T细胞疫苗对BXSB鼠免疫系统影响的实验研究

发布时间：2018-06-22 18:44

本文选题：T淋巴细胞疫苗 + BXSB鼠　；参考：《南方医科大学》2008年硕士论文

【摘要】： 研究背景系统性红斑狼疮(systemic lupus erythematosus，SLE)是一种常见的自身免疫性疾病，累及全身多系统和器官，发病机制复杂，临床变化多端，继发感染发生率高，诊治困难，是一种严重危害人类健康的疾病。其广泛分布于世界各地，全世界的患病率大约是70／10万-100／10万。如果以全国13亿人口计算，我国的SLE患者已达112-160万之多，且多见于年轻女性。 SLE的发病与多种因素有关，一般认为是遗传素质、性激素、感染、药物和环境等相互间错综复杂的作用引起免疫调节功能紊乱，导致SLE的发生、持续和不易缓解。由于病因尚未明确，为其根治带来困难。近20年来，随着现代医学的迅速发展，早期诊断的手段增多和治疗水平的提高，SLE的10年生存率已达92％，预后明显改善。然而糖皮质激素结合免疫抑制剂药物仍然是目前治疗SLE的首选药物。一般病例采用大剂量糖皮质激素治疗后小剂量维持，对急性暴发性危重SLE患者用激素冲击疗法，效果不理想时联合使用细胞毒药物(主要是环磷酰胺和硫唑嘌呤)。采用这种常规治疗方法可使大部分患者病情稳定，但用药量大、时间长，药物的副作用不容忽视，如诱发或加重感染、糖尿病、消化道溃疡、穿孔或消化道出血，长期应用可引起高血压和动脉粥样硬化、骨折及骨缺血性坏死、精神异常、柯兴综合征、痤疮、多毛等。60％以上患者最后死于激素的副作用和／或并发症。严重影响患者的生存期和生活质量。因此寻求SLE新的治疗方法一直是国内外研究的热点。大剂量免疫球蛋白冲击疗法、血浆置换疗法等，虽能使病情得到暂时缓解，同样由于未能针对病因治疗，且花费高，不能广泛应用。新型免疫抑制剂包括B细胞治疗、抗CD20抗单克隆抗体治疗、T细胞治疗、抗细胞因子治疗、补体治疗等大部分处于动物实验阶段，其确切治疗效果尚须大量临床对照试验证实。近年来造血干细胞移植治疗取得了令人鼓舞的疗效，但存在高风险和技术条件要求高，部分患者在动员阶段不能耐受，价格昂贵，较难推广。由于SLE是以T和B淋巴细胞功能异常为主要特点的自身免疫性疾病，自身反应性T淋巴细胞的活化及由此介导的自身免疫反应是本病发生的中心环节。研究发现SLE发病过程中自身抗原应答性辅助性T细胞(help T cell,Th)增加。从狼疮鼠克隆的T细胞系能向B细胞提供协助，使之产生IgG型抗DNA抗体。50％的这些T细胞克隆中能识别核小体中的T细胞抗原表位。在人及鼠SLE的Th细胞克隆中，识别核小体抗原表位的TCRα链能结合不同TCRβ链，形成TCRαβ二肽链，识别多种T细胞抗原表位，协助多种B细胞克隆，产生多种自身抗体。在SLE迁延过程中，T细胞反复受自身抗原激活而增值，从而使突变率增加，其突变的T细胞能促使自身应答B细胞产生抗DNA抗体。这些抗体与相应自身抗原结合，形成免疫复合物，沉积于肾小球基底膜、肝脏、中枢神经系统、小血管壁等多种器官及组织，活化补体、多型核白细胞及血小板，致局部炎症及小血管炎，使血管阻塞，而显示为多种临床病症。如能通过T细胞免疫抑制自身反应性T淋巴细胞的活化，控制SLE的发展，可能是治疗SLE的理想方法。 1981年Bennun等对Lewis大鼠实验性自身免疫性脑脊髓炎(Experimentalautoimmune encephalomyelitis，EAE)的研究时，从发病鼠淋巴组织分离到一株髓鞘碱性蛋白(Myelin basic protein，MBP)特异性的自身反应性T细胞。该细胞输给正常大鼠可诱发EAE，但若在转输前用放射线照射或用丝裂霉素处理的方法灭火该细胞，则受鼠反而获得对MBP免疫诱导的EAE的抵抗力。此时第一次提出T细胞疫苗(T cell vaccination)的概念，即用灭活或低于致病剂量的自身反应性T细胞或其T细胞受体(T cell receptor,TCR)多肽作为疫苗接种，诱导或触发调节网络产生特异性抑制作用，从而清除自身反应性T细胞，达到防治自身免疫性疾病的作用。近年来T细胞疫苗与自身免疫性疾病的关系得到广泛研究，T细胞疫苗在自身免疫性疾病中的应用也得到广泛关注。在多发性硬化、类风湿性关节炎、器官、组织移植术后，自身免疫性甲状腺炎，免疫性心肌炎等疾病的临床研究中均取得了令人满意的结果，为治疗难治性自身免疫性疾病的治疗提供了新思路。本课题即运用T细胞疫苗免疫的方法对SLE动物模型BXSB鼠进行试验性治疗，分析有关实验室指标变化，旨在为T细胞疫苗临床治疗系统性红斑狼疮提供实验依据。目的 1、用BXSB鼠脾细胞制备T细胞疫苗。 2、观察T细胞疫苗免疫前后BXSB鼠24小时尿蛋白定量、血清ANA抗体、ds-DNA抗体水平的动态变化，从而探讨T细胞疫苗免疫在一定时间内对BXSB鼠病情的影响。 3、观察T细胞疫苗免疫前后BXSB鼠血清IL-18水平动态变化，从而探讨T细胞疫苗免疫在一定时间内对BXSB鼠免疫状态影响的有关机制。方法 1、实验动物雄性BXSB鼠12只。随机分为2组。