B7-1单克隆抗体对小鼠狼疮样肾炎模型的免疫干预效应及分子机制研究

发布时间：2018-04-24 17:07

本文选题：B7-1分子 + 单克隆抗体　；参考：《苏州大学》2013年硕士论文

【摘要】：B7-1是表达于抗原提呈细胞(antigen presenting cell, APC)表面的重要协同刺激分子，其相应的受体是表达在T细胞表面的CD28家族分子。B7-1与CD28结合是介导T细胞活化、增殖、分化及发挥免疫效应所必需的协同刺激信号。若缺乏该信号，T细胞将进入无反应状态（Anergy）或免疫耐受（Tolerance）甚至引起程序性死亡（Apoptosis）。自身免疫性疾病（autoimmune disease，AID）是自身反应T、B细胞过度活化，产生大量自身抗体，进而导致多系统、多器官受损的一类严重疾病。系统性红斑狼疮（systemic lupus erythematosus，SLE）是其典型代表。目前研究发现，B7-CD28协同刺激信号参与了SLE的发生发展，阻断或削弱该信号，可减轻此类疾患的病理损伤。通过建立动物疾病模型模拟人类疾病的发生过程及临床表现是研究疾病发生、发展、转归及寻找新的诊治途径的重要手段。本课题在建立慢性移植物抗宿主病（cGVHD）小鼠狼疮样肾炎模型并对其进行生物学鉴定的基础上，运用自行研制的B7-1单克隆抗体对疾病模型进行干预。通过免疫学、血清学及肾脏病理损伤评价等指标考察抗体对疾病模型的逆转效应及分子机制。以期寻找新的对SLE及相关疾患具有防治作用的特异、高效、低毒的生物制剂。一、B7-1单克隆抗体的制备及鉴定目的：制备鼠抗人B7-1单克隆抗体并对其生物学特性进行鉴定。方法：采用体内诱生腹水法制备单抗；Protein G免疫亲和层析法纯化抗体；流式细胞术分析单抗对人及小鼠细胞膜型B7-1分子抗原表位的识别。结果：经体内诱生腹水法制备单抗，小鼠腹水形成阳性率约为80%，腹水收获量平均为3.5ml/只小鼠；经免疫亲和层析法纯化，腹水中抗体蛋白的得率为3.2mg/ml；B7-1单克隆抗体与转人B7-1基因小鼠成纤维细胞株L929-B7-1以及小鼠脾脏细胞的阳性结合率分别为99.3%及65.4%。结论： B7-1单克隆抗体能够特异性识别相应的抗原分子。二、慢性移植物抗宿主病小鼠狼疮样肾炎模型的建立及生物学鉴定目的：构建慢性移植物抗宿主病（cGVHD）小鼠狼疮样肾炎模型并对其进行生物学鉴定。方法：将8周龄雌性(C57BL/6×BALB/c) F1小鼠随机分为2组，造模组于0、3、7和11d经尾静脉注射亲代雌性BALB/c小鼠脾脏细胞107/100μL/只。正常对照组给予等量生理盐水。末次注射后第7天，免疫荧光及流式细胞术分析小鼠脾脏中巨噬细胞（CD11b+）、树突状细胞（CD11c+）、中性粒细胞（Gr1+）及抗体产生细胞（CD21+等）的阳性百分率；每2周定期检测血清中抗双链DNA抗体(ds-DNA)、抗核抗体(ANA)含量；每月定期检测尿蛋白含量。3个月后处死小鼠，经HE染色观测肾脏病理变化，石蜡切片直接免疫荧光法分析免疫复合物（IC）的沉积以及透射电镜观察肾小球超微结构的改变。结果：造模组小鼠脾脏中CD11b+、CD11c+及Gr1+细胞的百分率较对照组明显升高（p0.05），同时B细胞协同刺激分子CD21+、CD23+、CD80+及CD86+的表达上调明显（p0.05）；建模2周，70%的小鼠血清中可检测到抗dsDNA抗体和ANA，4周时达90%；建模3个月，70%的小鼠出现蛋白尿，蛋白含量为++~++++；HE染色及镜下观察，出现肾小球肿胀及淋巴细胞浸润；直接免疫荧光法呈现大量IC沉积；透射电镜下可见造模组小鼠肾基底膜节段性增厚等病理改变。结论：成功建立慢性移植物抗宿主病小鼠狼疮样肾炎模型。三、B7-1单克隆抗体对小鼠狼疮样肾炎的免疫干预效应及分子机制研究目的：探讨B7-1单克隆抗体通过阻断B7-1/CD28信号通路对狼疮样肾炎模型的免疫干预效应及其分子机制。方法：按上述建模方法，取F1代小鼠随机分为3组，即造模组、抗体干预组及对照组。每组15只。干预组在末次注射淋巴细胞后第1、3、5、8及15d分别尾静脉注射B7-1抗体(克隆4E5)200μg/只，然后每月再注射1次，连续注射2次。造模组同一时间给予等剂量的Ig同型对照。按照上述时间点分析脾脏中巨噬细胞（CD11b+）、树突状细胞（CD11c+）、中性粒细胞（Gr1+）及抗体产生细胞（CD21+等）的阳性百分率，抗双链DNA抗体(ds-DNA)、抗核抗体(ANA)含量，尿蛋白含量，免疫复合物（IC），肾小球超微结构等指标。结果：免疫荧光及流式细胞术分析结果显示，抗体干预组小鼠脾脏细胞CD11b+、CD11c+及GR1+等分子的表达及脾脏B细胞CD21+、CD23+、CD80+及CD86+等分子的表达均低于造模组（p0.05）；4周后，造模组90%的小鼠血清能检测到抗dsDNA抗体和ANA的表达，而抗体干预组仅40%出现阳性，且抗体滴度明显低于造模组（p0.05）；3个月时70%的造模组小鼠出现蛋白尿，蛋白含量为++~++++，抗体干预组仅有20%，且蛋白含量为+~++； HE染色后镜下观察，造模组肾脏组织出现肾小球肿胀，淋巴细胞浸润等典型的炎性改变，，但抗体干预组仅部分出现肾小球体积轻度增大，少许淋巴细胞浸润，毛细血管腔及球囊腔体积基本未变窄；免疫荧光法及透射电镜检查均发现造模组IC沉积，抗体干预组的肾小球轮廓较清晰，IC的沉积明显少于造模组；通过透射电镜对超微结构的观察，发现造模组出现基底膜节段性增厚等病理改变，抗体干预组基底膜厚度均匀，无明显病理改变。结论：B7-1单克隆抗体通过与相应抗原分子结合可抑制免疫细胞的活化，逆转狼疮样肾炎的病理损伤，提示该类特异性抗体对SLE或cGVHD等疾患具有潜在的防治作用。
