内毒素脂多糖受体CD14单克隆抗体免疫毒素的研制及临床意义
发布时间：2019-06-24 12:45
【摘要】： 研究背景: 小儿脓毒性休克是儿科重症监护的常见疾病,也是死亡的主要原因。脓毒性休克是由微生物及其毒素等产物引起微循环障碍和器官功能不全的危急重症,临床治疗十分棘手。目前,它的发病机制尚未完全明确。过去很长时间,人们从微循环学说来解释休克的发病机制,认为休克是一个以急性微循环障碍为主的综合征,是由于循环血容量减少,引起器官血液灌流不足和细胞功能紊乱。近年来,有学者对脓毒性休克发病机制提出了炎症失控学说,脓毒性休克并非细菌感染直接作用,而是机体对感染性因素的反应,反应启动后不再依赖原触发因素,常规的抗感染难以遏制这一进程。在脓毒性休克发生发展过程中,体内出现大量作用广泛而复杂的细胞因子,它们相互作用形成许多正反馈环而导致“瀑布效应”,使细胞因子过度产生,促进炎症反应。目前,该病的发病机制尚未完全明确,但是CD14作为LPS(脂多糖)受体,介导LPS性细胞反应,在脓毒性休克病理反应中对炎症介质的产生和释放起着关键的作用,越来越被人们重视。CD14能识别、结合LPS或LPS/LBP(脂多糖/月旨多糖结合蛋白)复合物,介导LPS所致的细胞反应,在LPS性炎症反应、脓毒性休克等病理反应中起重要作用。目前发现,内毒素是诱导休克和器官衰竭的主要介质之一。国外研究表明,阻滞内毒素的产生,抑制其活性,能减轻机体对细菌感染的一系列反应,降低脓毒性休克的病死率。但目前,国内类似研究及文献较少,因此我们进行了本课题研究。 研究目的: 本课题拟通过双功能光敏偶合剂Sulfo-SANPAH在体外进行2F9(我院自行研制的CD14单抗)与Genistein的交联来完成2F9-SANPAH-Genistein(简称2F9-Gen)的研制,研究其对表达mCD14单核细胞体外的靶向杀伤作用,可能为脓毒性休克的抗介质治疗研究提供一种治疗工具,同时为进一步研究脓毒性休克的发病机制奠定一定的理论基础。 材料与方法: 1.2F9亚类的鉴定 用正常人的外周血单个核细胞,2F9杂交瘤培养上清,通过间接荧光标记法,流式细胞术检测2F9亚类。 2.2F9腹水的制备、纯化和鉴定 按常规方法制备大量腹水并经硫酸钱盐析初步纯化,并将初步纯化的2F9过Econo-Pac~R Protein A Cartridge纯化。纯化后2F9样品采用十二烷基磺酸钠-聚丙稀酸胺凝胶不连续电泳(SDS-PAGE),考马斯亮蓝染色后,分析单抗重链和轻链分子量及抗体纯度估计。 3.2F9-FITC的制备 参考改良Marsshall法,用异硫氰酸荧光素(Fluoresceini sothiocyanate,FITC)标记纯化的2F9单抗,多余FITC经PBS充分透析除去,并用紫外分光光度计测定OD495及OD280值。 4.2F9基础抗体的阻滞试验 取2F9杂交瘤细胞培养上清1ml,按照常规流式细胞术检测法,观察其对自身直标单抗2F9-FITC和进口标准直标单抗CD14-FITC的阻滞效果。 5.2F9-Gen的研制 将直标纯化单抗2F9-FITC与Genistein(酪氨酸激酶抑制剂4′,5,7-三羟基异黄酮)在体外通过双功能光敏偶合剂Sulfo-SANPAH交联。 6.2F9-Gen对单核细胞体外的靶向杀伤 取适量正常人的外周血,分离出淋巴细胞并分别加入不同量的PBS,2F9,2F9-Gen及Gen,培养24小时后用Annexin V-FITC细胞凋亡检测试剂盒检测。 结果: 1.2F9单抗亚型鉴定: 鼠抗人抗体2F9的重链是亚型为IgG_1;其轻链亚型为κ。2F9抗体亚型为鼠IgG_1κ。 2.2F9腹水的制备、纯化和鉴定 经色谱分析柱过柱分离,杂蛋白与2F9单抗被完全分开。纯化后的2F9单抗经SDS-PAGE凝胶电泳,可见分子量为57.92KD的重链及30.29KD的轻链,未见其他杂蛋白条带,得到纯化的2F9单抗。 3.直标单抗2F9-FITC的成功制备: 采用改良Marsshall法,成功研制2F9-FITC,通过FCM检测发现,2F9-FITC与标准的CD14-FITC具有相当的特异性和敏感性。 4.2F9-SANPAH浓度的检测 2F9-SANPAH的浓度为0.35mg/ml(0.0019886mmol/L)。 5.2F9-Gen的成功研制 2F9-Gen的浓度为0.15mg/ml(0.0008571mmol/L)。 6.2F9-Gen对单核细胞体外的靶向杀伤 2F9-Gen和Gen在体外对单核细胞均有杀伤作用,但2F9-Gen杀伤作用大于Gen。 结论: 1.成功制备并纯化了2F9抗体。 2.成功制备并纯化了直标抗体2F9-FITC。 3.成功制备免疫毒素2F9-Gen。 4.2F9-Gen在体外有较好的靶向杀伤单核细胞作用,可能为脓毒性休克提供了抗介质治疗的新途径,但是仍需进一步研究。
[Abstract]:Study Background: Septic shock in children is a serious disease in the pediatric intensive care, and is the main cause of death. For reasons, septic shock is a critical, critical, clinical treatment of microcirculatory disorders and organ dysfunction caused by the products of microorganisms and their toxins. It's a tricky one. At present, its pathogenesis has not yet been completed. It's all clear. It used to be a long time to explain the pathogenesis of shock from the theory of micro-circulation. It is considered that shock is a syndrome with an acute microcirculatory disturbance. It is due to the decrease of circulating blood volume, the insufficient perfusion of the blood and the work of the cells. In recent years, some scholars have put forward the theory of inflammation of the septic shock, the septic shock is not the direct effect of the bacterial infection, it is the reaction of the body to the infectious factors, the response of the reaction is no longer dependent on the original trigger, and the conventional anti-infection is difficult to contain. A process. In the course of the development of septic shock, a large number of broad and complex cytokines are present in the body, which interact to form a number of positive feedback loops, leading to the "waterfall effect", the excessive production of cytokines, and the promotion of inflammation. At present, the pathogenesis of the disease is not completely