A组为2只，用来制备T细胞疫苗。B组10只，实验组，用来观察T细胞疫苗对BXSB鼠的影响。 2、BXSB鼠脾脏淋巴细胞分离：无菌条件下将BXSB鼠脾脏取下，制成脾细胞悬液，加入淋巴细胞分离液，离心，取淋巴细胞，用Hanks液清洗2次，最后用RPMI1640培养液调整细胞浓度到1×10~8／ml。台盼兰染法鉴定活细胞数95％。 3、T细胞疫苗的制备用完全培养液调整细胞至2×10~5／ml，加伴刀豆球蛋白A(ConA)混合后移置5％CO_237℃孵箱中培养48h后收集细胞，然后加入丝裂霉素C(mitomycin C)处理细胞，PBS洗涤后，用RPMI1640培养液调整细胞至1×10~8／ml备用。 4、T细胞疫苗皮下免疫：取1×10~7细胞皮下免疫BXSB鼠，每周1次，连续3次，并于免疫前和首次免疫后第1w、2w、4w观察有关指标变化。 5、观察指标及检测方法：于免疫前和首次免疫后第1w、2w、4w将BXSB鼠断尾取血，分离血清，ELISA法测血清ANA抗体、ds-DNA抗体和IL-18水平。用代谢笼收集BXSB鼠24小时尿量，考马斯量蓝染色法检测24小时尿蛋白定量。结果 1、T细胞疫苗免疫前后BXSB鼠24小时尿蛋白定量差异有显著意义(F=22．177，，P=0．000)，呈递减趋势。免疫后1w、2w、4w均低于免疫前。免疫后1w与免疫前无显著差异(P=0．519)，而免疫后2w和4w水平显著低于免疫前和免疫后1w(P0．05)，且免疫后4w水平低于免疫后2w(P=0．037)。 2、T细胞疫苗免疫前后BXSB鼠血清ANA抗体水平差异有显著意义(F=15．757，P=0．000)，呈下降趋势。免疫后1w、2w、4w均低于免疫前。免疫后1w与免疫前无显著差异(P=0．317)，而免疫后2w和4w水平明显低于免疫前和免疫后1w(P0．05)，但两者之间无显著差异(P=0．072)。 3、T细胞疫苗免疫前后BXSB鼠血清ds-DNA抗体水平有显著差异(F=16．735，P=0．000)，呈下降趋势。免疫后1w、2w、4w均低于免疫前。免疫后1w与免疫前无显著差异(P=0．569)，而免疫后2w和4w水平显著低于免疫前和免疫后1w(P0．05)，但两者之间无显著差异(P=0．056)。 4、T细胞疫苗免疫前后BXSB鼠血清IL-18水平差异有显著性意义(F=34．583，P=0．000)，呈递减趋势。免疫后1w、2w、4w均低于免疫前。免疫后1w与免疫前无显著差异(P=0．130)，而免疫后2w和4w水平显著低于免疫前和免疫后1w(P0．05)，免疫后4w水平继续降低，显著低于免疫后2w(P=0．006)。 5、首次免疫后4w，其中1只BXSB鼠死亡。结论 1、T细胞疫苗免疫BXSB鼠后2周其24小时尿蛋白定量、自身抗体水平即开始下降，免疫后4w仍维持较低水平。从而推测T细胞疫苗在一定时间内可抑制BXSB鼠B细胞活化，减少自身抗体的产生，减轻肾脏损伤，延缓病情发展。 2、T细胞疫苗免疫BXSB鼠后2周其血清IL-18水平开始显著降低，免疫后4w继续降低。提示T细胞疫苗在调节免疫细胞及细胞因子的相互联系中有重要作用。 3、T细胞疫苗治疗BXSB鼠有一定的有效性和安全性。为T细胞疫苗的临床应用提供了实验依据。
[Abstract]:Research background
Systemic lupus erythematosus (systemic lupus erythematosus, SLE) is a common autoimmune disease involving multiple systems and organs of the whole body. The pathogenesis is complex, clinical variable, high incidence of secondary infection and difficulty in diagnosis and treatment. It is a disease which is seriously harmful to human health. It is widely distributed all over the world and the prevalence rate in the world. It is about 70 / 100 thousand -100 / 100 thousand. If we calculate 1 billion 300 million people in China, the number of SLE patients in China is up to 112-160, and most of them are in young women.
