本文選題：骨髓腔內(nèi)注射 + 免疫耐受　； 參考：《第四軍醫(yī)大學(xué)》2017年博士論文

【摘要】：背景:大面積的創(chuàng)傷需要大量健康組織修復(fù)缺損,自體組織往往無(wú)法彌補(bǔ)這樣的缺陷,異體復(fù)合組織的移植使得解決這個(gè)問(wèn)題成為了可能。但是異體復(fù)合組織移植仍面臨一些問(wèn)題,其中最為關(guān)鍵的是如何延長(zhǎng)移植物的存活時(shí)間,降低免疫抑制劑的毒副作用。免疫耐受是指受體的免疫系統(tǒng)不再攻擊供體來(lái)源的組織。異體造血干細(xì)胞或骨髓移植的方法是公認(rèn)的誘導(dǎo)免疫耐受的重要方法。異體骨髓細(xì)胞中的供體來(lái)源的造血干細(xì)胞如果可以在受體骨髓腔內(nèi)定植,它們會(huì)在受體內(nèi)發(fā)育成熟為具有正常免疫功能以及抗原提呈功能的細(xì)胞。接著,這些異體來(lái)源的細(xì)胞不會(huì)攻擊同一供體來(lái)源的細(xì)胞及組織器官,從而延長(zhǎng)異體移植物的存活時(shí)間,甚至免疫耐受狀態(tài)的建立。異體骨髓移植仍存在著兩個(gè)問(wèn)題。第一,異體骨髓細(xì)胞仍可能誘發(fā)受體對(duì)其免疫排斥反應(yīng),所以仍然需要大劑量的免疫抑制、照射等副作用較大的免疫誘導(dǎo)方案。第二,異體造血干細(xì)胞在受體體內(nèi)定植需要供體來(lái)源的骨髓微環(huán)境,而通過(guò)靜脈注射的方法,難以成功移植供體來(lái)源的造血干細(xì)胞微環(huán)境。骨髓腔內(nèi)注射可以提高異體造血干細(xì)胞的定植效率,延長(zhǎng)異體實(shí)體器官的存活時(shí)間,降低移植物抗宿主反應(yīng)的發(fā)生。雖然已經(jīng)有學(xué)者對(duì)于這一現(xiàn)象進(jìn)行了初步的探索,但是對(duì)于骨髓腔內(nèi)注射是否可以延長(zhǎng)異體皮膚的存活時(shí)間,誘導(dǎo)局部骨髓中微環(huán)境的改變?nèi)匀蝗狈μ剿�。這些研究對(duì)于異體骨髓移植中異體造血干細(xì)胞功能和局部骨髓免疫微環(huán)境調(diào)節(jié)仍然缺乏深入的闡述。此外,局部照射聯(lián)合骨髓腔內(nèi)注射可否延長(zhǎng)異體皮片的存活時(shí)間,并降低免疫誘導(dǎo)方案對(duì)于受體的毒副作用呢?對(duì)于這些問(wèn)題的探索有助于我們更好的改變局部骨髓微環(huán)境,促進(jìn)異體造血干細(xì)胞的定植并延長(zhǎng)異體皮片的存活時(shí)間,為促進(jìn)異體復(fù)合組織的長(zhǎng)期存活提供新的治療方案。目標(biāo):1)探索骨髓腔內(nèi)注射異體骨髓細(xì)胞是否可以誘導(dǎo)異體皮片的長(zhǎng)期存活;2)探索骨髓腔內(nèi)注射異體骨髓細(xì)胞是否可以誘導(dǎo)局部骨髓中間質(zhì)微環(huán)境和免疫微環(huán)境改變,這些改變是否可以延長(zhǎng)異體皮膚的長(zhǎng)期存活;3)探索骨髓腔內(nèi)注射異體骨髓細(xì)胞聯(lián)合局部照射可否延長(zhǎng)異體皮片的長(zhǎng)期存活。方法:1)預(yù)處理模型以及皮片移植的模型的建立:受體小鼠經(jīng)過(guò)4 Gy X射線全身照射并給予骨髓腔內(nèi)注射供體來(lái)源的5×106的骨髓細(xì)胞。骨髓細(xì)胞移植后3周,受體接受同一供體來(lái)源1x1cm的鼠尾全厚皮片移植。待排斥終點(diǎn),取材并進(jìn)行病理切片。2)外周血嵌合體水平的測(cè)定:骨髓移植后1周、3周、12周、18周分別從小鼠內(nèi)眥靜脈叢取血,使用流式細(xì)胞技術(shù)測(cè)量供體細(xì)胞在外周血白細(xì)胞、粒細(xì)胞、T細(xì)胞、B細(xì)胞中的比例。3)骨髓及脾臟中供體來(lái)源造血干細(xì)胞、間質(zhì)細(xì)胞、間質(zhì)干細(xì)胞、調(diào)節(jié)性T細(xì)胞比例:分別在術(shù)后3周、12周測(cè)定受體注射側(cè)骨髓以及未注射側(cè)骨髓供體來(lái)源造血干細(xì)胞(H-2Kb+c-kit+sca-1+lineage-)、供體間質(zhì)細(xì)胞(H-2Kb+CD45-CD31-TER119-)、供體間質(zhì)干細(xì)胞(H-2Kb+CD45-CD31-TER119-PDGFR-α+)、調(diào)節(jié)性T細(xì)胞(CD4+Foxp3+)比例。4)測(cè)定骨髓及脾臟中細(xì)胞因子的表達(dá):分別在術(shù)后3周、12周使用實(shí)時(shí)定量PCR以及Western Blotting等測(cè)定骨髓和脾臟細(xì)胞中IL-2、IL-10、TGF-β等的表達(dá)水平。5)手術(shù)去除局部注射骨髓:在異體骨髓移植后,分別在術(shù)后7天和21天手術(shù)去除局部注射脛骨,術(shù)后3周、12周測(cè)定外周血及脾臟中供體細(xì)胞比例,并測(cè)定脾臟中調(diào)節(jié)性T細(xì)胞比例,術(shù)后3周接受異體來(lái)源的皮片移植。6)局部照射模型:使用3mm厚度的鉛板,并且修剪鉛板露出左側(cè)脛骨,給予局部脛骨30 Gy的X射線照射。局部照射后,通過(guò)病理切片觀察局部照射的效果。小鼠體重作為小鼠健康的指標(biāo)并進(jìn)行長(zhǎng)期監(jiān)測(cè)。7)統(tǒng)計(jì)學(xué)分析:使用student t檢驗(yàn)進(jìn)行靜脈注射組、骨髓腔內(nèi)注射組、假手術(shù)組等兩兩之間的比較。使用配對(duì)t檢驗(yàn)比較注射側(cè)骨髓及對(duì)側(cè)骨髓的數(shù)據(jù)。Log-rank檢驗(yàn)方法用于分析生存曲線數(shù)據(jù)。P值小于0.05認(rèn)為有統(tǒng)計(jì)學(xué)意義。結(jié)果:1)骨髓腔內(nèi)注射可以較靜脈注射骨髓細(xì)胞延長(zhǎng)異體皮片存活時(shí)間,并且誘導(dǎo)各系細(xì)胞的嵌合體水平增高。骨髓腔內(nèi)注射異體骨髓細(xì)胞可以較靜脈注射和假手術(shù)組延長(zhǎng)異體皮片的存活時(shí)間(中位存活時(shí)間:骨髓腔內(nèi)注射組,60天;假手術(shù)組,32.5天;靜脈注射組,29.5天)并升高外周血中B細(xì)胞、T細(xì)胞、粒細(xì)胞中供體細(xì)胞的水平,尤其升高了早期外周血中供體來(lái)源粒細(xì)胞水平。2)骨髓腔內(nèi)注射主要改變了局部注射骨髓的間質(zhì)細(xì)胞微環(huán)境。相較于靜脈注射組和假手術(shù)組,骨髓腔內(nèi)注射異體骨髓細(xì)胞可以將大多數(shù)局部注射骨髓的受體來(lái)源間質(zhì)細(xì)胞替換為供體來(lái)源的細(xì)胞(術(shù)后12周,注射側(cè)骨髓76.2%,未注射側(cè)骨髓33.3%,P0.05),并提高了局部注射骨髓中間質(zhì)干細(xì)胞的比例。