發(fā)布時(shí)間：2018-01-09 07:10

  本文關(guān)鍵詞：體外高糖狀態(tài)LPS對(duì)血管內(nèi)皮細(xì)胞分泌t-PA和PAI-1的影響　出處：《江西醫(yī)學(xué)院》2005年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 臍靜脈 內(nèi)皮細(xì)胞 分離 內(nèi)毒素 葡萄糖 纖溶酶原激活物抑制劑-1 組織型纖溶酶原激活物

【摘要】：目的:探討人臍靜脈內(nèi)皮細(xì)胞(Human umbilical endothelial cell,HUVEC)的分離培養(yǎng)及鑒定方法,在建立體外血管內(nèi)皮細(xì)胞原代培養(yǎng)模型的基礎(chǔ)上觀察不同濃度細(xì)菌內(nèi)毒素(Lipopolyssacarides,LPS)和葡萄糖(Glucose,GLU) 對(duì)血管內(nèi)皮細(xì)胞組織型纖溶酶原激活物(Tissue plasminogen activator, t-PA)和纖溶酶原激活物抑制劑-1(Plasminogen activator inhibitor-1,PAI-1)表達(dá)的影響,為進(jìn)一步研究各種血栓性疾病的形成機(jī)制提供實(shí)驗(yàn)依據(jù)。方法:采用5 號(hào)帶柄一次性靜脈輸液針灌注消化液,分別用0.125%、0.25%胰蛋白酶和0.1%膠原酶消化臍靜脈內(nèi)膜,比較三種酶液消化的結(jié)果,并在倒置相差顯微鏡下觀察消化產(chǎn)物形態(tài)特點(diǎn),同時(shí)用免疫組織化學(xué)的方法對(duì)所得細(xì)胞進(jìn)行鑒定。采用酶聯(lián)免疫吸附試驗(yàn)(ELISA)檢測(cè)各組HUVEC 培養(yǎng)液中PAI-1 和t-PA 含量。并用逆轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(RT-PCR)逆轉(zhuǎn)錄擴(kuò)增PAI-1 和內(nèi)參照三磷酸甘油醛脫氫酶(GAPDH)mRNA,擴(kuò)增產(chǎn)物經(jīng)瓊脂糖凝膠電泳分離后,使用凝膠分析軟件測(cè)定各條帶光密度值。結(jié)果:1. 0.125% 、0.25%胰蛋白酶和0.1%膠原酶的最佳消化時(shí)間分別為15min,10min,12min。2. 倒置相差顯微鏡下觀察內(nèi)皮細(xì)胞為單層生長(zhǎng),鵝卵石樣鑲嵌排列。免疫組織化學(xué)方法檢測(cè)表明細(xì)胞內(nèi)存在Ⅷ因子相關(guān)抗原。3. 1μg/ml LPS 能明顯促進(jìn)HUVEC 分泌PAI-1,2h開(kāi)始升高,24h 達(dá)最大效應(yīng)。4. 5.5mmol/LGLU 作用HUVEC 后,各不同時(shí)間段PAI-1 分泌量無(wú)明顯變化。40mmol/LGLU 作用HUVEC 24 h,PAI-1 蛋白分泌量達(dá)高峰,并明顯上調(diào)PAI-1 mRNA 水平。5.各濃度GLU 和LPS 對(duì)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.

【學(xué)位授予單位】：江西醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類(lèi)號(hào)】：R363

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本文編號(hào)：1400449