗原刺激的对照组间无显著差异。 6.两种MHC抗原的皮肤同时移植组皮片存活时间及皮下淋巴细胞浸润情况与单一MHC抗原组无显著差异。皮下浸润的炎性细胞中Foxp3+细胞比率在各组中无显著差异。 7. Qa-1基因敲除（KO）组中，多抗原刺激的MLR反应中的淋巴细胞增殖率较单一抗原刺激组无显著差异。Qa-1KO受体单一MHC皮肤移植物存活时间较WT受体显著缩短（p=0.041，0.032），但两种MHC抗原的皮肤同时移植的排斥时间较WT组均无显著差异。结论： 1.成功构建了“差异化”CTA移植物移植模型。随着CTA移植物组织成分复杂性的增加，CTA存活时间有延长的趋势。但该作用并不由受体的淋巴细胞对供体来源淋巴细胞的增殖反应的抑制引起。在多种MHC抗原表型的皮肤移植模型中，两种MHC表型的异基因皮肤同时移植较单一MHC表型皮肤移植并不能延长移植物的存活时间。 2.不同MHC抗原表型的淋巴细胞引起的MLR反应中存在Qa-1依赖的淋巴细胞克隆间相互抑制作用，该作用可能与多抗原刺激诱导IL-10的分泌增加有关，而Foxp3+调节性T细胞未参与MHC抗原的克隆竞争作用。
[Abstract]:Objective and background:
It is difficult to obtain a good morphological and functional reconstruction by traditional autologous tissue transplantation, which is caused by burn, trauma, and tumor. The complex plant, that is, allogeneic complex tissue transplantation (Composite T), is made up of a variety of tissues, such as heterologous skin, muscles, bones, nerves, tendons, and blood vessels. IssueAllotransplantation, CTA) may be one of the most effective solutions to this problem. Although many cases of CTA transplantation have been successfully carried out in clinical practice, the medical community is still controversial about the risks of long-term application of immunosuppressive agents, the risk of cancer and the benefits of complex tissue transplantation. How to induce the specific immune tolerance of the body to CTA may be an important issue to promote the clinical application of CTA. However, the understanding of the dynamic characteristics and mechanism of the immune response to CTA is not yet fully understood by the medical community. Therefore, the in-depth study of the immune mechanism of CTA may provide a new way to induce the long-term specific immune tolerance of CTA. The idea and theoretical basis.
In the study of animal models and clinical cases, the immune rejection caused by CTA is often lower than that of single tissue or organ transplantation, but the reason for this low immune response is not clear. Among them, the antigen competition of immune cells (antigen competition) may be one of the mechanisms of CTA low immunogenicity.
The antigen competition theory was first derived from the secondary histocompatibility (mH) antigen stimulation found in the transplant experiment to inhibit the immune response of the body to another mH antigen. After the study of the immunoreaction of the model antigen and the virus antigen peptide, the antigen of the immune response of different clones has been identified. In the process, inhibitory signals are delivered to APC and other lymphocytes by competitive binding antigen, and the activation of other cloned lymphocytes can be inhibited by secreting immunosuppressive cytokines. However, this phenomenon has significant effects only on some of the T cell clones that identify the same antigen in the TCR cell clone. The intercellular inhibition of T cells is rarely reported.
The low immunogenicity of.CTA, a good model for the study of lymphocyte clones, is also a good model for the interaction of lymphocyte clones by CTA grafts, which also suggests the possibility of mutual inhibition between lymphocyte clones for different tissue antigens in their immune responses. However, the competitive inhibition of lymphocyte in CTA transplantation The role of immune tolerance is also mainly derived from the speculations of the phenomenon, but the lack of direct evidence support. Whether multiple tissue antigens can inhibit the immune rejection of lymphocytes to allogeneic grafts and prolong the survival time of the grafts has not been reported.
Therefore, in this study, we designed a "differentiated" CTA transplantation model with various tissue antigen stimuli, as well as in vitro and in vivo models of a variety of MHC antigens, in order to explore the interaction and possible mechanisms of lymphocyte clones in the immune response to transplantation, especially in CTA transplantation, so as to further understand the immunization of CTA. The mechanism provides a new theoretical basis for inducing the immune tolerance of CTA.