3)骨髓腔內(nèi)注射可以升高局部注射骨髓中受體來(lái)源調(diào)節(jié)性T細(xì)胞的比例。相較于未注射骨髓,骨髓腔內(nèi)注射可以在早期(術(shù)后3周)升高局部注射骨髓中受體來(lái)源調(diào)節(jié)性T細(xì)胞的比例,而在晚期這種差異逐漸消失。同時(shí),調(diào)節(jié)性T細(xì)胞比例的升高還伴有IL-10等細(xì)胞因子表達(dá)的升高,而TGF-β、IL-2等卻沒(méi)有升高。4)早期(術(shù)后7天)去除局部注射骨髓可以使得供體來(lái)源細(xì)胞比例降低。在術(shù)后7天而非21天去除局部注射骨髓,可以使得供體來(lái)源細(xì)胞及造血干細(xì)胞的比例明顯降低,但是還不足以影響供體皮片的存活時(shí)間。5)局部照射聯(lián)合免疫抑制劑及骨髓腔內(nèi)注射異體骨髓細(xì)胞可以提高外周血中供體細(xì)胞的比例,但是不足以誘導(dǎo)免疫耐受或者延長(zhǎng)異體皮片的存活時(shí)間。局部照射(30Gy X射線)聯(lián)合免疫抑制劑(腹腔注射雷帕霉素2mg/kg/天14天)及骨髓腔內(nèi)注射異體骨髓細(xì)胞可以顯著提高外周血中供體細(xì)胞的比例,并一定程度延長(zhǎng)異體皮片的存活時(shí)間,但兩組之間并無(wú)統(tǒng)計(jì)學(xué)差異。6)局部創(chuàng)傷的作用不足以改變局部骨髓的微環(huán)境。在以上的實(shí)驗(yàn)中,局部注射PBS并沒(méi)有導(dǎo)致局部注射骨髓與假手術(shù)組之間出現(xiàn)明顯供體細(xì)胞比例、造血干細(xì)胞比例、間質(zhì)細(xì)胞比例、間質(zhì)干細(xì)胞比例、調(diào)節(jié)性T細(xì)胞比例及相關(guān)細(xì)胞因子的變化的統(tǒng)計(jì)學(xué)上的差異。結(jié)論:我們的研究中有以下發(fā)現(xiàn):1)在本實(shí)驗(yàn)室建立了骨髓腔內(nèi)注射聯(lián)合非致死劑量的全身照射延長(zhǎng)異體皮片長(zhǎng)期存活的小鼠模型,并證實(shí)了骨髓腔內(nèi)注射可以較靜脈注射延長(zhǎng)異體皮片的存活時(shí)間。2)本研究首次證實(shí)了骨髓腔內(nèi)注射異體骨髓細(xì)胞可以替換局部注射骨髓的間質(zhì)微環(huán)境并且升高局部注射骨髓中調(diào)節(jié)性T細(xì)胞及免疫抑制相關(guān)細(xì)胞因子的表達(dá)。3)骨髓腔內(nèi)注射異體骨髓細(xì)胞可以早期(7天)促進(jìn)異體造血干細(xì)胞在受體局部注射骨髓中定植,但是這種作用在術(shù)后21天消失。4)我們首次試圖使用聯(lián)合局部照射以及骨髓腔內(nèi)注射延長(zhǎng)異體皮片的存活時(shí)間,這種方法可以減少對(duì)于受體的毒副作用,該方法雖然可以提高外周血中供體細(xì)胞的比例,但仍然不足以誘導(dǎo)異體皮膚的長(zhǎng)期存活,具體的誘導(dǎo)方案仍然需要進(jìn)一步的實(shí)驗(yàn)進(jìn)行探索。5)骨髓腔內(nèi)注射引起的局部骨髓創(chuàng)傷不是骨髓腔內(nèi)注射可以較常規(guī)靜脈注射延長(zhǎng)異體皮片存活以及促進(jìn)異體造血干細(xì)胞定植的原因。
[Abstract]:Background: large area of trauma requires a large number of healthy tissues to repair defects. Autologous tissue often fails to make up for such defects. Allograft transplantation makes it possible to solve this problem. However, the allograft transplantation is still facing some problems. The most important thing is how to prolong the survival time of the graft and reduce the immunity. The toxic side effects of pestilence inhibitors. Immune tolerance means that the immune system of the receptor no longer attacks donor sources. Allogeneic hematopoietic stem cells or bone marrow transplants are recognized as an important method to induce immune tolerance. The receptor is developed into mature cells with normal immune function and antigen presenting function. Then, these allogenic cells do not attack the cells and organs of the same donor, thus prolonging the survival time of allograft, even the establishment of immune tolerance state. There are still two problems in allograft bone marrow transplantation. First, Allogeneic bone marrow cells may still induce the rejection of the recipient, so it is still necessary for a large dose of immunosuppression and irradiation to induce a larger side effect. Second, allogeneic hematopoietic stem cells are implanted in the recipient's bone marrow microenvironment, while intravenous injection is difficult to successfully transplant donor. Intramedullary hematopoietic stem cell microenvironment. Intramedullary injection can improve the efficiency of allogeneic stem cell colonization, prolong the survival time of the allogenic organs and reduce