人源化CD52单克隆抗体对食蟹猴淋巴细胞作用的研究

发布时间：2018-01-17 16:04

本文关键词：人源化CD52单克隆抗体对食蟹猴淋巴细胞作用的研究　出处：《南京大学》2011年博士论文　论文类型：学位论文

【摘要】：人源化CD52单克隆抗体(Campath-1H)对表达CD52抗原的T、B淋巴细胞、单核细胞、NK细胞等具有强大的清除作用,最初用于治疗慢性淋巴细胞白血病、多发性硬化等疾病,近年来常用于肾移植、肝移植、小肠移植等实体器官移植中诱导产生免疫耐受。虽然Campath-1H应用于器官移植已十余年,但缺少适宜的动物模型评价其在器官移植中的安全性和有效性,因此限制了其在器官移植中的进一步应用。Campath-1H只与非人灵长类动物中的某些种属具有交叉反应,而不能识别啮齿类动物细胞上的抗原。非人灵长类动物在亲缘关系上与人最接近,是研究人类疾病的理想动物模型。在临床移植中应用Campath-1H诱导方案后,可清除移植病人外周血中99%以上的淋巴细胞,并维持很长一段时间低淋巴细胞水平状态。肠道中的淋巴细胞数量比外周组织中的多,肠道是全身最大的免疫器官。目前国内外对Campath-1H的研究多集中于对外周血淋巴细胞的清除作用,尚缺乏对肠道淋巴细胞作用的研究。本研究中,我们采用食蟹猴作为动物模型,给予Campath-1H处理后,观察给药后不同时间食蟹猴外周血、脾脏、淋巴结淋巴细胞的变化,肠道上皮内及固有层淋巴细胞数量及亚群的变化,以及肠道粘膜地址素细胞粘附分子与淋巴细胞归巢受体的相互关系。研究Campath-1H对淋巴细胞的清除作用及归巢机制,为临床小肠移植中更好的应用Campath-1H提供实验依据。第一部分动物模型的建立及人源化CD52单克隆抗体对食蟹猴外周血、脾脏、淋巴结淋巴细胞的作用目的:人源化CD52单克隆抗体运用于器官移植已十余年,但缺少适宜的动物模型评价其在器官移植中的安全性和有效性,限制了其在器官移植中的进一步应用。本实验首先建立人源化CD52单克隆抗体诱导食蟹猴动物模型,并观察其对食蟹猴外周血、脾脏、淋巴结淋巴细胞的作用。方法:雄性食蟹猴,3-5岁龄,体重3-5.5 kg,使用流式细胞仪筛选红细胞表面不表达CD52抗原的食蟹猴,将筛选出的食蟹猴随机分为实验组和对照组,实验组按3 mg/kg给予静脉滴注Campath-1H,对照组给予等量生理盐水。分别于给药后9、14、35、56天处死实验组动物取材,给药组中的第56天处死的3只食蟹猴分别于第3、6、9、14、21、35、56天取外周血进行淋巴细胞计数及流式细胞仪分析淋巴细胞亚群,观察给药前后不同时间血中淋巴细胞数量变化情况。数据采用SPSS 13.0统计软件作单因素方差分析(ANOVA,LSD),两组间比较采用t检验,P0.05为差异有显著性统计学意义。结果:从40只食蟹猴中筛选出15只红细胞表面不表达CD52抗原的食蟹猴,筛选出的与未被筛选出的食蟹猴在血液生理和生化指标方面无显著性差异。给药后食蟹猴血中单核细胞、淋巴细胞呈现出快速被清除及恢复的过程,单核细胞在给药后第6天降至最低点,T、B淋巴细胞在给药后第9天降至最低点。单核细胞在给药后第14天恢复至给药前水平,CD20+B淋巴细胞、CD8+T淋巴细胞、CD4+T淋巴细胞分别在给药后21、35、56天恢复至给药前水平。给药后第9天,脾脏淋巴细胞、淋巴结淋巴细胞减少,给药后第35天,淋巴细胞数量逐渐增多,给药后第56天,脾脏、淋巴结组织形态恢复至给药前水平。结论:成功建立人源化CD52单克隆抗体诱导食蟹猴动物模型。应用Campath-1H后食蟹猴外周血中淋巴细胞清除效果强大,并逐渐恢复,脾脏、淋巴结淋巴细胞均呈现清除与恢复的过程。本实验模型可用于人源化CD52单克隆抗体临床前移植模型的研究。第二部分人源化CD52单克隆抗体对食蟹猴肠道淋巴细胞的作用目的:观察应用人源化CD52单克隆抗体后,食蟹猴肠道上皮内及固有层淋巴细胞的变化,评价该抗体对食蟹猴肠道淋巴细胞的作用,以及食蟹猴肠道粘膜地址素细胞粘附分子与淋巴细胞归巢受体的变化,研究两者相互关系。方法:雄性食蟹猴,3-5岁龄,体重3-5.5 kg,红细胞表面不表达CD52抗原15只,随机分为实验组和对照组,实验组按3mg/kg给予静脉滴注Campath-1H,对照组给予等量生理盐水。分别于给药后9、14、35、56天处死实验组动物取材,切取长约10 cm小肠,分离上皮内及固有层淋巴细胞,进行计数、流式细胞仪分析淋巴细胞亚群。取近回盲部回肠组织,进行病理、免疫荧光染色检查。采用RIPA法提取肠粘膜总蛋白,进行Western Blot检测肠粘膜MAdCAM-1蛋白的表达变化。流式细胞仪检测外周血T淋巴细胞β7整合素变化情况。数据采用SPSS 13.0统计软件作单因素方差分析(ANOVA,LSD),P0.05为差异有显著性统计学意义。结果:给药后第9天肠道IEL、LPL数量降至最低点,给药后14天仍处于低水平,给药后第35天数量逐渐增加,至给药后第56天恢复至给药前水平。免疫荧光染色证实了上述变化。给药后第14天,肠道IEL、LPL亚群发生改变,IEL中CD4-CD8+、TCRαβ+T淋巴细胞比例显著降低,而CD4+CD8+T淋巴细胞比例显著升高;LPL中CD3-CD20+B淋巴细胞、TCRyδ+T淋巴细胞比例显著降低,而CD4+CD8-、CD4-CD8+、CD4+CD8+T淋巴细胞比例显著升高。给药后第56天,肠道IEL、LPL各亚群比例恢复至给药前水平。病理结果显示对照组回肠粘膜完整,绒毛呈柱状,高度正常,给药后第9天回肠绒毛萎缩,高度变短,上皮细胞形态紊乱,有脱落,至给药后第56天回肠绒毛高度恢复正常,粘膜完整,上皮细胞排列整齐。Western Blot检测表明给药后第9天肠粘膜MAdCAM-1蛋白减少,给药后14天仍处于低水平,给药后第35天恢复至给药前水平。免疫荧光染色证实了上述变化。给药后第9天外周血T淋巴细胞β7整合素表达比例增高,给药后14天仍处于较高水平,给药后第35天恢复至给药前水平。结论:Campath-1H对肠道IEL、LPL具有清除作用,同时对其亚群也产生影响。肠道MAdCAM-1表达的变化与外周血T淋巴细胞α4β7整合素表达的变化趋势有相关性。在食蟹猴模型上,α4β7整合素与MAdCAM-1的特异性结合,可能是肠道淋巴细胞归巢的机制之一。
[Abstract]:Humanized CD52 monoclonal antibody (Campath-1H) on the expression of CD52 antigen T, B lymphocytes, monocytes, NK cells have strong scavenging effect, originally used for the treatment of chronic lymphocytic leukemia, multiple sclerosis and other diseases, in recent years, commonly used in kidney transplantation, liver transplantation, immune tolerance induced by intestinal transplantation in solid organ transplantation. Although the application of Campath-1H in organ transplantation has been more than ten years, but the lack of a suitable animal model for the evaluation in organ transplantation is safe and effective, thus limiting its in organ transplantation should be further used.Campath-1H only with nonhuman primates in some animal species has cross reaction, but not identification of rodent animal cells. The antigen in non-human primate animal genetic relationship with people most close, is an ideal animal model for the study of human disease. Application of Campath-1H induction in clinical transplantation After the program can be more than 99% of the lymphocytes in peripheral blood of patients with clearance of transplantation, and maintained for a long period of time. Low lymphocyte levels in the intestinal tract