【作者】 杨子浩；

【导师】 张晨美；

【作者基本信息】 浙江大学 ， 儿科学， 2008， 硕士

【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IgG₁;其轻链亚型为κ。2F9抗体亚型为鼠IgG₁κ。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。更多还原

【Abstract】 Research background:Septic shock is the main reason of death in pediatric intensive care unit,it mainly includes microcirculation disturbance caused by toxins of Microorganism and multi-organs function failure due to severe disease.Septic shock is very troublesome in clinic treatment,and it’s pathogenesy is not yet identified.People explained the pathogenesy of shock by micro-circulation theory in past.They think that shock is a syndrome of acute micro-circulation disturbance.Shock will cause organ’s blood hypoperfusion and cell’s functional disorder.Some scholars suggest the theory of inflammation out of control for the past few years.They think that septic shock is organism response to infectivity factor and routine anti-infection can not contain this processes.There are a great quantity of cytokines in the development of septic shock.They interact to format a lot of positive feedback loop,and induce "water fall effect".CD14,the receptor of LPS,plays capital role in pathologic response of septic shock.It recognizes and combines with LPS or LPS/LBP compound,thus mediates cell response inducded by LPS.Endotoxin is one of these main mediator that can induce shock and organ failure.Some abroad studies indicated that blocking endotoxic generation or inhibiting it’s activity can alleviat a series of reaction of organism,and decrease fatality rate of this disease.We carried out the study on immunotoxin against CD14 due to less study on this field in domestic now.Objective:This study plans to crosslink 2F9 and Genistein in vitro by Sulfo-SANPAH,and completes preparation of 2F9-Gen.To study 2F9-Gen’s targetic lethal effect to mononuclear cell in vitro and provide a mediator treatment tool for septic shock possibly.Material and methods:1.Identificat the subtype of 2F9To detIect the subtype of 2F9 with flow cytometry by fluorescent labeling.2.Preparation and purify and identification of 2F9 ascitesTo prepare ascites with routine methods,and to purify 2F9.To analyses the molecular weight of 2F9’s heavy chain and light chain after SDS-PAGE,and to estimate antibodie’s purity.3.Preparation of 2F9-FITCTo mark pure 2F9 with FITC by improvement Marsshall method,and to remove redundant FITC by dialysis.To determine it’s OD495 and OD280 with ultraviolet spectrophotometer.4.Blockade test of 2F9 antibodyTo observe the blocking result of 2F9 to 2F9-FITC and standard CD14-FITC,1ml supernatant of 2F9 hybridoma cell was used during the experiment the through FCM.5.Preparation of 2F9-GenTo crosslink 2F9 and Genistein in vitro by Sulfo-SANPAH. 6.2F9-Gen’s targetic lethal effect to mononuclear cell in vitro.To obtain appropriate amount peripheral blood of health child,and dissociate lymphocyte,and add in PBS,2F9,2F9-Gen,Gen,and eventually use Annexin VFITC apoptosis kit to detect after cultivated 24 hours.Result:1.Identification of the subtype of 2F9The heavy chain subtype of 2F9 is IgG1,the light chain belongs toκ.2F9 antibody subtype is mouse IgG1κ.2.Preparation and purification of 2F9 ascitesHybridprotein was separated from 2F9 antibody by chromatographic analytical column.Through SDS-PAGE gel electrophoresis,a heavy chain(molecular weight 57.92KDa) and a light chain(30.29KDa) were detected.The purified 2F9 was obtained.3.Preparation of 2F9-FITC and blocking test of 2F9 antibody2F9-FITC was prepared successfully by improved Marsshall method with the same Specificity and sensitivity compared with standard CD14-FITC by FCM.4.Detection of the concentration of 2F9-SANPAHThe concentration of 2F9-SANPAH is 0.35mg/ml(0.0019886mmol/L).5.Preparation of 2F9-GemThe concentration of 2F9-Gen is 0.15mg/ml(0.0008571mmol/L).6.2F9-Gen’s targetic lethal effect to mononuclear cell in vitro2F9-Gen and Gen both have lethal effect to mononuclear cell in vitro,but 2F9-Gen has better effect than Gen.Conclusions:1.Success to prepare and purify 2F9 antibody.2.Success to prepare and purify 2F9-FITC. 3.Success to prepare the immunotoxin(2F9-Gen).4.2F9-Gen has good effect of killing monocytes in vitro,and provide a new way of antimediator therapy to septic shock possibly,but the mechanism needs further research.更多还原