骨髓腔内注射异体骨髓细胞诱导异体皮肤长期存活的机理探索

发布时间：2018-06-19 08:17

  本文选题：骨髓腔内注射 + 免疫耐受　； 参考：《第四军医大学》2017年博士论文

【摘要】：背景:大面积的创伤需要大量健康组织修复缺损,自体组织往往无法弥补这样的缺陷,异体复合组织的移植使得解决这个问题成为了可能。但是异体复合组织移植仍面临一些问题,其中最为关键的是如何延长移植物的存活时间,降低免疫抑制剂的毒副作用。免疫耐受是指受体的免疫系统不再攻击供体来源的组织。异体造血干细胞或骨髓移植的方法是公认的诱导免疫耐受的重要方法。异体骨髓细胞中的供体来源的造血干细胞如果可以在受体骨髓腔内定植,它们会在受体内发育成熟为具有正常免疫功能以及抗原提呈功能的细胞。接着,这些异体来源的细胞不会攻击同一供体来源的细胞及组织器官,从而延长异体移植物的存活时间,甚至免疫耐受状态的建立。异体骨髓移植仍存在着两个问题。第一,异体骨髓细胞仍可能诱发受体对其免疫排斥反应,所以仍然需要大剂量的免疫抑制、照射等副作用较大的免疫诱导方案。第二,异体造血干细胞在受体体内定植需要供体来源的骨髓微环境,而通过静脉注射的方法,难以成功移植供体来源的造血干细胞微环境。骨髓腔内注射可以提高异体造血干细胞的定植效率,延长异体实体器官的存活时间,降低移植物抗宿主反应的发生。虽然已经有学者对于这一现象进行了初步的探索,但是对于骨髓腔内注射是否可以延长异体皮肤的存活时间,诱导局部骨髓中微环境的改变仍然缺乏探索。这些研究对于异体骨髓移植中异体造血干细胞功能和局部骨髓免疫微环境调节仍然缺乏深入的阐述。此外,局部照射联合骨髓腔内注射可否延长异体皮片的存活时间,并降低免疫诱导方案对于受体的毒副作用呢?对于这些问题的探索有助于我们更好的改变局部骨髓微环境,促进异体造血干细胞的定植并延长异体皮片的存活时间,为促进异体复合组织的长期存活提供新的治疗方案。目标:1)探索骨髓腔内注射异体骨髓细胞是否可以诱导异体皮片的长期存活;2)探索骨髓腔内注射异体骨髓细胞是否可以诱导局部骨髓中间质微环境和免疫微环境改变,这些改变是否可以延长异体皮肤的长期存活;3)探索骨髓腔内注射异体骨髓细胞联合局部照射可否延长异体皮片的长期存活。方法:1)预处理模型以及皮片移植的模型的建立:受体小鼠经过4 Gy X射线全身照射并给予骨髓腔内注射供体来源的5×106的骨髓细胞。骨髓细胞移植后3周,受体接受同一供体来源1x1cm的鼠尾全厚皮片移植。待排斥终点,取材并进行病理切片。2)外周血嵌合体水平的测定:骨髓移植后1周、3周、12周、18周分别从小鼠内眦静脉丛取血,使用流式细胞技术测量供体细胞在外周血白细胞、粒细胞、T细胞、B细胞中的比例。3)骨髓及脾脏中供体来源造血干细胞、间质细胞、间质干细胞、调节性T细胞比例:分别在术后3周、12周测定受体注射侧骨髓以及未注射侧骨髓供体来源造血干细胞(H-2Kb+c-kit+sca-1+lineage-)、供体间质细胞(H-2Kb+CD45-CD31-TER119-)、供体间质干细胞(H-2Kb+CD45-CD31-TER119-PDGFR-α+)、调节性T细胞(CD4+Foxp3+)比例。4)测定骨髓及脾脏中细胞因子的表达:分别在术后3周、12周使用实时定量PCR以及Western Blotting等测定骨髓和脾脏细胞中IL-2、IL-10、TGF-β等的表达水平。5)手术去除局部注射骨髓:在异体骨髓移植后,分别在术后7天和21天手术去除局部注射胫骨,术后3周、12周测定外周血及脾脏中供体细胞比例,并测定脾脏中调节性T细胞比例,术后3周接受异体来源的皮片移植。6)局部照射模型:使用3mm厚度的铅板,并且修剪铅板露出左侧胫骨,给予局部胫骨30 Gy的X射线照射。