T细胞疫苗诱导大鼠胰肾联合移植免疫耐受的实验研究

发布时间：2018-06-13 11:01

本文选题：T细胞疫苗 + 免疫耐受　；参考：《第四军医大学》2007年博士论文

【摘要】： 器官移植对许多终末期疾病的治疗是十分重要的手段,但器官移植导致的排斥反应是影响其成功率的主要原因。为了降低排斥反应的发生率,提高器官移植的成功率,目前需要病人终生使用免疫抑制剂。然而,长期使用免疫抑制剂除了会引起免疫抑制剂本身的毒副作用(诸如升高血压、肝肾毒性等不良反应)外,也可引起严重感染、恶性肿瘤的发生,从而影响器官移植的成功率和受者的长期存活率。若能造成移植宿主对移植抗原的免疫耐受状态或低免疫应答状态,同时保持对其它抗原免疫应答的反应能力是器官移植领域人们追求的理想目标。同种器官移植后,产生抗同种移植物的排斥反应,主要是因为受体免疫系统识别供体不相容的MHC抗原,引起免疫应答,触发了同种排斥反应的结果。其中同种反应性T细胞在排斥反应中发挥着重要作用,将其灭活后作为T细胞疫苗免疫,可诱导抗原特异性的免疫耐受或免疫低反应性。T细胞疫苗(T Cell Vaccine,TCV)是指用化学或物理方法灭活的致病性T细胞。其失去致病能力,但保持了活化致病性T细胞的免疫特征。研究证明,把引起自身免疫性疾病的自身反应性T细胞或导致同种移植排斥反应的同种反应性T细胞制备成TCV,免疫动物后能够诱导机体产生针对致病性T细胞的免疫应答。可以消除或减轻这些细胞的致病作用或反应能力,表现为对自身免疫性疾病的防治作用和诱导同种移植物质延长存活。糖尿病肾病是糖尿病中危害最严重、致死率最高的一种并发症,我国糖尿病病人中糖尿病肾病总发生率为47.7%。尤其以I型糖尿病病人为主。若I型糖尿病病程过20年,10%会有肾病,最终会发展为终末期肾病即尿毒症。胰肾联合移植(simultaneous pancreas-kidney transplantation,SPK)是临床治疗糖尿病肾病的唯一有效途径。1966年美国明尼苏达大学的Kelly和Lillehei成功实施了首例胰肾联合移植手术。根椐全美器官分配网络(UNOS)和国际胰腺移植登记中心(IPTR)记录,胰腺移植的总数正不断的增加,从1966年12月至2002年10月,总数达18843例,其中绝大多数在美国(13951例)。自1987年到2002年,UNOS报道的13330个胰腺移植病例中,其中78%采用SPK。迄今为止,SPK被公认为治疗I型糖尿病( IDDM)并发尿毒症的最理想的方法。20世纪80年代以来,随着移植技术的发展,以及新型免疫抑制剂的临床应用,使得该手术的安全性和成功率不断提高。本课题旨在研究T细胞疫苗是否能够诱导胰肾联合移植时的免疫耐受,为临床提高胰肾联合移植的成功率提供实验依据和理论参考。首先制备了T细胞疫苗并对其进行功能鉴定。实验选用近交系AS大鼠、SD大鼠和Wistar大鼠,利用AS大鼠和SD大鼠分离脾细胞后免疫Wistar大鼠,分离Wistar大鼠的脾细胞。在体外用ConA将其活化,使其表达多种活化受体,从而增强其抗原性,然后用丝裂霉素处理,灭活后制成T细胞疫苗。实验表明两种T细胞疫苗免疫Wistar大鼠后,都能够诱导脾细胞对ConA诱导的增殖能力低下,表明T细胞疫苗免疫后引起一定程度的T细胞非特异性免疫功能低下,部分解释了T细胞疫苗作用的抗原非特异性;T细胞疫苗能够诱导针对同种抗原发生免疫耐受,该效应既具有特异性,也有非特异性效应;T细胞疫苗能够诱导外周血T细胞发生凋亡,使CD4/CD8比值减小。建立稳定可靠的胰肾联合移植动物模型对于进行胰肾联合移植的生理及免疫方面的研究具有重要意义。大鼠由于基因型恒定,可利用纯系品种建立模型,证明模型稳定、可行,再用同种异体大鼠进行理论探索。因此大鼠是较理想的胰肾十二指肠联合移植的实验动物。我们在STZ诱导的糖尿病大鼠基础上建立了大鼠胰肾联合移植模型,术后生化指标、病理结果均正常。手术成功率为76%,为以后的免疫耐受试验奠定了基础。我们建立的大鼠胰肾联合移植模型采用了改进的血管吻合技术,大大减少了手术并发症,并缩短了手术时间。采用的胰液肠内引流术式更合乎生理,减少了并发症的发生。利用第一部分和第二部分的工作基础,制备同种抗原特异性T细胞疫苗,接种给胰肾联合移植受体大鼠,通过不同实验分组于不同时点进行免疫接种,以观察特异性免疫耐受的建立及排斥发生情况。我们用T细胞疫苗对同种异基因胰肾联合移植的排斥反应进行了治疗,实验中设立了免疫抑制剂FK506作为对照。研究结果表明,T细胞疫苗可部分抑制CD4和CD8细胞的比值增高,能够降低移植排斥反应时血清中白细胞介素-2和肿瘤坏死因子水平,并且能够在RNA水平和蛋白水平下调选择素的表达。根据现有的实验结果判断,我们认为T细胞疫苗对于胰肾联合移植的排斥反应具有一定的抑制效应,因而有一定的临床应用价值。综上所述,我们成功制作了T细胞疫苗,经验证明具有一定的诱导免疫耐受的作用,建立了稳定可靠的大鼠胰肾联合移植动物模型,并利用制作的T细胞疫苗观察了其对大鼠胰肾联合移植过程中免疫耐受的诱导,结果表明T细胞疫苗能够抑制排斥反应的发生,希望今后在临床胰肾联合移植的过程中发挥一定的作用,进一步提高移植成功率。
