Unlicensed NK细胞亚群与狼疮小鼠发病机制的研究

发布时间：2018-05-23 08:10

  本文选题：系统性红斑狼疮 + 狼疮性肾炎　； 参考：《郑州大学》2017年硕士论文

【摘要】：背景系统性红斑狼疮(systemic lupus erythematosus,SLE)是一种最常见的累及机体多系统、多器官的自身免疫性疾病,狼疮性肾炎(Lupus nephritis,LN)是其最常见的并发症。有关SLE的发病机制十分复杂,研究显示遗传、免疫紊乱、激素、感染和理化环境等均与SLE及LN的发生发展密切相关。基于SLE的特点之一产生以ANA、抗ds-DNA抗体为主的多种自身抗体及各型LN均有免疫复合物沉积,使得参与适应性免疫反应的T、B淋巴细胞在SLE的发病过程中最早受到关注并得到阐明,而有关NK细胞等固有免疫反应细胞研究较少。自然杀伤细胞(natural killer cell,NK细胞)是固有免疫反应中的主要细胞,可通过细胞-细胞之间的相互作用和分泌多种细胞因子和趋化因子来调节适应性免疫反应中的T、B淋巴细胞,被认为是连接固有免疫反应和适应性免疫反应的“桥梁”。越来越多的研究显示NK细胞与器官特异性自身免疫性疾病有相关性。依据Yokoyama等提出的“licensing”学说,可将NK细胞分为“licensed”和“unlicensed”两种亚群。有研究发现在骨髓移植急性排斥期,“unlicensed”NK细胞可促进同种异体移植物细胞在宿主体内的存活。本研究旨在使用Pristane(降植烷)诱导狼疮肾模型,观察“unlicensed”NK细胞在狼疮鼠发病过程中的作用。目的采用Pristane(降植烷)诱导狼疮小鼠模型,探讨unlicensed NK细胞亚群在SLE及狼疮性肾炎发病过程中的作用,为SLE的发病机制研究提供新思路。方法1.建立Pristane(降植烷)诱导的狼疮小鼠模型:将18只雌性、SPF级、9-10周龄的BALB/c小鼠随机分为两组:(1)诱导组(9只):腹腔注射0.5ml Pristane;(2)对照组(9只):腹腔注射0.5ml PBS。观察7个月,观察指标有小鼠外观变化、体重、尿蛋白、自身抗体(ANA、抗ds-DNA抗体、抗nRNP/Sm抗体和抗ss-DNA抗体)水平、肾脏病理(普通光镜和免疫荧光)等。比较两组上述指标间的变化。2.研究unlicensed NK细胞亚群与Pristane诱导的狼疮小鼠发病机制的关系。将27只雌性、SPF级、9-10周龄的BALB/c小鼠随机分为3组:(1)“U-NK”组:9只,实验开始前腹腔注射抗Ly49G2+的单克隆抗体4D11;(2)“non-NK”组:实验开始前腹腔注射Anti-asialoGM1;(3)“model”组:9只。实验开始当天,3组小鼠均一次性腹腔注射0.5ml Pristane。实验观察时间为7个月,指标有:小鼠外观变化、体重及尿蛋白情况,自身抗体(ANA、抗ds-DNA抗体、抗nRNP/Sm抗体和抗ss-DNA抗体)水平、肾脏病理改变(普通光镜和免疫荧光)以及流式细胞仪检测NK细胞亚群数量变化。结果1.诱导组部分小鼠出表现了红斑和脱毛等狼疮样表现,尿蛋白多为+1~+2,血清中出现四种自身抗体(ANA、抗ds-DNA抗体、抗nRNP/Sm抗体和抗ss-DNA抗体),肾脏组织病理光镜下示部分肾小球体积增大,系膜细胞及内皮细胞增生,肾间质伴炎症细胞浸润;免疫荧光提示IgG和IgM延肾小球毛细管襻和系膜区沉积。对照组无上述变化。2.实验观察结束后,3组间小鼠存活情况及外观变化无差异;“U-NK”组尿蛋白、自身抗体水平及肾脏病理改变均较“non-NK”和“model”组明显好转;而相对于“model”组,“non-NK”组抗nRNP/Sm抗体、抗ss-DNA抗体水平及IgG、IgM荧光强度均升高,情况较差。流式检测小鼠脾脏NK细胞数量显示,“U-NK”组unlicensed NK细胞数量较“model”组增多。结论Pristane(降植烷)可以诱导雌性BALB/c小鼠形成狼疮模型;unlicensed NK细胞在SLE及狼疮性肾炎的发生发展过程中有抑制作用,为SLE的发病机制提供了新思路。
[Abstract]:Background systemic lupus erythematosus (systemic lupus erythematosus, SLE) is one of the most common autoimmune diseases involving multiple systems and multiple organs. Lupus nephritis (Lupus nephritis, LN) is the most common complication. The pathogenesis of SLE is very complex. The study shows heredity, immune disorder, hormone, infection, and physicochemical environment. All of them are closely related to the occurrence and development of SLE and LN. One of the characteristics of SLE based on the production of ANA, anti ds-DNA antibodies and various types of LN have immune complex deposition, making T, B lymphocytes involved in adaptive immune response to the earliest attention and clarified in the pathogenesis of SLE, and the inherent NK cells and other inherent There are few immunoreactive cells. Natural killer cells (natural killer cell, NK cells) are the main cells in the innate immune response. They can regulate the T, B lymphocytes in the adaptive immune response through the interaction between cells and cells and secreting a variety of cytokines and chemokines, which are considered to be linked to the inherent immune response and appropriate to the adaptive immune response. A growing number of studies have shown that NK cells are associated with organ specific autoimmune diseases. According to the "licensing" theory proposed by Yokoyama, NK cells can be divided into two subgroups of "licensed" and "unlicensed". Studies have found that "unlicensed" NK in the acute rejection period of bone marrow transplantation. Cells