clear, but the CD14 is used as the LPS (lipopolysaccharide) receptor to mediate the LPS-induced cell reaction, and plays a key role in the production and release of the inflammatory mediators in the pathological reaction of septic shock, and more and more people The CD14 can identify, bind to LPS or LPS/ LBP (lipopolysaccharide/ yue-polysaccharide-binding protein) complex, mediate the cellular response induced by LPS, and can be lifted in the pathological reactions such as LPS-induced inflammatory reaction and septic shock. It is found that endotoxins are the major agents for inducing shock and organ failure. Foreign studies have shown that the production of endotoxin is blocked, its activity is inhibited, a series of reactions to the bacterial infection of the body can be reduced, and septic shock can be reduced. The case fatality rate. However, at present, similar research and literature are available in China, so we conducted this lesson Research on the subject. The purpose of this study was to study the development of 2F9-SANPAH-Genistein (2F9-Gen) by double-functional photosensitive coupling agent Sulfo-SANPAH in vitro, and to study the expression of mCD14 single-core in the preparation of 2F9-SANPAH-Genistein (2F9-Gen). The targeted killing effect of the cells in vitro may provide a therapeutic tool for the study of the anti-media therapy of septic shock, and to further study the pathogenesis of septic shock. to lay a foundation for the mechanism The theoretical basis of the theory. Materials and Methods: 1.2 F9 Subclasses of Human Peripheral Blood Mononuclear Cells, 2F9 Hybridoma Culture Supernatants by indirect fluorescence labeling, flow cytometry, Preparation, purification and identification of 2F9 subclass 2.2.2 F9 ascites preparation, purification and identification of a large amount of ascites according to the conventional method and preliminary purification by salting out of sulfuric acid, and the preliminary purification of 2F9 The purified 2F9 samples were SDS-PAG with SDS-PAG. E), after the Coomassie brilliant blue is dyed, separating Analysis of the heavy chain and light chain molecular weight of the monoclonal antibody and the estimated antibody purity. The preparation of the F9-FITC was modified by Marsshall method, and the purified 2F9 monoclonal antibody was labeled with fluorescein isothiocyanate (FITC). The residual FITC was fully dialyzed by PBS. Go, and measure the OD495 and OD280 values with an ultraviolet spectrophotometer. The block test of the F9 base antibody takes 2F9 hybridoma cell culture supernatant 1ml and is fine according to the conventional flow The method of cell-operation is used to observe its self-interest. The blocking effect of the direct standard single anti-2F9-FITC and the import standard direct-labeled single anti-CD14-FITC. The development of 5.2 F9-Gen will direct the purified monoclonal antibody 2F9-FITC and Geni stein (Tyrosine Kinase Inhibitor 4),5,7- In vitro, trihydroxy isoflavone is cross-linked by a double-functional photosensitive couple agent Sulfo-SANPAH. the cells and adding different amounts of PBS, 2F9, 2F9-Gen and Gen, incubated for 24 hours with Annexin V-FITC cell apoptosis test reagent Box detection. Result:1 . 2F9 monoclonal antibody subtype identification: heavy chain of mouse anti-human antibody 2F9 Subtype is IgG _ 1; the subtype of the light chain of the IgG_1; the subtype of the light chain of the IgG_1. 