[Abstract]:Organ transplantation is an important means for the treatment of many end-stage diseases, but rejection caused by organ transplantation is the main cause of its success. In order to reduce the incidence of rejection and improve the success rate of organ transplantation, patients need to use immunosuppressive agents throughout the life. However, the long-term use of immunosuppressants is the exception. It can cause the toxic and side effects of the immunosuppressant itself (such as rising hypertension, liver and kidney toxicity and other adverse reactions), and can also cause severe infection, malignant tumor, and thus affect the success rate of organ transplantation and the long-term survival rate of the recipient. Maintaining the ability to respond to other antigens is an ideal target for organ transplantation.
After the allograft of the same organ, the rejection of the allograft is produced mainly because the receptor immune system identifies the incompatible donor MHC antigen, triggers the immune response and triggers the result of the allograft rejection, in which the allogenic T cells play an important role in the rejection reaction, and they are inactivated as a T cell vaccine. The induction of antigen specific immune tolerance or immune hypo response.T cell vaccine (T Cell Vaccine, TCV) is a pathogenic T cell inactivated by chemical or physical methods. It loses its pathogenicity but maintains the immune characteristics of activated pathogenetic T cells. T cells of allograft rejection are prepared to form TCV. Immune animals can induce immune responses to pathogenic T cells after immune animals. They can eliminate or mitigate the pathogenicity or reaction ability of these cells, which represent the prevention and control of autoimmune diseases and the prolongation of survival of the allograft material.