The incidence of SLE is related to a variety of factors. It is generally believed that the complex effects of sex hormones, infections, drugs and the environment cause the disorder of immune regulation, which lead to the occurrence of SLE, which is not clear and difficult to cure. In the past 20 years, with the rapid development of modern medicine The 10 year survival rate of SLE has reached 92%, and the prognosis is obviously improved. However, glucocorticoid combined with immunosuppressive drugs is still the first choice for the treatment of SLE. The general case adopts a small dose of glucocorticoid after the treatment of large dose of glucocorticoid, and the use of hormone for acute critical SLE patients. Combined use of cytotoxic drugs (mainly cyclophosphamide and azathioprine) when the effect is not satisfactory. The use of this routine treatment can make most patients stable, but with a large amount of medicine and long time, the side effects of the drug can not be ignored, such as induced or aggravated infection, diabetes, peptic ulcer, perforation or gastrointestinal bleeding. Period application can cause hypertension and atherosclerosis, fracture and osteonecrosis of bone, psychosis, Cushing's syndrome, acne, and hairy, and more than.60% in the final death of hormone side effects and / or complications. It seriously affects the life and quality of life of the patients.
Therefore, the search for new treatment methods of SLE has always been a hot spot at home and abroad. Large dose immunoglobulin shock therapy, plasma exchange therapy, etc., can make the disease be temporarily relieved, also because it can not be used for etiological treatment, and can not be widely used. The new immunosuppressive agents include B cell therapy, anti CD20 anti monoclonal antibody treatment. The treatment, T cell therapy, anti cytokine therapy, and complement therapy are mostly in the experimental stage of animal experiment. The exact therapeutic effects of the treatment are still confirmed by a large number of clinical trials. In recent years, the treatment of hematopoietic stem cells has achieved encouraging results, but high risk and technical requirements are high, and some patients are not tolerated at the mobilization stage. It is very expensive and difficult to promote.