2F9 antibody is the preparation, purification and identification of the ascites of the mouse IgG _ 1, and the purification and the identification are separated by the chromatographic column through column, and the heteroprotein It was completely separated from 2F9. The purified 2F9 single The heavy chain with the molecular weight of 57.92 KD and the light chain of 30.29 KD were found by SDS-PAGE gel electrophoresis. The successful preparation of monoclonal antibody 2F9-FITC: Using the modified Marshall method, 2F9-FITC was successfully developed and found by FCM. 2F9-FITC and standard CD 14-FITC has a comparable specificity and sensitivity. 4.2 F9-SANPAH concentration The detected concentration of 2F9-SANPAH is 0.35 mg/ ml (0.001988mmol/ L). system 2F9-Gen at a concentration of 0.1 5mg/ml(0.0008571mmol/L) . 6.2 F9-Gen to single core The targeted killing of 2F9-Gen and Gen in vitro has a killing effect on monocytes in vitro, but the killing effect of 2F9-Gen is greater than Gen.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392;R720.597
本文编号：2505066
[Abstract]:Study Background: Septic shock in children is a serious disease in the pediatric intensive care, and is the main cause of death. For reasons, septic shock is a critical, critical, clinical treatment of microcirculatory disorders and organ dysfunction caused by the products of microorganisms and their toxins. It's a tricky one. At present, its pathogenesis has not yet been completed. It's all clear. It used to be a long time to explain the pathogenesis of shock from the theory of micro-circulation. It is considered that shock is a syndrome with an acute microcirculatory disturbance. It is due to the decrease of circulating blood volume, the insufficient perfusion of the blood and the work of the cells. In recent years, some scholars have put forward the theory of inflammation of the septic shock, the septic shock is not the direct effect of the bacterial infection, it is the reaction of the body to the infectious factors, the response of the reaction is no longer dependent on the original trigger, and the conventional anti-infection is difficult to contain. A process. In the course of the development of septic shock, a large number of broad and complex cytokines are present in the body, which interact to form a number of positive feedback loops, leading to the "waterfall effect", the excessive production of cytokines, and the promotion of inflammation. At present, the pathogenesis of the disease is not completely clear, but the CD14 is used as the LPS (lipopolysaccharide) receptor to mediate the LPS-induced cell reaction, and plays a key role in the production and release of the inflammatory mediators in the pathological reaction of septic shock, and more and more people The CD14 can identify, bind to LPS or LPS/ LBP (lipopolysaccharide/ yue-polysaccharide-binding protein) complex, mediate the cellular response induced by LPS, and can be lifted in the pathological reactions such as LPS-induced inflammatory reaction and septic shock. It is found that endotoxins are the major agents for inducing shock and organ failure. Foreign studies have shown that the production of endotoxin is blocked, its activity is inhibited, a series of reactions to the bacterial infection of the body can be reduced, and septic shock can be reduced. The case fatality rate. However, at present, similar research and literature are available in China, so we conducted this lesson Research on the subject. The purpose of this study was to study the development of 