[Abstract]:B7-1 is an important synergistic stimulator expressed on the surface of antigen presenting cell (APC). Its corresponding receptor is the binding of CD28 family molecule.B7-1 and CD28 expressed on the surface of T cells as a synergistic stimulus required to mediate activation, proliferation, differentiation and immune effects of T cells. If the signal is lacking, T cells will be entered. No response (Anergy) or immune tolerance (Tolerance) or even programmed death (Apoptosis).
Autoimmune disease (autoimmune disease, AID) is a serious disease in which T, B cells excessively activate and produce a large number of autoantibodies, resulting in multiple systems and multiple organs damage. Systemic lupus erythematosus (systemic lupus erythematosus, SLE) is a typical representative. The present study found that B7-CD28 synergistic stimulus signals participated in SLE. The development, blocking or weakening of the signal can reduce the pathological damage of such diseases. The process and clinical manifestation of human disease through the establishment of animal disease model is an important means to study the occurrence, development, prognosis and search for new methods of diagnosis and treatment of disease in the chronic graft versus host disease (cGVHD) mice. On the basis of the nephritis model and its biological identification, the self developed B7-1 monoclonal antibody was used to intervene the disease model. The reverse effect and molecular mechanism of antibody against the disease model were investigated by immunology, serology and renal pathological damage evaluation. In order to find new prevention and cure effect on SLE and related diseases. Specific, efficient, low toxic biological agents.
Preparation and identification of B7-1 monoclonal antibody
Objective: to prepare the mouse anti human B7-1 monoclonal antibody and identify its biological characteristics. Methods: the monoclonal antibody was prepared by the induced ascites method in vivo; the antibody was purified by Protein G immunoaffinity chromatography; the flow cytometry was used to identify the epitope of the membrane type B7-1 antigen epitopes of human and mouse cells. The positive rate of mouse ascites was about 80%, and the average ascites was 3.5ml/ mice. The antibody protein in ascites was 3.2mg/ml by immuno affinity chromatography, and the positive binding rate of B7-1 monoclonal antibody and B7-1 mouse fibroblast cell strain L929-B7-1 and mouse spleen cells was 99.3% and 65.4%, respectively. Conclusion: B7-1 monoclonal antibody can specifically identify the corresponding antigen molecules.