the occurrence of graft versus host response. Although some scholars have explored this phenomenon initially, it is possible to prolong the skin allograft by intramedullary injection. The survival time of the skin and the change of the microenvironment in the local bone marrow still lack exploration. These studies are still lacking in the detailed description of the allogeneic hematopoietic stem cell function and local bone marrow immuno microenvironment regulation in the allograft bone marrow transplantation. The exploration of these problems will help us to better change the local bone marrow microenvironment, promote the transplantation of allogeneic hematopoietic stem cells and prolong the survival time of the allogeneic skin, and provide a new treatment for the long-term survival of the allogenic tissue. Objective: 1) to explore the intramedullary intramedullary injection. Whether allogeneic bone marrow cells can induce long-term survival of skin allograft; 2) whether intramedullary intramedullary injection of allogenic bone marrow cells can induce local marrow interstitial microenvironment and immune microenvironment changes, whether these changes can prolong the long-term survival of the skin allograft; 3) explore the combination of allogenic bone marrow cells in bone marrow. Method: 1) 1) preconditioning model and the establishment of a model of skin graft: the recipient mice were irradiated with 4 Gy X rays and injected 5 * 106 bone marrow cells from the donor bone marrow cavity. After 3 weeks of bone marrow transplantation, the receptor accepted the full thick skin graft of the rat tail of the same donor source 1x1cm. The level of peripheral blood chimerism: 1 weeks, 3 weeks, 12 weeks, 18 weeks after bone marrow transplantation, blood was taken from the inner canthus plexus of mice, and the donor cells were measured in peripheral blood white blood cells, granulocytes, T cells, B cells and the donor origin of the spleen in 1 weeks, 12 weeks and 18 weeks after bone marrow transplantation. Stem cells, stromal cells, mesenchymal stem cells, and regulatory T cells ratio: 3 weeks after the operation, 12 weeks respectively to determine the recipient bone marrow and uninjected bone marrow donor derived hematopoietic stem cells (H-2Kb+c-kit+sca-1+lineage-), donor mesenchymal cells (H-2Kb+CD45-CD31-TER119-), donor mesenchymal stem cells (H-2Kb+CD45-CD31-TER119-PDGFR- alpha +), and the transfer of donor mesenchymal stem cells (H-2Kb+CD45-CD31-TER119-PDGFR- alpha +). The expression of cytokine in bone marrow and spleen was measured by T cell (CD4+Foxp3+) ratio.4) in bone marrow and spleen: 3 weeks after operation, 12 weeks using real-time quantitative PCR and Western Blotting to determine the expression level of IL-2, IL-10, TGF- beta, etc. in bone marrow and spleen cells, and to remove the local injection of bone marrow: after allograft bone marrow transplantation, 7 days and 21 after the operation, respectively. In the day operation, the local injection of tibia was removed. The proportion of donor cells in peripheral blood and spleen was measured at 3 weeks after operation, and the