脂多糖致血管内皮细胞通透性增加的机制研究

发布时间：2018-06-08 06:08

本文选题：脂多糖 + 血管内皮细胞　；参考：《首都医科大学》2015年硕士论文

【摘要】：目的探讨脂多糖（LPS）对基于人脐静脉内皮细胞（HUVEC）的内皮通透性增加的作用机制. 方法将HUVEC分为以下各组:A组（对照组，无预处理），B组（LPS刺激组），C组（无功能RNAi慢病毒预处理HUVEC+LPS刺激），D组（慢病毒RNAi抑制HUVEC中ANXA2表达+LPS刺激），E组（Src激酶抑制剂预处理HUVEC+LPS刺激）。利用Bradford法测定各组HUVEC细胞通透性，qPCR测定各组细胞ANXA2及VE-Cadherin mRNA转录水平，Western-blot测定ANXA2及VE-Cadherin蛋白表达水平。结果 1.各组HUVEC通透性变化: 与A组相比，B组通透性增加，而D组通透性大于B组。E组通透性高于A组但小于其它各组。 2.各组间ANXA2及VE-Cadherin mRNA转录变化相对A组，B组加入LPS处理后ANXA2转录无明显变化，VE-Cadherin转录减少，而在D组利用RNAi沉默ANXA2基因后，ANXA2转录明显减少，VE-Cadherin转录亦减少。在E组内，经Src激酶抑制剂处理后，HUVEC内ANXA2转录较其它各组明显增加，而VE-Cadherin转录高于D组，低于B组。 3.各组间ANXA2及VE-Cadherin表达差异与A组相比，B处理组ANXA2蛋白浓度水平无变化，而VE-Cadherin蛋白浓度水平明显下降。而在D组内，利用RNAi沉默ANXA2基因后，ANXA2蛋白浓度明显减少，与B组相比,VE-Cadherin蛋白浓度减低，在E组内，经Src激酶抑制剂处理后，，HUVEC内ANXA2蛋白浓度较其它各组明显增加，而VE-Cadherin蛋白浓度亦高于其它各组。结论当LPS刺激HUVEC时会导致其构成的内皮通透性的增加，VE-Cadherin及ANXA2正常转录表达是LPS作用下基于HUVEC的内皮通透性增加的保护性因素。HUVEC内Src激酶的存在可能抑制了LPS刺激下ANXA2的过度表达，通过药物抑制Src激酶的功能可以增加ANXA2及VE-Cadherin的表达，从而减轻LPS对基于ANXA2及VE-Cadherin的细胞间连接的破坏作用。
[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.
【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R459.7

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