【作者】 张敕；

【导师】 陈元仲；

【作者基本信息】 福建医科大学 ， 内科学， 2008， 硕士

【摘要】 血管内皮细胞具有非常重要的生理功能,主要体现在:1.构成天然屏障,维持血管内膜光滑,防止血小板和白细胞粘附及有害物质侵入血管壁;2.组成渗透性屏障,调节物质交换和主动转运功能;3.参与血栓形成和止血过程,保持动态平衡,包括组织纤溶酶原激活物(t-PA)、纤溶酶原激活物抑制剂-1(PAI-1)等。许多病理状态如严重感染、DIC等均涉及血管内皮细胞的功能损害,继而加剧病理性过程,形成恶性循环。因此如何恢复病理状态下失衡的血管内皮细胞的形态与功能一直是学术界研究的重点。纤连蛋白(Fibronectin,FN)是一种广泛存在于细胞外液、细胞表面及结缔组织等处的糖蛋白。在抗感染、维持微血管完整性及通透性等方面具有重要的作用,故曾有“调理性α2表面结合蛋白”、“细胞表面蛋白”及“细胞粘附分子”之称。已知FN主要分为细胞型和血浆型两种,细胞型FN主要由内皮细胞及成纤维细胞合成,它对于内皮细胞之间的连接及内皮细胞附着于内皮下层的胶原纤维起重要作用,从而保证了微血管完整性。因此,研究外源性FN对某些病理因素造成血管内皮细胞损伤的修复作用是很有价值的,鉴于从血浆分离FN存在着血浆资源不足和可能传播血源性传染病的问题,同时还由于FN分子量大,达450KD,全分子表达又存在技术上的难题。因此利用基因工程技术表达FN的功能区多肽并研究其功能,来代替FN可以说是一种可行的办法。TNF-α是一种重要的炎性介质,感染等病理情况下TNF-α增加,我们以往的研究表明,FN及肝素结合域多肽可防治脂多糖内毒素小鼠DIC的发生,其机制就涉及抑制TNF-α的表达。因此,根据前期研究基础,本实验的目的在于建立TNF-α损伤血管内皮细胞损伤的模型及半乳糖胺增敏的内毒素血症小鼠模型,通过体外和体内实验,研究FN及肝素结构域多肽FNNHBSPP、FNCHBSPP对血管内皮细胞的影响。1.人脐静脉内皮细胞的培养和鉴定采用酶消化法培养人脐静脉内皮细胞;用免疫组化法检测Ⅷ因子鉴定内皮细胞。2.FN和两种肝素结合域多肽FNNHBSPP、FNCHBSPP制备用明胶亲和层析的方法从人新鲜血浆分离FN;经SDS-聚丙烯凝胶电泳和免疫印迹(Western blot)法鉴定FN纯度和特异性;两种肝素结合域多肽FNNHBSPP、FNCHBSPP由本研究所自行制备的酵母表达载体中表达和制备。3.建立TNF-α损伤人脐静脉内皮细胞模型3.1实验分组:(1)空白对照组:不加TNF-α处理的正常生长的HUVECs;(2)不同浓度TNF-α处理组:分别用5ng/ml,10 ng/ml,20 ng/mlTNF-α作用18h;(3)不同时间TNF-α处理组:用10 ng/mlTNF-α分别作用6h,12h,,18h,24h。3.2用ELISA法检测各组内皮细胞培养上清中PAI-1、tPA、sICAM-1的含量4.研究FN及两种肝素结合域多肽FNNHBSPP、FNCHBSPP对TNF-α作用的内皮细胞的影响4.1实验分组:(1)空白对照组:不加TNF-α处理的正常生长的HUVECs;(2)TNF-α作用组:10ng/mlTNF-α作用18h,加入与多肽等体积的培养液6h;(3)TNF-α+FNNHBSPP作用组:10ng/ml TNF-α作用18h,加入浓度为600ug/ml的FNNHBSPP作用6h;(4)TNF-α+FNCHBSPP作用组:10ng/mlTNF-α作用18h,加入浓度为600ug/ml的FNCHBSPP作用6h;(5)TNF-α+FN作用组:10ng/mlTNF-α作用18h,加入浓度为600ug/ml的FN作用6h。4.2用倒置显微镜和透射电镜观察FN及其两种肝素结合域多肽FNNHBSPP、FNCHBSPP对TNF-α损伤的内皮细胞作用下的形态和超微结构4.3用ELISA法检测各组内皮细胞培养上清中PAI-1、tPA、sICAM-1的含量5.研究FN及其两种肝素结合域多肽FNNHBSPP、FNCHBSPP对内毒素血症小鼠肝、肺组织血管内皮细胞的影响5.1建立半乳糖胺增敏的内毒素血症小鼠模型应用半乳糖胺增敏内毒素脂多糖建立内毒素血症小鼠模型,即给小鼠腹腔注射半乳糖胺(400mg/kg)和内毒素(100ug/kg)。5.2实验分组:正常组、内毒素血症组、FN作用组、FNNHBSPP作用组和FNCHBSPP作用组。在注射半乳糖胺及内毒素前半小时从其尾静脉分别注射FN、FNNHBSPP、FNCHBSPP(20mg/kg),内毒素血症组给等体积生理盐水。5.3病理形态学观察各实验组小鼠肝、肺组织血管内皮细胞腹腔注射药物9小时后,颈椎脱臼法处死小鼠,取小鼠的肝、肺组织,用10%福尔马林固定,石蜡包埋,切片,常规HE染色,做病理形态学观察。5.4免疫组织化学法检测肝、肺组织血管内皮细胞纤维连接蛋白(FN)表达取得了以下结果与结论:1.建立了稳定传代的人脐静脉内皮细胞培养体系。2.建立了TNF-α损伤内皮细胞的模型,不同浓度的TNF-α(5,10,20ng/ml)作用于HUVECs18h,培养液中PAI-1、sICAM-1的浓度与空白对照组相比,各组均增加(p﹤0.01),其中以10ng/ml的TNF-α作用最明显;用同一浓度10ng/ml的TNF-α作用6,12,18,24h,与空白对照组相比,PAI-1、sICAM-1的浓度均有明显增高(p﹤0.01),在18hTNF-α作用最明显;tPA各组无明显差异(p﹥0.05)。3.FN及其肝素结合域多肽FNNHBSPP、FNCHBSPP作用组的内皮细胞,培养液上清中PAI-1及sICAM-1的浓度均比TNF-α组明显减低(p﹤0.01),FN及两种多肽的作用无明显差异(p﹥0.05)。4.建立了内毒素血症小鼠模型,予FN及多肽FNNHBSPP、FNCHBSPP作用组的小鼠肝、肺组织血管内皮细胞病变明显减轻,血管内皮细胞的FN表达比内毒素血症小鼠增强。5.结论:FN及其多肽FNNHBSPP、FNCHBSPP对TNF-α及败血症所致的血管内皮细胞的损伤具有保护作用;FN及其两种肝素结合域多肽在这方面的作用无明显差异。更多还原

【Abstract】 Vascular endothelial cells are an interface between blood vessel walls and blood flow, and play important roles in physiological functions.1.Constitute a natural barrier to maintain endometrial vascular smooth, preventing platelet and leukocyte adhesion and harmful substances invasive vascular wall.2.Permeability barrier formed to regulate the exchange of material and active transport function .3.Thrombosis and hemostasis involved in the process, and maintain dynamic equilibrium ,which is including tissue