發(fā)布時(shí)間：2018-04-24 17:07

  本文選題：B7-1分子 + 單克隆抗體��； 參考：《蘇州大學(xué)》2013年碩士論文

【摘要】：B7-1是表達(dá)于抗原提呈細(xì)胞(antigen presenting cell, APC)表面的重要協(xié)同刺激分子，其相應(yīng)的受體是表達(dá)在T細(xì)胞表面的CD28家族分子。B7-1與CD28結(jié)合是介導(dǎo)T細(xì)胞活化、增殖、分化及發(fā)揮免疫效應(yīng)所必需的協(xié)同刺激信號。若缺乏該信號，T細(xì)胞將進(jìn)入無反應(yīng)狀態(tài)（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）是自身反應(yīng)T、B細(xì)胞過度活化，產(chǎn)生大量自身抗體，進(jìn)而導(dǎo)致多系統(tǒng)、多器官受損的一類嚴(yán)重疾病。系統(tǒng)性紅斑狼瘡（systemic lupus erythematosus，SLE）是其典型代表。目前研究發(fā)現(xiàn)，B7-CD28協(xié)同刺激信號參與了SLE的發(fā)生發(fā)展，阻斷或削弱該信號，可減輕此類疾患的病理損傷。通過建立動(dòng)物疾病模型模擬人類疾病的發(fā)生過程及臨床表現(xiàn)是研究疾病發(fā)生、發(fā)展、轉(zhuǎn)歸及尋找新的診治途徑的重要手段。本課題在建立慢性移植物抗宿主病（cGVHD）小鼠狼瘡樣腎炎模型并對其進(jìn)行生物學(xué)鑒定的基礎(chǔ)上，運(yùn)用自行研制的B7-1單克隆抗體對疾病模型進(jìn)行干預(yù)。通過免疫學(xué)、血清學(xué)及腎臟病理損傷評價(jià)等指標(biāo)考察抗體對疾病模型的逆轉(zhuǎn)效應(yīng)及分子機(jī)制。以期尋找新的對SLE及相關(guān)疾患具有防治作用的特異、高效、低毒的生物制劑。 一、B7-1單克隆抗體的制備及鑒定 目的：制備鼠抗人B7-1單克隆抗體并對其生物學(xué)特性進(jìn)行鑒定。方法：采用體內(nèi)誘生腹水法制備單抗；Protein G免疫親和層析法純化抗體；流式細(xì)胞術(shù)分析單抗對人及小鼠細(xì)胞膜型B7-1分子抗原表位的識別。結(jié)果：經(jīng)體內(nèi)誘生腹水法制備單抗，小鼠腹水形成陽性率約為80%，腹水收獲量平均為3.5ml/只小鼠；經(jīng)免疫親和層析法純化，腹水中抗體蛋白的得率為3.2mg/ml；B7-1單克隆抗體與轉(zhuǎn)人B7-1基因小鼠成纖維細(xì)胞株L929-B7-1以及小鼠脾臟細(xì)胞的陽性結(jié)合率分別為99.3%及65.4%。結(jié)論： B7-1單克隆抗體能夠特異性識別相應(yīng)的抗原分子。 二、慢性移植物抗宿主病小鼠狼瘡樣腎炎模型的建立及生物學(xué)鑒定 目的：構(gòu)建慢性移植物抗宿主�。╟GVHD）小鼠狼瘡樣腎炎模型并對其進(jìn)行生物學(xué)鑒定。方法：將8周齡雌性(C57BL/6×BALB/c) F1小鼠隨機(jī)分為2組，造模組于0、3、7和11d經(jīng)尾靜脈注射親代雌性BALB/c小鼠脾臟細(xì)胞107/100μL/只。正常對照組給予等量生理鹽水。末次注射后第7天，免疫熒光及流式細(xì)胞術(shù)分析小鼠脾臟中巨噬細(xì)胞（CD11b+）、樹突狀細(xì)胞（CD11c+）、中性粒細(xì)胞（Gr1+）及抗體產(chǎn)生細(xì)胞（CD21+等）的陽性百分率；每2周定期檢測血清中抗雙鏈DNA抗體(ds-DNA)、抗核抗體(ANA)含量；每月定期檢測尿蛋白含量。3個(gè)月后處死小鼠，經(jīng)HE染色觀測腎臟病理變化，石蠟切片直接免疫熒光法分析免疫復(fù)合物（IC）的沉積以及透射電鏡觀察腎小球超微結(jié)構(gòu)的改變。