lymphocytes than in peripheral tissues, intestinal tract is the body's largest immune organ. The scavenging effect of Campath-1H at home and abroad research focused on peripheral blood, is a lack of research on intestinal lymphocytes. In this study, we used cynomolgus monkeys as animal model, given after the Campath-1H treatment, were observed at different time after administration of cynomolgus monkey peripheral blood, spleen, lymph node lymphocytes changes, intestinal intraepithelial and lamina propria lymphocytes subsets the changes, as well as the relationship between intestinal mucosal addressin cell adhesion molecules and lymphocyte homing receptors. Campath-1H on lymphocyte clearance and homing mechanism for clinical small bowel transplantation Provide the experimental basis for the application of Campath-1H in the better. Establishment and humanized CD52 monoclonal antibody partithe animal model on the cynomolgus monkey peripheral blood, spleen, lymph node lymphocytes. Objective: using humanized CD52 monoclonal antibody in organ transplantation has been more than ten years, but the lack of suitable animal model to evaluate the organ in less in the transplantation of safety and effectiveness, limiting its further application in organ transplantation. The humanized CD52 monoclonal antibody induced animal model in cynomolgus monkeys, and observe the effect on cynomolgus monkey peripheral blood, spleen, lymph node lymphocytes. Methods: male cynomolgus monkeys, 3-5 age, weight 3-5.5 kg, using the screening of red blood cell surface expression of CD52 antigen in cynomolgus monkeys by flow cytometry, the selected cynomolgus monkeys were randomly divided into experimental group and control group, the experimental group received intravenous injection of Campath-1H at 3 mg/kg, The control group received normal saline respectively. Based on the experimental animal group were sacrificed at 9,14,35,56 days after administration, the drug group in fifty-sixth days were only 3 cynomolgus monkeys were in the 3,6,9,14,21,35,56 days from the peripheral blood lymphocyte count and analysis of lymphocyte subsets by flow cytometry, observed before and after administration of blood lymphocytes at different time the number of changes. The data using SPSS 13 statistical software for single factor analysis of variance (ANOVA, LSD), between the two groups using t test, P0.05 difference was statistically significant. Results: We screened out 15 red cell surface expression of CD52 antigen in cynomolgus monkeys from 40 cynomolgus monkeys. There was no significant difference between the screened and was not screened in cynomolgus monkeys in the blood physiological and biochemical indexes. After administration of fresh monocytes in blood lymphocytes of cynomolgus monkeys, showing a rapid removal process and recovery of mononuclear cells in Sixth days after the administration of the lowest, T, B lymphocytes fell to the lowest level in ninth days after administration. The mononuclear cells in Fourteenth days after administration and returned to the level before administration, CD20+B lymphocytes, CD8+T lymphocytes, CD4+T lymphocytes in 21,35,56 days after administration to recover before administration level to ninth. Days after administration of spleen lymphocytes, lymph node lymphocytes decreased thirty-fifth days after dosing, the number of lymphocytes gradually increased to fifty-sixth days after the administration of spleen, lymph node morphology restored to the level before administration. Conclusion: the successful establishment of humanized CD52 monoclonal antibody induced animal model in cynomolgus monkeys after application of Campath-1H food. Fascicularis in peripheral blood lymphocyte clearance effect is strong, and gradually restored, spleen, lymph node lymphocytes showed the process of removal and recovery. This model can be used to study the transplantation model of