Establishment and biological identification of lupus nephritis model in mice with chronic graft-versus-host disease
Objective: to construct the model of lupus like nephritis in mice with chronic graft versus host disease (cGVHD) and to make a biological identification. Methods: the 8 week old female (C57BL/6 x BALB/c) F1 mice were randomly divided into 2 groups. The model group was injected into the tail vein of 0,3,7 and 11d in the female BALB/c mice with the spleen cells 107/100 u L/ only. The normal control group was given the same amount of birth. Seventh days after the last injection, immunofluorescence and flow cytometry were used to analyze the macrophage (CD11b+), dendritic cells (CD11c+), neutrophils (Gr1+) and antibody producing cells (CD21+ and so on) in mice. The content of anti double chain DNA antibody (ds-DNA) and anti nuclear antibody (ANA) content in serum was regularly detected every 2 weeks; regular examination was performed on a monthly basis. After.3 months of urinary protein content, the mice were killed and renal pathological changes were observed by HE staining. The deposition of immunofluorescence (IC) and ultrastructural changes of glomeruli were observed by direct immunofluorescence of paraffin section and transmission electron microscopy. The results showed that the percentage of CD11b+, CD11c + and Gr1+ cells in the spleen of the model mice was significantly higher than that in the control group (p0. 05), at the same time, the expression of B cells co stimulator CD21+, CD23+, CD80+ and CD86+ increased significantly (P0.05); in the 2 week of modeling, 70% of the mice were able to detect anti dsDNA antibody and ANA, and 90% in 4 weeks; 70% mice appeared proteinuria, protein content was + + + + +, HE staining and microscopic observation, glomerular swelling and lymphocyte Infiltration; direct immunofluorescence (direct immunofluorescence) showed a large number of IC deposits; under transmission electron microscopy, the pathological changes in the segmental thickening of the renal basement membrane were seen in the model mice. Conclusion: the model of lupus nephritis in mice with chronic graft versus host disease was successfully established.
Effect and molecular mechanism of B7-1 monoclonal antibody on lupus nephritis in mice
Objective: To investigate the immune intervention effect and molecular mechanism of B7-1 monoclonal antibody by blocking the B7-1/CD28 signaling pathway in lupus nephritis model. Methods: according to the above modeling methods, the F1 generation mice were randomly divided into 3 groups, namely, the model group, the antibody intervention group and the control group, 15 rats in each group. After the last injection of lymphocyte, the group was 1,3,5,8 and 1. 5D was injected with B7-1 antibody (clone 4E5) 200 mu g/ respectively, and then injected 1 times a month and 2 times continuously. The module was given equal dose of Ig same type at the same time. In accordance with the time point, the spleen macrophages (CD11b+), dendritic cells (Gr1+) and antibody producing cells (CD21+ and so on) were positive. Fraction, anti double chain DNA antibody (ds-DNA), anti nuclear antibody (ANA) content, urine protein content, immune complex (IC) and glomerular ultrastructure. Results: the results of immunofluorescence and flow cytometry analysis showed that the expression of CD11b+, CD11c+ and GR1+ in the spleen cells of the antibody intervention group and the CD21+, CD23+, CD80+, CD86+ and so on of spleen B cells The expression of the molecule was lower than that of the model group (P0.05). After 4 weeks, the expression of anti dsDNA antibody and ANA was detected in the mouse serum of 90% of the model group, but only 40% of the antibody intervention group was positive, and the antibody titer was significantly lower than that of the model group (P0.05), and 70% of the model mice were proteinuria at 3 months, the protein content was + + + + +, and the antibody intervention group was only 20%, The protein content was + + +. After HE staining, the glomerular swelling, lymphocyte infiltration and other typical inflammatory changes were observed in the kidney tissue of the model group, but only part of the antibody intervention group had a slight enlargement of the glomerular volume, a little lymphocyte infiltration, and the volume of the capillary cavity and the balloon cavity were basically not narrowed; the immunofluorescence method and transmission were found. IC deposition in the model group was found by electron microscopy. The glomerular contour of the antibody intervention group was clearer, and the deposition of IC was obviously less than that of the model group. Through the observation of the ultrastructure by transmission electron microscopy, the pathological changes of the basal membrane segmental thickening were found in the model group. The thickness of the basement membrane in the antibody intervention group was uniform and no obvious pathological changes were found. Conclusion: B7-1 single gram. It can inhibit the activation of immune cells and reverse the pathological damage of lupus nephritis by binding to the corresponding antigen molecules, suggesting that these specific antibodies have potential preventive and therapeutic effects on SLE or cGVHD diseases.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R593.241;R392

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