本文選題：脂多糖 + 血管內(nèi)皮細(xì)胞��； 參考：《首都醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的 探討脂多糖（LPS）對(duì)基于人臍靜脈內(nèi)皮細(xì)胞（HUVEC）的內(nèi)皮通透性增加的作用機(jī)制. 方法 將HUVEC分為以下各組:A組（對(duì)照組，無(wú)預(yù)處理），B組（LPS刺激組），C組（無(wú)功能RNAi慢病毒預(yù)處理HUVEC+LPS刺激），D組（慢病毒RNAi抑制HUVEC中ANXA2表達(dá)+LPS刺激），E組（Src激酶抑制劑預(yù)處理HUVEC+LPS刺激）。利用Bradford法測(cè)定各組HUVEC細(xì)胞通透性，qPCR測(cè)定各組細(xì)胞ANXA2及VE-Cadherin mRNA轉(zhuǎn)錄水平，Western-blot測(cè)定ANXA2及VE-Cadherin蛋白表達(dá)水平。 結(jié)果 1.各組HUVEC通透性變化: 與A組相比，B組通透性增加，而D組通透性大于B組。E組通透性高于A組但小于其它各組。 2.各組間ANXA2及VE-Cadherin mRNA轉(zhuǎn)錄變化 相對(duì)A組，B組加入LPS處理后ANXA2轉(zhuǎn)錄無(wú)明顯變化，VE-Cadherin轉(zhuǎn)錄減少，而在D組利用RNAi沉默ANXA2基因后，ANXA2轉(zhuǎn)錄明顯減少，VE-Cadherin轉(zhuǎn)錄亦減少。 在E組內(nèi)，經(jīng)Src激酶抑制劑處理后，HUVEC內(nèi)ANXA2轉(zhuǎn)錄較其它各組明顯增加，而VE-Cadherin轉(zhuǎn)錄高于D組，低于B組。 3.各組間ANXA2及VE-Cadherin表達(dá)差異 與A組相比，B處理組ANXA2蛋白濃度水平無(wú)變化，而VE-Cadherin蛋白濃度水平明顯下降。而在D組內(nèi)，利用RNAi沉默ANXA2基因后，ANXA2蛋白濃度明顯減少，與B組相比,VE-Cadherin蛋白濃度減低，在E組內(nèi)，經(jīng)Src激酶抑制劑處理后，，HUVEC內(nèi)ANXA2蛋白濃度較其它各組明顯增加，而VE-Cadherin蛋白濃度亦高于其它各組。 結(jié)論 當(dāng)LPS刺激HUVEC時(shí)會(huì)導(dǎo)致其構(gòu)成的內(nèi)皮通透性的增加，VE-Cadherin及ANXA2正常轉(zhuǎn)錄表達(dá)是LPS作用下基于HUVEC的內(nèi)皮通透性增加的保護(hù)性因素。HUVEC內(nèi)Src激酶的存在可能抑制了LPS刺激下ANXA2的過(guò)度表達(dá)，通過(guò)藥物抑制Src激酶的功能可以增加ANXA2及VE-Cadherin的表達(dá)，從而減輕LPS對(duì)基于ANXA2及VE-Cadherin的細(xì)胞間連接的破壞作用。
[Abstract]:Objective to investigate the mechanism of lipopolysaccharide (LPS) on the increase of endothelial permeability based on human umbilical vein endothelial cells (HUVECs). Methods HUVEC was divided into the following groups: group A (control group). LPS-stimulated HUVEC was pretreated with lentivirus without preconditioning (lentivirus preconditioning HUVEC LPS / D) (lentivirus RNAi inhibited ANXA2 expression in HUVEC and LPS-stimulated LPS-stimulated HUVEC was pretreated with SRC kinase inhibitor. The transcriptional level of ANXA2 and VE-Cadherin mRNA in HUVEC cells was determined by Bradford method and the expression of ANXA2 and VE-Cadherin protein was detected by Western-blot. Results 1. The changes of HUVEC permeability in each group: compared with group A, the permeability of group B was increased, but the permeability of group D was higher than that of group B. E was higher than that of group A, but less than that of other groups. 2. The transcriptional changes of ANXA2 and VE-Cadherin mRNA in group A and VE-Cadherin had no significant change compared with group A (group B) treated with LPS. However, in group D, the transcription of ANXA2 decreased significantly after silencing ANXA2 gene by RNAi. In group E, the transcription of ANXA2 was also decreased after silencing ANXA2 gene. After treatment with SRC kinase inhibitor, ANXA2 transcription in HUVEC was significantly higher than that in other groups, while VE-Cadherin transcription was higher in group D than that in group B. There was no change in ANXA2 and VE-Cadherin expression among groups compared with group A, but the level of VE-Cadherin protein decreased significantly. In group D, the concentration of ANXA2 protein was significantly decreased after silencing ANXA2 gene by RNAi, and the concentration of ANXA2 protein in HUVEC was significantly higher in group E than that in group B after treatment with SRC kinase inhibitor. Conclusion when HUVEC was stimulated by LPS, the endothelial permeability of HUVEC was increased. The normal transcriptional expression of VE-Cadherin and ANXA2 was the protective factor of HUVEC-based endothelial permeability increase induced by LPS. The presence of SRC kinase may inhibit the overexpression of ANXA2 induced by LPS. Drug inhibition of Src kinase can increase the expression of ANXA2 and VE-Cadherin, thus attenuating the damage of LPS to the intercellular junctions based on ANXA2 and VE-Cadherin.
【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R459.7

【共引文獻(xiàn)】

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