局部照射后,通过病理切片观察局部照射的效果。小鼠体重作为小鼠健康的指标并进行长期监测。7)统计学分析:使用student t检验进行静脉注射组、骨髓腔内注射组、假手术组等两两之间的比较。使用配对t检验比较注射侧骨髓及对侧骨髓的数据。Log-rank检验方法用于分析生存曲线数据。P值小于0.05认为有统计学意义。结果:1)骨髓腔内注射可以较静脉注射骨髓细胞延长异体皮片存活时间,并且诱导各系细胞的嵌合体水平增高。骨髓腔内注射异体骨髓细胞可以较静脉注射和假手术组延长异体皮片的存活时间(中位存活时间:骨髓腔内注射组,60天;假手术组,32.5天;静脉注射组,29.5天)并升高外周血中B细胞、T细胞、粒细胞中供体细胞的水平,尤其升高了早期外周血中供体来源粒细胞水平。2)骨髓腔内注射主要改变了局部注射骨髓的间质细胞微环境。相较于静脉注射组和假手术组,骨髓腔内注射异体骨髓细胞可以将大多数局部注射骨髓的受体来源间质细胞替换为供体来源的细胞(术后12周,注射侧骨髓76.2%,未注射侧骨髓33.3%,P0.05),并提高了局部注射骨髓中间质干细胞的比例。3)骨髓腔内注射可以升高局部注射骨髓中受体来源调节性T细胞的比例。相较于未注射骨髓,骨髓腔内注射可以在早期(术后3周)升高局部注射骨髓中受体来源调节性T细胞的比例,而在晚期这种差异逐渐消失。同时,调节性T细胞比例的升高还伴有IL-10等细胞因子表达的升高,而TGF-β、IL-2等却没有升高。4)早期(术后7天)去除局部注射骨髓可以使得供体来源细胞比例降低。在术后7天而非21天去除局部注射骨髓,可以使得供体来源细胞及造血干细胞的比例明显降低,但是还不足以影响供体皮片的存活时间。5)局部照射联合免疫抑制剂及骨髓腔内注射异体骨髓细胞可以提高外周血中供体细胞的比例,但是不足以诱导免疫耐受或者延长异体皮片的存活时间。局部照射(30Gy X射线)联合免疫抑制剂(腹腔注射雷帕霉素2mg/kg/天14天)及骨髓腔内注射异体骨髓细胞可以显著提高外周血中供体细胞的比例,并一定程度延长异体皮片的存活时间,但两组之间并无统计学差异。6)局部创伤的作用不足以改变局部骨髓的微环境。在以上的实验中,局部注射PBS并没有导致局部注射骨髓与假手术组之间出现明显供体细胞比例、造血干细胞比例、间质细胞比例、间质干细胞比例、调节性T细胞比例及相关细胞因子的变化的统计学上的差异。结论:我们的研究中有以下发现:1)在本实验室建立了骨髓腔内注射联合非致死剂量的全身照射延长异体皮片长期存活的小鼠模型,并证实了骨髓腔内注射可以较静脉注射延长异体皮片的存活时间。2)本研究首次证实了骨髓腔内注射异体骨髓细胞可以替换局部注射骨髓的间质微环境并且升高局部注射骨髓中调节性T细胞及免疫抑制相关细胞因子的表达。3)骨髓腔内注射异体骨髓细胞可以早期(7天)促进异体造血干细胞在受体局部注射骨髓中定植,但是这种作用在术后21天消失。4)我们首次试图使用联合局部照射以及骨髓腔内注射延长异体皮片的存活时间,这种方法可以减少对于受体的毒副作用,该方法虽然可以提高外周血中供体细胞的比例,但仍然不足以诱导异体皮肤的长期存活,具体的诱导方案仍然需要进一步的实验进行探索。5)骨髓腔内注射引起的局部骨髓创伤不是骨髓腔内注射可以较常规静脉注射延长异体皮片存活以及促进异体造血干细胞定植的原因。
[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.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R622
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