体外高糖状态LPS对血管内皮细胞分泌t-PA和PAI-1的影响

发布时间：2018-01-09 07:10

本文关键词：体外高糖状态LPS对血管内皮细胞分泌t-PA和PAI-1的影响　出处：《江西医学院》2005年硕士论文　论文类型：学位论文

【摘要】：目的:探讨人脐静脉内皮细胞(Human umbilical endothelial cell,HUVEC)的分离培养及鉴定方法,在建立体外血管内皮细胞原代培养模型的基础上观察不同浓度细菌内毒素(Lipopolyssacarides,LPS)和葡萄糖(Glucose,GLU) 对血管内皮细胞组织型纤溶酶原激活物(Tissue plasminogen activator, t-PA)和纤溶酶原激活物抑制剂-1(Plasminogen activator inhibitor-1,PAI-1)表达的影响,为进一步研究各种血栓性疾病的形成机制提供实验依据。方法:采用5 号带柄一次性静脉输液针灌注消化液,分别用0.125%、0.25%胰蛋白酶和0.1%胶原酶消化脐静脉内膜,比较三种酶液消化的结果,并在倒置相差显微镜下观察消化产物形态特点,同时用免疫组织化学的方法对所得细胞进行鉴定。采用酶联免疫吸附试验(ELISA)检测各组HUVEC 培养液中PAI-1 和t-PA 含量。并用逆转录聚合酶链式反应(RT-PCR)逆转录扩增PAI-1 和内参照三磷酸甘油醛脱氢酶(GAPDH)mRNA,扩增产物经琼脂糖凝胶电泳分离后,使用凝胶分析软件测定各条带光密度值。结果:1. 0.125% 、0.25%胰蛋白酶和0.1%胶原酶的最佳消化时间分别为15min,10min,12min。2. 倒置相差显微镜下观察内皮细胞为单层生长,鹅卵石样镶嵌排列。免疫组织化学方法检测表明细胞内存在Ⅷ因子相关抗原。3. 1μg/ml LPS 能明显促进HUVEC 分泌PAI-1,2h开始升高,24h 达最大效应。4. 5.5mmol/LGLU 作用HUVEC 后,各不同时间段PAI-1 分泌量无明显变化。40mmol/LGLU 作用HUVEC 24 h,PAI-1 蛋白分泌量达高峰,并明显上调PAI-1 mRNA 水平。5.各浓度GLU 和LPS 对HUVEC
[Abstract]:Objective: To investigate the effect of human umbilical vein endothelial cells (Human umbilical endothelial cell, HUVEC) isolation and identification of the method in the establishment of vascular endothelial cells cultured in vitro model based on the observation of different concentrations of bacterial endotoxin (Lipopolyssacarides, LPS) and glucose (Glucose, GLU) on endothelial cell tissue plasminogen activator (Tissue plasminogen activator, t-PA) and plasminogen activator inhibitor -1 (PAI-1 Plasminogen activator inhibitor-1) expression, to provide experimental basis for further study of the mechanism of formation of various thrombotic diseases. Methods: using 5 shank disposable venous infusion needle infusion fluid, respectively 0.125%, 0.25% and 0.1% trypsin collagenase digestion umbilical vein, comparison of three kinds of enzyme digestion results, and observe the morphological characteristics of digestive products under the inverted microscope. At the same time for free Methods for chemical epizootics were identified on the cells. Using enzyme-linked immunosorbent assay (ELISA) were detected in HUVEC culture of PAI-1 and the content of t-PA in the solution. And using reverse transcription polymerase chain reaction (RT-PCR) amplification of three reverse transcriptase to glyceraldehyde phosphate dehydrogenase (GAPDH) in PAI-1 and mRNA, the PCR products were separated by agarose gel electrophoresis after using gel analysis software, determination of optical density value of each band. Results: 1. of 0.125%, the best digestion time of 0.25% trypsin and collagenase 0.1% respectively 15min, 10min, under the microscope of endothelial cell monolayer growth difference of 12min.2. inversion, cobblestone mosaic arrangement. Immunohistochemistry showed that cells in factor VIII related antigen.3. 1 g/ml LPS could promote the secretion of HUVEC PAI-1,2h 24h began to increase, reached the maximum effect of.4. 5.5mmol/LGLU HUVEC, all at the same time PAI- 1 secretion did not change significantly..40mmol/LGLU acted on HUVEC 24 h, PAI-1 protein secretion reached a peak, and significantly increased PAI-1 mRNA level,.5. concentration GLU and LPS to HUVEC.

【学位授予单位】：江西医学院
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R363

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