本文選題：異基因復合組織移植 + 免疫耐受　； 參考：《第三軍醫(yī)大學》2012年博士論文

【摘要】：目的及背景： 燒傷、創(chuàng)傷和腫瘤等造成大范圍的肢體組織缺損很難通過傳統(tǒng)的自體組織移植獲得良好的形態(tài)學與功能學重建。利用異基因皮膚、肌肉、骨骼、神經(jīng)、肌腱、血管等不同發(fā)育來源的多種組織構成的復合植物，即異基因復合組織移植（Composite TissueAllotransplantation, CTA）來替代缺損的肢體可能成為解決這一難題的有效手段之一。盡管目前臨床已成功進行了多例CTA移植，但醫(yī)學界對于權衡長期應用免疫抑制劑造成的感染、腫瘤的風險以及復合組織移植帶來的益處上尚存在爭議。如何誘導機體對于CTA的特異性免疫耐受可能成為推動CTA臨床應用的重要問題。但目前醫(yī)學界對于機體對CTA免疫反應的動力學特點以及其機制的認識尚不完全。因此，深入探討CTA的免疫機理，可能為誘導CTA的長期特異性免疫耐受提供新的思路和理論依據(jù)。 從動物模型及臨床病例的研究中發(fā)現(xiàn)，CTA引起的免疫排斥反應往往低于單一組織或器官移植，但這種低免疫應答強度的原因尚不清楚。其中，免疫細胞的抗原競爭（antigen competition）可能是CTA低免疫原性的機制之一。 抗原競爭理論最早源于移植實驗中所發(fā)現(xiàn)的一種次要組織相容性（mH）抗原刺激可抑制機體對另一種mH抗原的免疫反應的現(xiàn)象。之后，在模式抗原以及病毒抗原肽免疫反應的研究中已經(jīng)證實，不同克隆的淋巴細胞免疫反應的抗原識別過程中可通過競爭性結合抗原、向APC及其他淋巴細胞遞呈抑制性信號、分泌免疫抑制性細胞因子的方式抑制其他克隆淋巴細胞的活化。然而，這一現(xiàn)象僅在一些識別相同抗原的TCR轉基因的T細胞克隆的模型中具有顯著效應，而針對不同抗原的T細胞間的相互抑制作用則少有報道。 CTA移植物由于其具有多種組織抗原的特點使其成為研究淋巴細胞克隆間相互作用的良好模型。CTA的低免疫原性也提示在其免疫反應中存在針對不同組織抗原的淋巴細胞克隆間相互抑制的可能性。然而淋巴細胞的競爭性抑制在CTA移植免疫耐受中的作用亦主要源于現(xiàn)象的推測，而缺乏直接的證據(jù)支持。多種組織抗原刺激是否能夠抑制淋巴細胞對異基因移植物的免疫排斥反應，延長移植物的存活時間亦未見報道。 因此，我們在本研究中設計了“差異化”的多種組織抗原刺激的CTA移植模型，以及多種MHC抗原刺激的體外與在體模型，以探討在移植，特別是CTA移植的免疫反應中淋巴細胞克隆間相互抑制作用及其可能機制。從而為進一步了解CTA的免疫機理，誘導CTA的免疫耐受提供新的理論依據(jù)。 方法： 1.將BN大鼠來源的含不同組織成分的的“差異化”CTA移植物移植至F344大鼠，構建CTA模型。分組為：S組（腹股溝皮片），SL組（腹股溝皮瓣），SLM組（腹股溝肌皮瓣）。觀察不同組織成分移植物的存活時間。 2.在CTA術后第7天，于受體大鼠內眥靜脈取血后，分離外周血淋巴細胞，與供體來源的淋巴細胞進行混合淋巴細胞反應，利用WST-8比色法檢測淋巴細胞增殖率。 3.分離BalB/c（H-2d）、C3H（H-2k）、C57BL/6（H-2b）Qa-1WT及C57BL/6（H-2b）Qa-1KO三種不同MHC表型的近交系小鼠脾臟淋巴細胞。分別以單一MHC表型的淋巴細胞，或1：1混合的兩種MHC表型的淋巴細胞作為刺激細胞，在體外進行單向混合淋巴細胞反應。