Since SLE is an autoimmune disease characterized by T and B lymphocyte dysfunction, the activation of its own reactive T lymphocyte and its mediated autoimmune reaction are the central link of this disease. The study found that the autoantigen responsive T fine cell (help T cell, Th) increased during the pathogenesis of SLE. From the clone of lupus mice The T cell line can provide assistance to B cells to identify the T cell antigen epitopes in the nucleosomes of these T cell clones that produce IgG anti DNA antibody.50%. In the Th cell clone of human and mouse SLE, the TCR alpha chain identifying the nucleosome epitopes can combine with different TCR beta chains to form a TCR alpha beta two peptide chain and identify a variety of antigen epitopes. A variety of B cell clones contribute to the production of a variety of autoantibodies. In the process of SLE migration, T cells are repeatedly activated by their own antigens, which increase the mutation rate, and the mutant T cells can induce their own response to B cells to produce anti DNA antibodies. These antibodies combine with the corresponding autoantigens and form immune complexes, deposited in the glomerular basement membrane, and the liver A variety of organs and tissues such as dirty, central nervous system, small vessel wall, activating complement, polymorphonuclear leukocytes and platelets, causing local inflammation and small vasculitis, causing vascular obstruction, and showing a variety of clinical symptoms. It is possible to control the development of SLE by inhibiting the activation of the T lymphocyte and controlling the development of SLE. It may be ideal for the treatment of SLE. Method.
In the study of the experimental autoimmune encephalomyelitis (Experimentalautoimmune encephalomyelitis, EAE) of Lewis rats in 1981, Bennun isolated from the lymphoid tissue of the infected rat to a specific self reactive T fine cell of the myelin alkaline protein (Myelin basic protein, MBP). This cell could induce EAE, but if it was transferred to the normal rats When the cells were put out by radiation or treated with mitomycin, the mice received the resistance to EAE induced by MBP. At this time, the concept of the T cell vaccine (T cell vaccination) was first proposed, that is, the self reactive T fine cell or its T cell receptor (T cell receptor, TCR) peptides with inactivated or lower than the pathogenic dose. Vaccination, inducing or triggering the regulatory network to produce specific inhibitory effects, thus scavenging the self reactive T cells and achieving the role of preventing autoimmune diseases. In recent years, the relationship between T cell vaccine and autoimmune diseases has been widely studied. The application of T cell vaccine in autoimmune diseases has also been widely concerned. The clinical studies of multiple sclerosis, rheumatoid arthritis, organ and tissue transplantation, autoimmune thyroiditis, immune myocarditis and other diseases have obtained satisfactory results and provide new ideas for the treatment of refractory autoimmune diseases. This topic is the use of T cell vaccine immunization to the SLE animal model. BXSB rats were treated with experimental treatment, and the changes of laboratory indicators were analyzed. The aim was to provide experimental evidence for the treatment of systemic lupus erythematosus with T cell vaccine.
objective
1, T cell vaccine was prepared by BXSB mouse splenocytes.
2, to observe the 24 hour urine protein quantitative, serum ANA antibody and ds-DNA antibody level of BXSB mice before and after immunization of T cell vaccine, and to explore the effect of T cell vaccine immunization on the condition of BXSB mice in a certain time.
3, the dynamic changes of IL-18 level in serum of BXSB mice were observed before and after the immunization of T cell vaccine, and the mechanism of the effect of T cell vaccine immunization on the immune status of BXSB mice in a certain period of time was discussed.
Method
1, experimental animals
A total of 12 male BXSB rats were randomly divided into 2 groups, 2 in group.A, which were used to prepare 10 T cell vaccine group.B, and the experimental group was used to observe the effect of T cell vaccine on BXSB rats.