plasminogen activator, plasminogen activator inhibitor-1 (PAI-1). Many pathological conditions such as severe infection, DIC, are involved in vascular endothelial cells function, and then aggravate the pathological process, forming a vicious cycle. Fibronectin (FN) is a glycoprotein which is presence in the extracellular matrix, plasma and other body fluids .As an important adhesion molecule with several integrin receptor binding, FN participates in a number of important physiological and pathological processes including cell adhesion, migration and extracellular matrix assembly, embryonic differentiation, wound healing, immune and inflammatory. And various diseases are closely linked. FN not only has a function anti-infective and but also maintain the integrity and permeability of microvascular. It is also called"rationalα2 surface-binding protein", "cell surface protein" and "cell adhesion molecules." FN is known mainly divided into two types: cellular and plasma-fibronectin. cFN is secreted by endothelial cells and fibroblasts. It has play an important role over the endothelial cells surface and endothelial cells attached to the subendothelial layer collagen fibers .So cFN ensure the integrity of microvascular .It is great value to study how exogenous FN repair vascular endothelial cells on certain pathological .There are still some problems accompanying with extracting FN from human plasma ,for example lack of blood resource ,spread of blood-borne infectious disease , big molecular weight and the technical problems to expression whole molecule . Therefore using genetic engineering technology express the peptide functional areas of FN and study their functions. It is a practical ways using recombinant polypeptides to replace FN. TNF-αis an important inflammatory mediators. In some pathological cases, such as infection, there is an increased TNF-αlevel. Our previous studies showed that FN and recombinant heparin-binding domain peptide can control endotoxin lipopolysaccharide mice in the DIC and the mechanism involving the suppression of TNF-αexpression. Therefore the main purpose was to study the influence of FN and its polypeptides on vascular endothelial cells in vitro and in vivo experiments.The subjects of thesis were focused on the following aspects:1. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunohistochemistry;2. FN and its recombinant heparin-binding site polypeptides preparation. Purification of FN from plasma was achieved by gelatin-sepharose affinity chromatography. The purifed product was analyzed by SDS-PAGE and western blot, and then was quantified by ELISA. The high pure and specific FNNHBD and FNCHBD were obtained by expression and preparation of recombinant FN heparin-binding domain polypeptide in pichia expression system;3. Established a TNF-αdamage endothelial cells model.3.1 Experimental group :(1) the first group of HUVECs was blank group;(2) the second group were treated with 5ng/ml,10 ng/ml or 20 ng/ml TNF-αfor 18h;(3) the third group were treated with 10 ng/ml TNF-αfor 6h,12h,18h or 24h respectively.3.2 PAI-1, tPA, sICAM-1 levels in the