Method:
1. "differential" CTA grafts from BN rats were transplanted into F344 rats, and CTA models were constructed. Group S group (inguinal skin slice), SL group (inguinal flap), SLM group (inguinal myocutaneous flap). The survival time of different tissue components was observed.
2. after the seventh day after CTA, the peripheral blood lymphocytes were isolated from the canthus vein of the recipient rats, and the lymphocyte reaction was reacted with the donor lymphocytes, and the lymphocyte proliferation rate was detected by WST-8 colorimetric assay.
3. BalB/c (H-2d), C3H (H-2k), C57BL/6 (H-2b) Qa-1WT and C57BL/6 (H-2b) Qa-1KO three different MHC phenotype mice spleen lymphocytes. The rate of lymphocyte proliferation was detected by method.
4. the lymphocyte culture medium supernatant was collected for seventy-second hours, and the secretion of TGF- beta 1, TNF- alpha and IL-10 cytokines was detected by ELISA.
5. collecting lymphocytes from mixed culture for seventy-second hours, extracting total RNA, and using RT-qPCR to detect the expression level of IL-10 and Foxp3 gene mRNA.
6. a single BalB/c (H-2d) source, a single C3H (H-2k) source or a whole layer of BalB/c and C3H source were transplanted to C57BL/6 (H-2b) mice at the same time. The survival time of the skin slices and the infiltration of the lymphocyte in the basal portion of the skin were observed.
7. immunohistochemical staining was used to detect the distribution of Foxp3+ cells in the skin graft of each group.
Result:
1. the survival time of CTA was 6.25 + 0.25 days in group S, 9 + 0.707 days in group SL and 10.5 + 0.645 days in group SLM. The survival time of grafts in SL and SLM group was significantly longer than that in S group (p=0.006), but the survival time of group SLM was longer than that in SL group, but there was no significant difference (p=0.136).
2. the proliferation rates of CTA receptor on donor lymphocytes were 1.632 + 0.103 in group S, 1.588 + 0.294 in group SL and 1.508 + 0.232 in group SLM. There was no significant difference between the groups.
3. the proliferation of T lymphocytes induced by 1:1 stimulation was weaker than that of two stimulation cells.
4. the concentration of IL-10 in the two MHC antigen stimulation group was significantly higher than that in the single MHC antigen stimulation group (p0.001), while there was no significant difference in the concentration of TNF- alpha and TGF- beta 1 among the MLR reaction groups.
5. the IL-10mRNA expression of lymphocyte in the two MHC antigen stimulated group was significantly higher than that of the single MHC antigen stimulation group (p0.001), but there was no significant difference between the regulatory T cell marker Foxp3 gene expression in the reaction group and the antigenic control group.
There was no significant difference between the survival time of skin graft and the infiltration of subcutaneous lymphocyte in the skin graft of 6. two MHC antigens and the single MHC antigen group. There was no significant difference in the ratio of Foxp3+ cells in the subcutaneous infiltration of inflammatory cells.
In the 7. Qa-1 gene knockout (KO) group, there was no significant difference in the proliferation rate of lymphocyte in the MLR response stimulated by multiple antigens than that in the single antigen stimulus group. The survival time of the.Qa-1KO receptor single MHC skin graft was significantly shorter than that of the WT receptor (p=0.041,0.032), but the rejection time of the two MHC antigens in the same time was not significantly different from that of the WT group.
Conclusion:
1. the "differential" CTA graft model was successfully constructed. With the increase in the complexity of the CTA graft composition, the survival time of the CTA was prolonged. However, this effect was not caused by the inhibition of the receptor lymphocyte's proliferation response to the donor lymphocyte. In a variety of MHC antigen phenotype skin transplantation models, two MHC Phenotypic allogeneic skin transplantation is more than single MHC phenotype. Skin transplantation does not prolong graft survival.
2. the MLR response induced by lymphocytes with different MHC antigen phenotype exists in the interaction of Qa-1 dependent lymphocyte clones, which may be related to the increase of IL-10 secretion induced by multiple antigen stimulation, while Foxp3+ regulatory T cells do not participate in the competitive role of the clones of the MHC antigen.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

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