結(jié)果：造模組小鼠脾臟中CD11b+、CD11c+及Gr1+細(xì)胞的百分率較對照組明顯升高（p0.05），同時(shí)B細(xì)胞協(xié)同刺激分子CD21+、CD23+、CD80+及CD86+的表達(dá)上調(diào)明顯（p0.05）；建模2周，70%的小鼠血清中可檢測到抗dsDNA抗體和ANA，4周時(shí)達(dá)90%；建模3個(gè)月，70%的小鼠出現(xiàn)蛋白尿，蛋白含量為++~++++；HE染色及鏡下觀察，出現(xiàn)腎小球腫脹及淋巴細(xì)胞浸潤；直接免疫熒光法呈現(xiàn)大量IC沉積；透射電鏡下可見造模組小鼠腎基底膜節(jié)段性增厚等病理改變。結(jié)論：成功建立慢性移植物抗宿主病小鼠狼瘡樣腎炎模型。 三、B7-1單克隆抗體對小鼠狼瘡樣腎炎的免疫干預(yù)效應(yīng)及分子機(jī)制研究 目的：探討B(tài)7-1單克隆抗體通過阻斷B7-1/CD28信號通路對狼瘡樣腎炎模型的免疫干預(yù)效應(yīng)及其分子機(jī)制。方法：按上述建模方法，取F1代小鼠隨機(jī)分為3組，即造模組、抗體干預(yù)組及對照組。每組15只。干預(yù)組在末次注射淋巴細(xì)胞后第1、3、5、8及15d分別尾靜脈注射B7-1抗體(克隆4E5)200μg/只，然后每月再注射1次，連續(xù)注射2次。造模組同一時(shí)間給予等劑量的Ig同型對照。按照上述時(shí)間點(diǎn)分析脾臟中巨噬細(xì)胞（CD11b+）、樹突狀細(xì)胞（CD11c+）、中性粒細(xì)胞（Gr1+）及抗體產(chǎn)生細(xì)胞（CD21+等）的陽性百分率，抗雙鏈DNA抗體(ds-DNA)、抗核抗體(ANA)含量，尿蛋白含量，免疫復(fù)合物（IC），腎小球超微結(jié)構(gòu)等指標(biāo)。結(jié)果：免疫熒光及流式細(xì)胞術(shù)分析結(jié)果顯示，抗體干預(yù)組小鼠脾臟細(xì)胞CD11b+、CD11c+及GR1+等分子的表達(dá)及脾臟B細(xì)胞CD21+、CD23+、CD80+及CD86+等分子的表達(dá)均低于造模組（p0.05）；4周后，造模組90%的小鼠血清能檢測到抗dsDNA抗體和ANA的表達(dá)，而抗體干預(yù)組僅40%出現(xiàn)陽性，且抗體滴度明顯低于造模組（p0.05）；3個(gè)月時(shí)70%的造模組小鼠出現(xiàn)蛋白尿，蛋白含量為++~++++，抗體干預(yù)組僅有20%，且蛋白含量為+~++； HE染色后鏡下觀察，造模組腎臟組織出現(xiàn)腎小球腫脹，淋巴細(xì)胞浸潤等典型的炎性改變，，但抗體干預(yù)組僅部分出現(xiàn)腎小球體積輕度增大，少許淋巴細(xì)胞浸潤，毛細(xì)血管腔及球囊腔體積基本未變窄；免疫熒光法及透射電鏡檢查均發(fā)現(xiàn)造模組IC沉積，抗體干預(yù)組的腎小球輪廓較清晰，IC的沉積明顯少于造模組；通過透射電鏡對超微結(jié)構(gòu)的觀察，發(fā)現(xiàn)造模組出現(xiàn)基底膜節(jié)段性增厚等病理改變，抗體干預(yù)組基底膜厚度均勻，無明顯病理改變。結(jié)論：B7-1單克隆抗體通過與相應(yīng)抗原分子結(jié)合可抑制免疫細(xì)胞的活化，逆轉(zhuǎn)狼瘡樣腎炎的病理損傷，提示該類特異性抗體對SLE或cGVHD等疾患具有潛在的防治作用。
[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.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R593.241;R392

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