Diabetic nephropathy is the most serious and fatal complication in diabetes. The total incidence of diabetic nephropathy in diabetic patients in China is 47.7%. especially in type I diabetes. If the course of type I diabetes is over 20 years, 10% will have kidney disease, and eventually it will develop into final stage nephropathy, uremia. Simultaneous combined pancreas kidney transplantation (simultaneous). Pancreas-kidney transplantation, SPK) is the only effective way for clinical treatment of diabetic nephropathy..1966, University of Minnesota, Kelly and Lillehei, successfully implemented the first joint pancreas kidney transplantation. The total number of pancreas transplants is constantly being recorded according to the United States organ distribution network (UNOS) and the International Pancreas Transplantation Registry (IPTR) record. The increase from December 1966 to October 2002, the total number of 18843 cases, the vast majority of them in the United States (13951 cases), from 1987 to 2002, UNOS reported 13330 cases of pancreas transplantation, 78% of the use of SPK. so far, SPK is recognized as the treatment of I diabetes mellitus (IDDM) the most ideal method of uremia since 80s, since 80s, with With the development of transplantation technology and the clinical application of new immunosuppressive agents, the safety and success rate of the operation has been continuously improved.
The purpose of this study is to investigate whether T cell vaccine can induce immune tolerance in combined pancreas and kidney transplantation, and provide experimental basis and theoretical reference for the success rate of combined pancreas kidney transplantation. First, T cell vaccine was prepared and its function was identified. The experiment selected inbred AS rats, SD rats and Wistar rats, and AS rats and SD The spleen cells of Wistar rats were immunized with spleen cells isolated from the rats. The spleen cells of Wistar rats were isolated and activated by ConA in vitro. They were activated to express a variety of activated receptors, thus enhancing their antigenicity, and then treated with mitomycin and inactivated to make T cell vaccine. The experiment showed that the spleen cells were able to induce the spleen cells to Con after immunization with the two T cell vaccine. The proliferation ability induced by A was low, indicating that the T cell vaccine was immune to a certain degree of non specific immune function of T cells, partly explaining the non specificity of the antigen of the T cell vaccine, and the T cell vaccine could induce immune tolerance against the same antigen, which was both specific and non specific; T cell pestilence Seedlings can induce apoptosis of peripheral blood T cells and reduce CD4/CD8 ratio.
The establishment of a stable and reliable model of pancreas kidney transplantation is of great significance for the study of the physiological and immune aspects of the combined pancreas kidney transplantation. The rat model is stable and feasible because of the constant genotypes. It is proved that the model is stable and feasible. Therefore, the rat is the ideal pancreas kidney. We established the model of rat pancreas kidney transplantation on the basis of STZ induced diabetic rats. The biochemical indexes and pathological results were normal after the operation. The success rate of the operation was 76%, which laid the foundation for the immune tolerance test in the future. The model of rat pancreas kidney transplantation in rats was improved. Vascular anastomosis technique greatly reduces the complications of the operation and reduces the time of the operation. The use of the enteric drainage of the pancreatic fluid is more physiologic and reduces the incidence of complications.
Using the work basis of the first and second parts, the specific antigen specific T cell vaccine was prepared and inoculated to the pancreas kidney transplantation recipient rats. The immunization was carried out at different points in different experiments to observe the establishment of specific immune tolerance and the condition of rejection. We used T cell vaccine to combine the allogeneic pancreas kidney. The allograft rejection was treated. In the experiment, the immunosuppressant FK506 was established as a control. The results showed that T cell vaccine could partly inhibit the increase of the ratio of CD4 and CD8 cells, and could reduce the level of serum interleukin -2 and tumor necrosis factor in the serum of transplant rejection, and could be at the level of RNA and protein. The expression of the selectin. According to the existing experimental results, we think that T cell vaccine has certain inhibitory effects on the rejection of the combined pancreas and kidney transplantation, so it has certain clinical value.
To sum up, we successfully produced the T cell vaccine. Experience proved that it has a certain role in inducing immune tolerance. A stable and reliable rat model of pancreas kidney transplantation was established, and the T cell vaccine was used to observe the induction of immune tolerance in the process of combined pancreas kidney transplantation in rats. The results showed that the T cell vaccine could be suppressed. The occurrence of rejection is expected to play a certain role in the future process of simultaneous pancreas kidney transplantation and further improve the success rate of transplantation.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R617;R392.4

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