proportion of donor cells in the spleen was measured at 12 weeks. The proportion of regulatory T cells in the spleen was measured, and the skin graft of the allogeneic source was transplanted for.6) at 3 weeks after the operation. The local irradiation model was used for the 3mm thickness of the lead plate, and the left tibia was pruned and the X ray of the local tibia of 30 Gy was given. The effect of local irradiation was observed by pathological section. The body weight of mice was used as the index of mice health and the long-term monitoring of.7) was statistically analyzed. The comparison between the intravenous group, the intramedullary intramedullary injection group and the sham operation group was compared with the student t test. The paired t test was used to compare the bone marrow and contralateral side of the injection side. The.Log-rank test of bone marrow data was used to analyze the data of the survival curve and the.P value was less than 0.05. Results: 1) intramedullary injection can prolong the survival time of the allograft compared with the intravenous injection of bone marrow cells, and induce the increase of the chimerism level of each cell. The intramedullary marrow injection of allogenic bone marrow cells can be compared with the vein. The injection and sham groups extended the survival time of the skin allograft (median survival time: intramedullary injection group, 60 days, sham operation group, 32.5 days, 29.5 days of intravenous injection, 29.5 days) and increased the level of B cells, T cells, granulocyte donor cells in peripheral blood, especially in the early peripheral blood donor granulocyte level.2) intramedullary injection Intramedullary injection of allogeneic bone marrow cells can replace most of the recipient derived mesenchymal cells from the bone marrow into the donor cells (12 weeks after the operation, 76.2% of the bone marrow at the side of the injection, 33.3% without side marrow, P0.05), and in comparison with the intravenous injection group and the sham operation group (12 weeks after the operation). Intrumedullary intramedullary injection can increase the proportion of receptor source regulatory T cells in the bone marrow by local injection of bone marrow mesenchymal stem cells (.3). Compared to uninjected marrow, intramedullary intramedullary injection can increase the proportion of the receptor source regulatory T cells in the bone marrow at the early stage (3 weeks after the operation), while in the late period this difference is a difference. At the same time, the increase in the proportion of regulatory T cells was accompanied by an increase in the expression of IL-10 and other cytokines, while TGF- beta, IL-2, etc. did not increase.4) early (7 days after the operation) removal of the local injection of bone marrow could reduce the proportion of donor cells. The donor cells could be caused by the removal of the local injection of bone marrow at the 7 day after the operation, but not 21 days after the operation. The proportion of hematopoietic stem cells decreased significantly, but it was not enough to affect the survival time of donor