內(nèi)毒素脂多糖受體CD14單克隆抗體免疫毒素的研制及臨床意義
發(fā)布時(shí)間：2019-06-24 12:45
【摘要】： 研究背景: 小兒膿毒性休克是兒科重癥監(jiān)護(hù)的常見(jiàn)疾病,也是死亡的主要原因。膿毒性休克是由微生物及其毒素等產(chǎn)物引起微循環(huán)障礙和器官功能不全的危急重癥,臨床治療十分棘手。目前,它的發(fā)病機(jī)制尚未完全明確。過(guò)去很長(zhǎng)時(shí)間,人們從微循環(huán)學(xué)說(shuō)來(lái)解釋休克的發(fā)病機(jī)制,認(rèn)為休克是一個(gè)以急性微循環(huán)障礙為主的綜合征,是由于循環(huán)血容量減少,引起器官血液灌流不足和細(xì)胞功能紊亂。近年來(lái),有學(xué)者對(duì)膿毒性休克發(fā)病機(jī)制提出了炎癥失控學(xué)說(shuō),膿毒性休克并非細(xì)菌感染直接作用,而是機(jī)體對(duì)感染性因素的反應(yīng),反應(yīng)啟動(dòng)后不再依賴原觸發(fā)因素,常規(guī)的抗感染難以遏制這一進(jìn)程。在膿毒性休克發(fā)生發(fā)展過(guò)程中,體內(nèi)出現(xiàn)大量作用廣泛而復(fù)雜的細(xì)胞因子,它們相互作用形成許多正反饋環(huán)而導(dǎo)致“瀑布效應(yīng)”,使細(xì)胞因子過(guò)度產(chǎn)生,促進(jìn)炎癥反應(yīng)。目前,該病的發(fā)病機(jī)制尚未完全明確,但是CD14作為L(zhǎng)PS(脂多糖)受體,介導(dǎo)LPS性細(xì)胞反應(yīng),在膿毒性休克病理反應(yīng)中對(duì)炎癥介質(zhì)的產(chǎn)生和釋放起著關(guān)鍵的作用,越來(lái)越被人們重視。CD14能識(shí)別、結(jié)合LPS或LPS/LBP(脂多糖/月旨多糖結(jié)合蛋白)復(fù)合物,介導(dǎo)LPS所致的細(xì)胞反應(yīng),在LPS性炎癥反應(yīng)、膿毒性休克等病理反應(yīng)中起重要作用。目前發(fā)現(xiàn),內(nèi)毒素是誘導(dǎo)休克和器官衰竭的主要介質(zhì)之一。國(guó)外研究表明,阻滯內(nèi)毒素的產(chǎn)生,抑制其活性,能減輕機(jī)體對(duì)細(xì)菌感染的一系列反應(yīng),降低膿毒性休克的病死率。但目前,國(guó)內(nèi)類似研究及文獻(xiàn)較少,因此我們進(jìn)行了本課題研究。 研究目的: 本課題擬通過(guò)雙功能光敏偶合劑Sulfo-SANPAH在體外進(jìn)行2F9(我院自行研制的CD14單抗)與Genistein的交聯(lián)來(lái)完成2F9-SANPAH-Genistein(簡(jiǎn)稱2F9-Gen)的研制,研究其對(duì)表達(dá)mCD14單核細(xì)胞體外的靶向殺傷作用,可能為膿毒性休克的抗介質(zhì)治療研究提供一種治療工具,同時(shí)為進(jìn)一步研究膿毒性休克的發(fā)病機(jī)制奠定一定的理論基礎(chǔ)。 材料與方法: 1.2F9亞類的鑒定 用正常人的外周血單個(gè)核細(xì)胞,2F9雜交瘤培養(yǎng)上清,通過(guò)間接熒光標(biāo)記法,流式細(xì)胞術(shù)檢測(cè)2F9亞類。 2.2F9腹水的制備、純化和鑒定 按常規(guī)方法制備大量腹水并經(jīng)硫酸錢鹽析初步純化,并將初步純化的2F9過(guò)Econo-Pac~R Protein A Cartridge純化。純化后2F9樣品采用十二烷基磺酸鈉-聚丙稀酸胺凝膠不連續(xù)電泳(SDS-PAGE),考馬斯亮藍(lán)染色后,分析單抗重鏈和輕鏈分子量及抗體純度估計(jì)。 3.2F9-FITC的制備 參考改良Marsshall法,用異硫氰酸熒光素(Fluoresceini sothiocyanate,FITC)標(biāo)記純化的2F9單抗,多余FITC經(jīng)PBS充分透析除去,并用紫外分光光度計(jì)測(cè)定OD495及OD280值。 4.2F9基礎(chǔ)抗體的阻滯試驗(yàn) 取2F9雜交瘤細(xì)胞培養(yǎng)上清1ml,按照常規(guī)流式細(xì)胞術(shù)檢測(cè)法,觀察其對(duì)自身直標(biāo)單抗2F9-FITC和進(jìn)口標(biāo)準(zhǔn)直標(biāo)單抗CD14-FITC的阻滯效果。 5.2F9-Gen的研制 將直標(biāo)純化單抗2F9-FITC與Genistein(酪氨酸激酶抑制劑4′,5,7-三羥基異黃酮)在體外通過(guò)雙功能光敏偶合劑Sulfo-SANPAH交聯(lián)。 6.2F9-Gen對(duì)單核細(xì)胞體外的靶向殺傷 取適量正常人的外周血,分離出淋巴細(xì)胞并分別加入不同量的PBS,2F9,2F9-Gen及Gen,培養(yǎng)24小時(shí)后用Annexin V-FITC細(xì)胞凋亡檢測(cè)試劑盒檢測(cè)。 結(jié)果: 1.2F9單抗亞型鑒定: 鼠抗人抗體2F9的重鏈?zhǔn)莵喰蜑镮gG_1;其輕鏈亞型為κ。2F9抗體亞型為鼠IgG_1κ。 2.2F9腹水的制備、純化和鑒定 經(jīng)色譜分析柱過(guò)柱分離,雜蛋白與2F9單抗被完全分開(kāi)。純化后的2F9單抗經(jīng)SDS-PAGE凝膠電泳,可見(jiàn)分子量為57.92KD的重鏈及30.29KD的輕鏈,未見(jiàn)其他雜蛋白條帶,得到純化的2F9單抗。 3.直標(biāo)單抗2F9-FITC的成功制備: 采用改良Marsshall法,成功研制2F9-FITC,通過(guò)FCM檢測(cè)發(fā)現(xiàn),2F9-FITC與標(biāo)準(zhǔn)的CD14-FITC具有相當(dāng)?shù)奶禺愋院兔舾行浴?4.2F9-SANPAH濃度的檢測(cè) 2F9-SANPAH的濃度為0.35mg/ml(0.0019886mmol/L)。 5.2F9-Gen的成功研制 2F9-Gen的濃度為0.15mg/ml(0.0008571mmol/L)。 6.2F9-Gen對(duì)單核細(xì)胞體外的靶向殺傷 2F9-Gen和Gen在體外對(duì)單核細(xì)胞均有殺傷作用,但2F9-Gen殺傷作用大于Gen。 結(jié)論: 1.成功制備并純化了2F9抗體。 2.成功制備并純化了直標(biāo)抗體2F9-FITC。 3.成功制備免疫毒素2F9-Gen。 4.2F9-Gen在體外有較好的靶向殺傷單核細(xì)胞作用,可能為膿毒性休克提供了抗介質(zhì)治療的新途徑,但是仍需進(jìn)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392;R720.597
本文編號(hào)：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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392;R720.597
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