本文選題：間充質(zhì)干細(xì)胞 + 免疫耐受�。� 參考：《第四軍醫(yī)大學(xué)》2012年博士論文

【摘要】：研究意義 異體器官移植已成為臟器功能衰竭治療中的一種常規(guī)治療方案。在器官移植領(lǐng)域，目前主要使用免疫抑制藥物來避免、減輕和逆轉(zhuǎn)排斥反應(yīng)，但是使用這些藥物的同時也帶來了一系列問題，如感染幾率增加、惡性腫瘤發(fā)病率提高、費(fèi)用昂貴等。盡管如此，這些方法仍不能避免慢性移植物功能衰竭，因此，大量研究致力于尋求一種更好的方法建立免疫耐受。經(jīng)骨髓移植建立混合嵌合體被證實是建立異體移植免疫耐受的理想方法之一。然而，在誘導(dǎo)混合嵌合體的過程中，受體必須經(jīng)過輻射、放療及化療等預(yù)處理方案，以提高造血干細(xì)胞移植成活率，這點(diǎn)很大程度上限制了該技術(shù)的推廣應(yīng)用。到目前為止，仍沒有一種切實可靠、適用于臨床的預(yù)處理方案。 BMSC在體外易于培養(yǎng)擴(kuò)增，BMSCs的低免疫原性與免疫調(diào)節(jié)兩方面特性使得它在治療移植排斥方面具有潛在優(yōu)勢。目前，人們已經(jīng)利用BMSCs的免疫調(diào)節(jié)特性對自身免疫性疾病、全身炎癥反應(yīng)綜合癥及GVHD等多種疾病展開治療，并取得良好的效果�；谀壳巴ㄟ^骨髓移植建立嵌合體尚無低毒性預(yù)處理方案，本研究擬嘗試用BMSCs對受體進(jìn)行預(yù)處理，然后進(jìn)行骨髓移植，觀察是否可以成功建立混合嵌合體并形成免疫耐受�；赥h17/Treg細(xì)胞在免疫排斥與耐受中的重要地位，本研究進(jìn)一步通過體外實驗研究BMSCs對Th17/Treg細(xì)胞分化的影響，并對其機(jī)制進(jìn)行探索。 材料與方法 1.將供體（Balb/c）小鼠來源BMSCs進(jìn)行體外培養(yǎng)擴(kuò)展，通過尾靜脈向受體輸注，然后將供體骨髓向受體移植，觀察在不使用免疫毒性藥物的情況下是否可以移植成活形成嵌合體；在此基礎(chǔ)上，將供體及第三方（ICR）小鼠來源皮片向受體移植，觀察其存活情況，分析是否形成免疫耐受； 2.體外培養(yǎng)受體CD4~+T細(xì)胞作為反應(yīng)細(xì)胞，獲取供體脾細(xì)胞為刺激細(xì)胞，二者進(jìn)行單向混合淋巴細(xì)胞反應(yīng)，觀察在異體抗原刺激下初始T細(xì)胞向Th17/Treg細(xì)胞分化情況；進(jìn)一步在該體系中加入BMSCs進(jìn)行混合培養(yǎng)，觀察BMSCs對Th17/Treg細(xì)胞分化的影響，通過對細(xì)胞因子等進(jìn)行檢測，初步探討其可能機(jī)制； 3.通過1-D-MT抑制BMSCs細(xì)胞內(nèi)IDO功能，觀察BMSCs對Th17/Treg細(xì)胞分化的影響，分析IDO在BMSCs干預(yù)Th17/Treg細(xì)胞分化中的作用。 結(jié)果 1.BMSCs對受體預(yù)處理后進(jìn)行骨髓移植，受體外周血中可持續(xù)檢測到供體來源細(xì)胞，在移植4周后，供體細(xì)胞占外周血總細(xì)胞約20-25%；在該模型上進(jìn)行皮膚移植，發(fā)現(xiàn)供體來源皮膚可長期存活，值得關(guān)注的是，供體來源皮膚的毛發(fā)在移植后可持續(xù)正常生長，提示未發(fā)生移植物慢性功能喪失。病理切片顯示，供體來源皮片結(jié)構(gòu)完整，，僅有少量單個核細(xì)胞浸潤，而第三方來源皮片則出現(xiàn)表皮脫落，基底層大量單個核細(xì)胞浸潤等；通過取脾細(xì)胞進(jìn)行單項混合淋巴細(xì)胞反應(yīng)，發(fā)現(xiàn)成功建立嵌合體的小鼠脾細(xì)胞對于供體抗原具有低反應(yīng)性，而對第三方抗原反應(yīng)性正常； 2.通過單項混合淋巴細(xì)胞反應(yīng)結(jié)果發(fā)現(xiàn)，異體抗原刺激初始T細(xì)胞，可促使其向Th17/Treg細(xì)胞分化；BMSCs干預(yù)后，反應(yīng)性T細(xì)胞增殖明顯受抑制，同時Th17細(xì)胞比例減少，而Treg細(xì)胞增多，進(jìn)一步分析發(fā)現(xiàn)，BMSCs可降低反應(yīng)體系中IL-1β、IL-2、IL-6及IL-17，同時提高IL-10、TGFβ1及IDO； 3.通過1-D-MT預(yù)孵育可抑制BMSCs細(xì)胞內(nèi)IDO功能，該BMSCs對T細(xì)胞增殖及Th17/Treg細(xì)胞分化的影響作用明顯降低，但培養(yǎng)體系內(nèi)介導(dǎo)TGFβ1及IL-6水平與正常BMSCs組比較未見明顯差異。 結(jié)論 在不使用任何免疫毒性藥物的情況下，通過BMSCs聯(lián)合骨髓移植可成功建立混合嵌合體并形成供體特異性免疫耐受，在此模型上進(jìn)行供體皮膚移植可長期存活；BMSCs可促使異體抗原刺激引起的Th17/Treg細(xì)胞分化向Treg細(xì)胞方向偏移，IDO在其中可能具有關(guān)鍵性作用。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 劉麗敏;接英;潘志強(qiáng);;供體骨髓細(xì)胞輸注誘導(dǎo)免疫耐受延長豬-猴異種角膜移植植片存活的研究[J];首都醫(yī)科大學(xué)學(xué)報;2010年01期

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