纤连蛋白及其重组肝素结合域多肽对血管内皮细胞损伤的影响

发布时间：2018-01-12 17:39

本文关键词：纤连蛋白及其重组肝素结合域多肽对血管内皮细胞损伤的影响　出处：《福建医科大学》2008年硕士论文　论文类型：学位论文

【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㩳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㩳0.01),在18hTNF-α作用最明显;tPA各组无明显差异(p㧐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㩳0.01),FN及两种多肽的作用无明显差异(p㧐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。
[Abstract]:Vascular endothelial cells have very important physiological functions, mainly reflected in: 1. form a natural barrier, maintain intimal smooth, prevent the adhesion of platelets and leukocytes and harmful substances into the vessel wall; 2. permeability barrier, regulating the material exchange and active transport function; 3. involved in thrombosis and hemostasis, maintain dynamic balance, including tissue plasminogen activator (t-PA), plasminogen activator inhibitor -1 (PAI-1). Many pathological conditions such as severe infection, dysfunction of DIC is involved in vascular endothelial cells, and then aggravate the pathological process, forming a vicious spiral. So how to restore the morphology and function of vascular endothelial cells under pathological conditions has been imbalance the focus of academic research. Fibronectin (Fibronectin, FN) is a widely exist in the extracellular fluid, cell surface glycoproteins and connective tissue and so on. In the dimension of anti infection. Has an important role to microvascular integrity and permeability, so it had "conditioning alpha 2 surface binding protein", "cell surface proteins and cell adhesion molecules called". Known FN is mainly divided into two types of cells and plasma cells, FN is mainly composed of endothelial cells and fibroblasts cell synthesis, for connection between endothelial cells and endothelial cell adhesion to subendothelial collagen fibers play an important role, so as to ensure the microvascular integrity. Therefore, the research of exogenous FN on some pathological factors to repair the injury of vascular endothelial cells with is very valuable, given the separation of FN from plasma exists the plasma resource shortage and problems may spread of infectious diseases, but also because the molecular weight of FN, up to 450KD, the expression and technical problems. Therefore, using genetic engineering expression of FN multi function area Peptide and study its function, it can be said is a feasible way for.TNF- alpha is a kind of important inflammatory mediators instead of FN, infection and other pathological conditions TNF- increased, our previous study showed that FN and heparin binding domain polypeptide can prevent the occurrence of DIC in mice with LPS, the mechanism involves inhibition of expression of TNF- alpha. Therefore, based on the previous research, the purpose of this experiment is that mouse model of endotoxemia model and galactosamine establish damage vascular endothelial cell injury alpha TNF- sensitization, by in vitro and in vivo experiments, the research of FN and heparin domain polypeptide FNNHBSPP, effects of FNCHBSPP on vascular endothelial cells.
1. culture and identification of human umbilical vein endothelial cells by enzyme digestion and cultured human umbilical vein endothelial cells; Determination of factor VIII characterization of endothelial cells by immune group.
2.FN and two kinds of heparin binding domain polypeptide FNNHBSPP, FNCHBSPP method for preparation of gelatin affinity chromatography for separating FN from fresh human plasma; by SDS- polyacrylamide gel electrophoresis and immunoblotting (Western blot) method for identification of FN purity and specificity; two heparin binding domain polypeptide FNNHBSPP, FNCHBSPP by the self prepared yeast expression preparation and expression vector.
3. the establishment of TNF- alpha injury human umbilical vein endothelial cell model
3.1 experiment group: (1) blank control group: normal growth HUVECs without TNF- alpha treatment; (2) different concentrations of TNF- alpha treatment group: 5ng/ml, 10 ng/ml, 20 ng/mlTNF- alpha acting 18h respectively; (3) TNF- TNF- treatment group at different time: 6h, 12h, ng/mlTNF-, 10 with ng/mlTNF- ng/mlTNF-.