利用WST-8比色法檢測淋巴細胞增殖率。 4.收集混合培養(yǎng)第72小時的淋巴細胞培養(yǎng)基上清液，，用ELISA法檢測TGF-β1、TNF-α與IL-10細胞因子的分泌。 5.收集混合培養(yǎng)第72小時的淋巴細胞，提取總RNA，利用RT-qPCR檢測其IL-10、Foxp3基因mRNA的表達水平。 6.將單一BalB/c（H-2d）來源、單一C3H（H-2k）來源或同時將BalB/c與C3H來源的全層皮片移植至C57BL/6（H-2b）小鼠，觀察皮片存活時間及皮片基底部淋巴細胞浸潤情況。 7.免疫組織化學染色檢測Foxp3+細胞在各組皮膚移植物基底部的分布情況。 結果： 1.不同組織成分CTA的存活時間分別為：S組6.25±0.25天，SL組為9±0.707天，SLM組為10.5±0.645天，SL及SLM組移植物存活時間較S組存活時間顯著延長（p=0.006），而SLM組存活時間較SL組有延長趨勢，但無顯著統(tǒng)計學差異（p=0.136）。 2.不同組織成分CTA受體對供體來源淋巴細胞的增殖率分別為：S組1.632±0.103，SL組1.588±0.294，SLM組1.508±0.232，組間無顯著差異。 3.兩種刺激細胞以1:1混合后同時刺激引起的T淋巴細胞增殖反應弱于相同數(shù)量的單一刺激細胞引起的細胞增殖。 4.兩種MHC抗原刺激組的IL-10濃度顯著高于單一MHC抗原刺激組（p0.001）。而TNF-α、TGF-β1濃度在各MLR反應組間無顯著差異。 5.兩種MHC抗原刺激組淋巴細胞IL-10mRNA表達顯著高于單一MHC抗原刺激組（p0.001），而調節(jié)性T細胞標志Foxp3基因表達在各反應組及無抗原刺激的對照組間無顯著差異。 6.兩種MHC抗原的皮膚同時移植組皮片存活時間及皮下淋巴細胞浸潤情況與單一MHC抗原組無顯著差異。皮下浸潤的炎性細胞中Foxp3+細胞比率在各組中無顯著差異。 7. Qa-1基因敲除（KO）組中，多抗原刺激的MLR反應中的淋巴細胞增殖率較單一抗原刺激組無顯著差異。Qa-1KO受體單一MHC皮膚移植物存活時間較WT受體顯著縮短（p=0.041，0.032），但兩種MHC抗原的皮膚同時移植的排斥時間較WT組均無顯著差異。 結論： 1.成功構建了“差異化”CTA移植物移植模型。隨著CTA移植物組織成分復雜性的增加，CTA存活時間有延長的趨勢。但該作用并不由受體的淋巴細胞對供體來源淋巴細胞的增殖反應的抑制引起。在多種MHC抗原表型的皮膚移植模型中，兩種MHC表型的異基因皮膚同時移植較單一MHC表型皮膚移植并不能延長移植物的存活時間。 2.不同MHC抗原表型的淋巴細胞引起的MLR反應中存在Qa-1依賴的淋巴細胞克隆間相互抑制作用，該作用可能與多抗原刺激誘導IL-10的分泌增加有關，而Foxp3+調節(jié)性T細胞未參與MHC抗原的克隆競爭作用。
[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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R392

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相關期刊論文 前1條

1 肖博;郭樹忠;;同種異體復合組織移植的免疫研究進展[J];中國修復重建外科雜志;2011年10期

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