skin.5). Local irradiation combined with immunosuppressive agents and intramedullary injection of allogeneic bone marrow cells could increase the proportion of donor cells in peripheral blood, but it was not sufficient to induce immune tolerance or to prolong the survival time of skin allograft. Local irradiation (3 0Gy X ray) combined immunosuppressant (intraperitoneal injection of rapamycin 2mg/kg/ days 14 days) and intramedullary injection of allogenic bone marrow cells can significantly increase the proportion of donor cells in the peripheral blood, and extend the survival time of the skin allograft to a certain extent, but there is no statistical difference between the two groups. The effect of local trauma is not sufficient to change the local bone. Intramedullary microenvironment. In the above experiments, local injection of PBS did not lead to a significant difference in the proportion of donor cells, the proportion of hematopoietic stem cells, the proportion of mesenchymal cells, the proportion of mesenchymal stem cells, the proportion of mesenchymal stem cells, the proportion of mesenchymal stem cells, the proportion of regulatory T cells and the changes of related cytokines between local injection of bone marrow and sham operation group. The following findings are as follows: 1) in this laboratory, a mouse model was established to prolong the long-term survival of the allogeneic skin with an intracavitary injection of bone marrow combined with a non lethal dose of the skin, and the survival time of the intramedullary intramedullary injection can be compared with the prolongation of the survival time of the skin allograft compared with the intravenous injection. This study was the first to confirm that the allogenic bone marrow cells were injected into the marrow cavity for the first time. Intramedullary injection of allogeneic bone marrow cells in bone marrow by replacing the interstitial microenvironment with local injection of bone marrow and increasing local injection of regulatory T cells and expression of immunosuppressive cytokine.3) can promote the early transplantation of allogeneic hematopoietic stem cells in the bone marrow by local injection of the recipient, but this effect disappeared at the 21 day after the operation of.4). For the first time, we tried to use combined local irradiation and intramedullary intramedullary injection to prolong the survival time of the skin allograft. This method can reduce the toxic side effects on the receptor. Although the method can improve the proportion of donor cells in the peripheral blood, it is still not sufficient to induce the long-term survival of the skin allograft, and the specific induction scheme is still needed. Further experiments are conducted to explore.5) local bone marrow trauma caused by intramedullary intramedullary injection is not the reason for prolonging the survival of the allograft and promoting the allogeneic stem cell colonization by intramedullary injection of the bone marrow.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R622
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本文編號(hào)：2039227