3.2 the content of PAI-1, tPA, sICAM-1 in cultured supernatant of endothelial cells was detected by ELISA
4. study the effect of FN and two heparin binding domain polypeptide FNNHBSPP, FNCHBSPP on the endothelial cells of TNF- alpha
4.1 experimental groups: (1) blank control group: without the normal growth of TNF- treated HUVECs; (2) TNF- group: alpha 10ng/mlTNF- alpha 18h, 6h liquid added to the culture and peptide volume; (3) +FNNHBSPP group: 10ng/ml TNF- alpha TNF- alpha 18h, concentration of FNNHBSPP was added the role of 6h 600ug/ml; (4) TNF- group: 10ng/mlTNF- alpha +FNCHBSPP alpha 18h, concentration of FNCHBSPP was added 6h 600ug/ml; (5) TNF- group: 10ng/mlTNF- alpha alpha +FN 18h, adding concentration of FN 6h. 600ug/ml
4.2, we observed the morphology and ultrastructure of FN and its two heparin binding domains FNNHBSPP and FNCHBSPP on TNF- injured endothelial cells under inverted microscope and transmission electron microscope.
4.3 the content of PAI-1, tPA, sICAM-1 in cultured supernatant of endothelial cells was detected by ELISA
5. study the effect of FN and its two heparin binding domain polypeptide FNNHBSPP, FNCHBSPP on the vascular endothelial cells of liver and lung tissue of mice with endotoxemia
5.1 a Galacto amine sensitized endotoxemia mouse model was established. Galactoamine sensitized endotoxin lipopolysaccharide (LPS) was used to establish endotoxemia mice model, namely, intraperitoneal injection of galactoamine (400mg/kg) and endotoxin (100ug/kg).
5.2, the experimental group was divided into normal group, endotoxemia group, FN action group, FNNHBSPP action group and FNCHBSPP action group. FN, FNNHBSPP and FNCHBSPP (20mg/kg) were injected from the caudal vein half an hour before injecting galactoamine and endotoxin, and the volume of physiological saline was given to the endotoxemia group.
5.3 pathomorphological observation. After 9 hours of intraperitoneal injection of liver and lung endothelium in each experimental group, the mice were killed by cervical dislocations, and the liver and lung tissues of mice were fixed with 10% formalin and paraffin embedded. Slices were stained with conventional HE staining for pathomorphological observation.
5.4 immunohistochemical method to detect the expression of fibronectin (FN) in the vascular endothelial cells of the liver and lung
The following results and conclusions are obtained.
1. a cultured human umbilical vein endothelial cell culture system was established.
2. the establishment of the endothelial cell injury TNF- alpha model, different concentrations of TNF- alpha (5,10,20ng/ml) in HUVECs18h, medium PAI-1, compared with the blank control group the concentration of sICAM-1, were increased (P? 0.01), of which the most obvious effect of 10ng/ml TNF- alpha TNF- alpha 6,12,18,24h; with the same concentration 10ng/ml, compared with the control group PAI-1, sICAM-1 concentrations were significantly higher (P? 0.01), the most obvious effect of 18hTNF- alpha had no significant difference in tPA group; (P? 0.05).
The concentrations of PAI-1 and sICAM-1 in endothelial cells of 3.FN and heparin binding domain FNNHBSPP and FNCHBSPP groups were significantly lower than those in TNF- group (P? 0.01), but there was no significant difference in FN and two peptides (P 0.05).
4. the establishment of a mouse model of endotoxemia, with FN and FNNHBSPP peptides, mouse liver FNCHBSPP group, lung tissue vascular endothelial cell lesion significantly reduced, vascular endothelial cells, enhanced expression of FN than endotoxaemia in mice.
5. conclusion: FN and its polypeptide FNNHBSPP and FNCHBSPP have protective effects on the injury of vascular endothelial cells induced by TNF- alpha and septicemia. FN and its two heparin binding domain peptides have no significant difference in this aspect.

【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363

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