humanized CD52 monoclonal antibody before clinic. Second Division of humanized CD52 monoclonal antibody on cynomolgus monkey intestinal lymphocyte function objective: To observe the application of humanized CD52 monoclonal antibody, changes in cynomolgus monkey intestinal intraepithelial and lamina propria lymphocytes, evaluation of the role of the antibody on the cynomolgus monkey intestinal lymphocytes, as well as changes in cynomolgus monkey intestinal mucosal addressin cell adhesion molecule with the lymphocyte homing receptor, studies the relationship between the two. Methods: male cynomolgus monkeys, aged 3-5 years old, weighing 3-5.5 kg, red cell surface expression of CD52 antigen in 15, were randomly divided into experimental group and control group, the experimental group received intravenous infusion of Campath-1H by 3mg /kg, the control group received normal saline respectively. Based on the experimental animal group were sacrificed at 9,14,35,56 days after dosing, cut a length of about 10 cm small intestine intraepithelial and lamina propria lymphocytes separation, counting, analysis of lymphocyte subsets by flow cytometry. From ileocecal junction back Intestinal tissue, pathology, immunofluorescence staining. The intestinal mucosa extract total protein by RIPA method, the expression of Western Blot of intestinal mucosa and the detection of MAdCAM-1 protein. T lymphocyte beta peripheral blood flow cytometry 7 integrin changes. Data using SPSS 13 statistical software for single factor analysis of variance (ANOVA, LSD P0.05), there was significant difference statistically significant. Results: ninth days after administration of intestinal IEL, LPL number dropped to the lowest point, 14 days after administration is still at a low level, thirty-fifth days after administration, the number gradually increased to fifty-sixth days after administration, restored to the level before administration. Immunofluorescence staining confirmed the above changes. For fourteenth days, after the administration of intestinal IEL subgroup LPL change, IEL CD4-CD8+, TCR alpha and beta +T lymphocyte ratio decreased significantly, and the percentage of CD4+CD8+T cells was significantly increased; CD3-CD20+B lymphocyte LPL, TCRy lymphocyte ratio was significantly +T. Reduce, and CD4+CD8-, CD4-CD8+, CD4+CD8+T lymphocyte ratio increased significantly. For fifty-sixth days, after the administration of intestinal IEL, LPL subsets restored to the level before administration. The pathological results showed that the control group of ileal mucosa integrity, villous columnar, normal height, Ninth days after administration of ileal villous atrophy, height shorter that epithelial cells form disorder, dropped to fifty-sixth days after administration of ileum mucosa intact, normal epithelial cells arranged in.Western Blot assay showed that MAdCAM-1 protein of intestinal mucosa decreased ninth days after administration, 14 days after administration is still at a low level, thirty-fifth days after the administration restored to the level before administration. Immunofluorescence staining confirmed the changes. After administration of T lymphocytes in peripheral blood of 7 day beta ninth integrin expression ratio increased after administration of 14 days is still at a high level, thirty-fifth days after the administration restored to the level before administration. Conclusion: Campath-1H on intestinal IEL, L PL has a scavenging effect, but also affect the subsets. There is correlation between the changes of expression of 4 integrin beta 7 trends on expression of intestinal MAdCAM-1 alpha and T lymphocytes in peripheral blood. In the cynomolgus monkey model, specific binding of alpha 4 beta 7 integrin and MAdCAM-1, may be one of the mechanisms of intestinal lymph cells homing.

【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392

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