2, splenic lymphocyte separation in BXSB rats:
Under the aseptic condition, the spleen of BXSB rat was taken down to make the splenic cell suspension, add the lymphocyte separation liquid, centrifuge, take the lymphocyte and clean the cell with Hanks solution for 2 times. Finally, the cell concentration was adjusted by the RPMI1640 culture solution to the 1 x 10~8 / ml. table and the number of living cells was identified by the trypan blue staining method.
3, preparation of T cell vaccine
The cells were adjusted to 2 x 10~5 / ml with complete culture medium and mixed with concanavin A (ConA) and then transferred to 5%CO_237 centigrade incubator to collect the cells. Then the cells were treated with mitomycin C (mitomycin C). After the PBS was washed, the cells were adjusted to 1 * 10~ 8 / reserve by the RPMI1640 culture solution.
4, subcutaneous immunization of T cell vaccine:
1 BXSB 10~7 cells were subcutaneously immunized with BXSB mice 1 times a week for 3 consecutive times, and the changes of the indexes were observed before and after 1W, 2W and 4W after the first immunization.
5, observation index and detection method:
The blood was taken from the tail of BXSB rats before and after the first immunization, 1W, 2W, and 4W. Serum ANA, ds-DNA antibody and IL-18 level were measured by ELISA. The urine volume of BXSB rats was collected for 24 hours by metabolic cage, and the urine protein was measured by Kaumas blue staining method for 24 hours.
Result
1, before and after immunization of T cell vaccine, the quantitative difference of 24 hours urine protein in 24 hours was significant (F=22.177, P=0.000), and decreased. 1W, 2W, 4W were lower than before immunization. There was no significant difference between 1W and immune before immunization (P=0.519), while the 2W and 4W levels after immunization were significantly lower than those before and after immunization, and the level of immune 1W was lower than that of immunization. After immunization, 2W (P=0.037).
2, there was a significant difference in the level of ANA antibody in serum of BXSB mice before and after immunization with T cell vaccine (F=15.757, P=0.000), and decreased. 1W, 2W, 4W were lower than before immunization. There was no significant difference between immune 1W and pre immunization (P=0.317), but the 2W and 4W levels were significantly lower than those before and after immunization, but there was no significant difference between them. Difference (P=0.072).
3, the level of ds-DNA antibody in serum of BXSB mice before and after immunization with T cell vaccine was significantly different (F=16.735, P=0.000), and decreased. 1W, 2W, 4W were lower than before immunization. There was no significant difference between immune 1W and pre immunization (P=0.569), but 2W and 4W levels were significantly lower than those before and after immunization, but there was no significant difference between the two groups. P=0.056).
4, there was a significant difference in the serum IL-18 level of BXSB mice before and after immunization with T cell vaccine (F=34.583, P=0.000), which decreased. 1W, 2W, 4W were all lower than before immunization. There was no significant difference between immune 1W and pre immunization (P=0.130), while the 2W and 4W levels after immunization were significantly lower than those before and after immunization, and the level of immune after immunization continued to decrease. It was significantly lower than 2W (P=0.006) after immunization.
5, after the first immunization of 4W, 1 of the BXSB mice died.
conclusion
1, T Cell Vaccine Immunized BXSB mice for 24 hours after 2 weeks of urine protein quantitative, the level of autoantibody began to decrease, and the 4W remained low after immunization. Thus, it was suggested that T cell vaccine could inhibit the activation of B cells in BXSB mice, reduce the production of autoantibodies, reduce the injury of kidney and postpone the development of the disease.
2, the serum IL-18 level of BXSB mice was significantly reduced after immunization with T cell vaccine for 2 weeks, and the 4W continued to decrease after immunization. It suggested that the T cell vaccine played an important role in regulating the interaction of immune cells and cytokines.
3, T cell vaccine is effective and safe in the treatment of BXSB mice. It provides experimental evidence for the clinical application of T cell vaccine.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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