can promote the survival of allograft cells in the host. This study aims to use Pristane (descending) to induce lupus kidney model and observe the role of "unlicensed" NK cells in the pathogenesis of lupus mice. Objective to induce lupus mouse model with Pristane (descending alkane) and to explore the unlicensed NK cell subgroup in SLE and lupus. The role of the pathogenesis of sexual nephritis provides new ideas for the study of the pathogenesis of SLE. Method 1. a mouse model of lupus induced by Pristane was established: 18 female, SPF, and 9-10 week old BALB/c mice were randomly divided into two groups: (1) the induction group (9 rats): intraperitoneal injection of 0.5ml Pristane; (2) the control group (9): intraperitoneal injection 0.5ml PBS. observation 7 The changes of appearance, weight, urine protein, autoantibodies (ANA, anti ds-DNA, nRNP/Sm and ss-DNA), renal pathology (common light and immunofluorescence) were observed, and the changes between the two groups were compared with the.2. study on the pathogenesis of unlicensed NK cell subsets and Pristane induced lupus mice. 27 female, SPF, 9-10 weeks old BALB/c mice were randomly divided into 3 groups: (1) "U-NK" group: 9, before the experiment began to intraperitoneal injection of anti Ly49G2+ monoclonal antibody 4D11; (2) "non-NK" group: before the experiment began to intraperitoneal injection of Anti-asialoGM1; (3) "model" group: 9. The same day, 3 groups of mice were intraperitoneally injected 0.5ml Prista. Ne. experiment was observed for 7 months. The indexes were: changes in appearance of mice, body weight and urine protein, the level of autoantibodies (ANA, anti ds-DNA, anti nRNP/Sm and anti ss-DNA), renal pathological changes (common light and immunofluorescence) and the quantitative change of NK cell subsets by flow cytometry. Results the 1. induced mice were out of the table. Erythema and hair removal, such as lupus like manifestations, urinary protein is +1~+2, there are four kinds of autoantibodies (ANA, anti ds-DNA, anti nRNP/Sm, and anti ss-DNA) in serum. The renal tissue pathological light microscopy shows that partial glomerular volume increases, mesangial cells and endothelial cells increase, renal interstitium with inflammatory cells infiltration; immunofluorescence suggests IgG and I GM delayed glomerular capillary loop and mesangial region deposition. There was no difference in survival and appearance between the 3 groups without the above changes in the control group. The urine protein, autoantibody level and renal pathological changes in the "U-NK" group were significantly better than those in the "non-NK" and "model" group, while the "non-NK" group was resistant to the "model" group. NRNP/Sm antibody, anti ss-DNA antibody level and IgG, IgM fluorescence intensity increased, and the number of NK cells in splenic NK cells in flow test mice showed that the number of unlicensed NK cells in "U-NK" group was higher than that of the "model" group. Conclusion Pristane (descending alkanes) can induce female BALB/c mice to form lupus model. Glomerulonephritis has inhibitory effect in the course of occurrence and development, and provides a new idea for the pathogenesis of SLE.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R593.241
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本文编号：1923951