2F9-SANPAH-Genistein (2F9-Gen) by double-functional photosensitive coupling agent Sulfo-SANPAH in vitro, and to study the expression of mCD14 single-core in the preparation of 2F9-SANPAH-Genistein (2F9-Gen). The targeted killing effect of the cells in vitro may provide a therapeutic tool for the study of the anti-media therapy of septic shock, and to further study the pathogenesis of septic shock. to lay a foundation for the mechanism The theoretical basis of the theory. Materials and Methods: 1.2 F9 Subclasses of Human Peripheral Blood Mononuclear Cells, 2F9 Hybridoma Culture Supernatants by indirect fluorescence labeling, flow cytometry, Preparation, purification and identification of 2F9 subclass 2.2.2 F9 ascites preparation, purification and identification of a large amount of ascites according to the conventional method and preliminary purification by salting out of sulfuric acid, and the preliminary purification of 2F9 The purified 2F9 samples were SDS-PAG with SDS-PAG. E), after the Coomassie brilliant blue is dyed, separating Analysis of the heavy chain and light chain molecular weight of the monoclonal antibody and the estimated antibody purity. The preparation of the F9-FITC was modified by Marsshall method, and the purified 2F9 monoclonal antibody was labeled with fluorescein isothiocyanate (FITC). The residual FITC was fully dialyzed by PBS. Go, and measure the OD495 and OD280 values with an ultraviolet spectrophotometer. The block test of the F9 base antibody takes 2F9 hybridoma cell culture supernatant 1ml and is fine according to the conventional flow The method of cell-operation is used to observe its self-interest. The blocking effect of the direct standard single anti-2F9-FITC and the import standard direct-labeled single anti-CD14-FITC. The development of 5.2 F9-Gen will direct the purified monoclonal antibody 2F9-FITC and Geni stein (Tyrosine Kinase Inhibitor 4),5,7- In vitro, trihydroxy isoflavone is cross-linked by a double-functional photosensitive couple agent Sulfo-SANPAH. the cells and adding different amounts of PBS, 2F9, 2F9-Gen and Gen, incubated for 24 hours with Annexin V-FITC cell apoptosis test reagent Box detection. Result:1 . 2F9 monoclonal antibody subtype identification: heavy chain of mouse anti-human antibody 2F9 Subtype is IgG _ 1; the subtype of the light chain of the IgG_1; the subtype of the light chain of the IgG_1. 2F9 antibody is the preparation, purification and identification of the ascites of the mouse IgG _ 1, and the purification and the identification are separated by the chromatographic column through column, and the heteroprotein It was completely separated from 2F9. The purified 2F9 single The heavy chain with the molecular weight of 57.92 KD and the light chain of 30.29 KD were found by SDS-PAGE gel electrophoresis. The successful preparation of monoclonal antibody 2F9-FITC: Using the modified Marshall method, 2F9-FITC was successfully developed and found by FCM. 2F9-FITC and standard CD 14-FITC has a comparable specificity and sensitivity. 4.2 F9-SANPAH concentration The detected concentration of 2F9-SANPAH is 0.35 mg/ ml (0.001988mmol/ L). system 2F9-Gen at a concentration of 0.1 5mg/ml(0.0008571mmol/L) . 6.2 F9-Gen to single core The targeted killing of 2F9-Gen and Gen in vitro has a killing effect on monocytes in vitro, but the killing effect of 2F9-Gen is greater than Gen.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392;R720.597
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