endothelial cells culture supernatant were detected by ELISA.4. We investigate the effect of FN and the two polypeptides on HUVECs following treatment with TNF-α.4.1 Experimental group :(1) the first group of HUVECs was blank group;(2) the second group were treated with 10 ng/ml TNF-αfor 18h,and then mediam which has the same volume as peptides was added;(3) the third group were treated with 10ng/ml TNF-αfor 18h,and then 600 ug/ml FNNHBSPP was added;(4) the fourth group were treated with 10ng/ml TNF-αfor 18h,and then 600 ug/ml FNCHBSPP was added;(5) the last group were treated with 10ng/ml TNF-αfor 18h,and then 600 ug/ml FN was added.4.2 Observe the endothelial cells morphology and ultrastructure using an inverted microscope and transmission electron microscopy.4.3 PAI-1, tPA, sICAM-1 levels in the endothelial cells culture supernatant were detected by ELISA;5.Study Fibronectin and Recombinant Heparin-binding Site Polypeptides of Human Fibronectin’ effects on sepsis mouse VECs.5.1 Establishment of sepsis mouse model.5.2 Experimental group : the normal group、the blank group、FN-treated group、FNNHBSPP-treated group and FNCHBSPP-treated group. We established the septic mouse model by the injuection of 100ug/kg lipopolysaccharide(LPS)and 400mg/kg D-Galactosamine(GalN) into the mice which is according to A.-H. Kwon,s and our test condiction. We indicated that FN、FNNHBSPP and FNCHBSPP administrated intravenously 30min before LPS/GalN injection at the base of 20mg/kg. The control group gave the volume saline.5.3 Pathological observation of the liver, lung vascular changes . 9h after intraperitoneal injection drug, the mice were killed by cervical dislocation. The mice liver and lung tissues fixed with 10% formalin, embedded in paraffin, slicing, conventional HE staining.5.4 Immunohistochemical detect the vascular endothelial cells fibronectin (FN) expression.The major results and conclusions were obtained as follows:1. Isolation and culure human umbilical vein endothelial cells;2. Established TNF-αdamage endothelial cells model.Our results shew that TNF-alpha increased the levels of PAI-1 and sICAM-1 in cultured human umbilical vein endothelial cells (HUVECs) compared HUVECs in normal conditions(p﹤0.01); And the maximum stimulatory effect of 10ng/ml of TNF-alpha was observed after 18h. There was no significant difference of tPA level(p﹥0.05);3. The levels of PAI-1 and sICAM-1 in cultured HUVECs which were treated with FN、FNNHBSPP or FNCHBSPP were decreased than the TNF-alpha -treated group; Also, there was no significant difference between the FN、FNNHBSPP and FNCHBSPP treated groups(p﹥0.05);4. We established the septic mouse model. The Vascular endothelial cells injury risks in septic mouse’s liver and lung were reduced which were treated with FN and FNNHBSPP, FNCHBSPP. Also, the expression of FN in vascular endothelial cells was more than that of the septic mouse;5. Conclusion: FN and its polypeptides FNNHBSPP、FNCHBSPP had a protective effect on VECs which were damaged by TNF-αor LPS/GalN.Also, there was no significant difference between the FN、FNNHBSPP and FNCHBSPP treated groups.更多还原