硫丹引起HUVEC-C细胞骨架重构的机制分析

发布时间：2018-06-25 13:12

本文选题：硫丹 + 细胞骨架　；参考：《大连海事大学》2017年硕士论文

【摘要】：硫丹属于持久性有机污染物,具有难降解性、生物蓄积性、长距离迁移性以及高毒性。本课题前期研究发现硫丹具有内皮细胞毒性,但硫丹对内皮细胞屏障功能的影响尚不清楚。本研究以人脐静脉内皮细胞(HUVEC-C)为实验对象,主要探讨了硫丹对血管内皮细胞骨架的影响及其信号通路,并着重分析了由于细胞骨架重构而引起的血管内皮通透性改变的分子机制。本研究利用免疫染色法分析了硫丹暴露对HUVEC-C细胞骨架的微丝结构(F-actin)和粘着斑激酶(FAK)分布的影响,用qRT-PCR方法测定了骨架相关基因ROCK2和PXN的mRNA表达水平。用蛋白印记法(Western blot)分析了基质金属蛋白酶(MMP-3)、层粘连蛋白(LAMC1)、FAK和pFAK的表达。用RNAi干扰的方法探讨了 LAMC1对HUVEC-C细胞存活率、细胞骨架和内皮细胞通透性的影响。用Western blot和qRT-PCR方法分别检测了细胞间粘附连接蛋白E-cadherin和β-catenin及缝隙连接关键因子Cx43的变化。最后利用Coaster transwell system探讨了硫丹暴露影响HUVEC-C细胞通透性的时间和剂量效应。结果发现,硫丹暴露能够引起HUVEC-C细胞骨架破坏,ROCK2和PXN的mRNA表达降低,MMP-3蛋白表达升高而降解LAMC1,引起FAK和pFAK的表达降低,这些提示硫丹暴露可能通过影响MMP3/LAMC1/pFAK信号通路而破坏内皮细胞骨架。LAMC1 siRNAs显著降低HUVEC-C细胞生存率,引起细胞骨架破坏和内皮细胞通透性升高,说明LAMC1在维持血管内皮细胞结构与功能中起重要作用。硫丹通过降低E-cadherin和β-catenin表达而影响内皮细胞间连接。硫丹使缝隙连接功能减弱,与Cx43的表达降低有关。硫丹暴露48h导致内皮细胞通透性的升高,具有剂量效应关系。本研究揭示了硫丹能够通过破坏血管内皮细胞骨架的微丝结构,减弱细胞间粘附连接和缝隙连接功能,从而引起了内皮细胞的通透性增加,影响血管内皮细胞的屏障作用。首次发现LAMC1参与了硫丹暴露引起的血管内皮细胞骨架重构和通透性变化。本研究为硫丹是否作为危险因素导致血管稳态失衡而引起心血管疾病提供了重要的理论依据。
[Abstract]:Endosulfan is a persistent organic pollutant, which has difficult degradation, bioaccumulation, long distance migration and high toxicity. The previous study found that endosulfan has endothelial cell toxicity, but the effect of endosulfan on endothelial cell barrier function is not clear. This study was mainly focused on human umbilical vein endothelial cells (HUVEC-C) as the experimental object. The effect of endosulfan on vascular endothelial cytoskeleton and its signaling pathway, and the molecular mechanism of vascular endothelial permeability change caused by cytoskeleton reconstruction were emphatically analyzed. The effects of endosulfan exposure on the distribution of F-actin and FAK of HUVEC-C cytoskeleton were analyzed by immunostaining, and qR was used to analyze the effects of endosulfan exposure on the distribution of the microwire structure of the cytoskeleton and the adhesion kinase (FAK). T-PCR method was used to determine the mRNA expression level of skeleton related genes ROCK2 and PXN. The expression of matrix metalloproteinase (MMP-3), laminin (LAMC1), FAK and pFAK were analyzed by protein imprinting (Western blot). The effects of LAMC1 on the survival rate, cytoskeleton and endothelial cell permeability were investigated by RNAi interference. The changes in the intercellular adhesion connexin E-cadherin, beta -catenin and the key factor Cx43 of gap junction were detected by RN blot and qRT-PCR methods. Finally, the time and dose effects of endosulfan exposure on the permeability of HUVEC-C cells were investigated using Coaster Transwell system. The results showed that endosulfan exposure could cause the cytoskeleton of HUVEC-C cells. The mRNA expression of ROCK2 and PXN decreased, the expression of MMP-3 protein was increased and LAMC1 was degraded, and the expression of FAK and pFAK decreased. These suggest that endosulfan exposure may reduce the.LAMC1 siRNAs of the endothelial cytoskeleton by affecting the MMP3/LAMC1/pFAK signaling pathway and significantly reduce the survival rate of HUVEC-C cells, and cause cytoskeleton destruction and endothelial cell permeability. It is suggested that LAMC1 plays an important role in maintaining the structure and function of vascular endothelial cells. Endosulfan affects the connection between endothelial cells by reducing the expression of E-cadherin and beta -catenin. Endosulfan weakens the gap junction function and is related to the decrease of Cx43 expression. Endosulfan exposes 48h to increase the endothelial cell permeability, and has a dose effect relationship. This study reveals that endosulfan can weaken the microfilament structure of the endothelial cytoskeleton, weaken the adhesion and connexion of the cells, and thus cause the permeability of endothelial cells to increase and affect the barrier effect of vascular endothelial cells. It is the first time that LAMC1 has been found to be involved in the remodeling of endothelial cytoskeleton caused by endosulfan exposure. This study provides an important theoretical basis for endosulfan as a risk factor leading to cardiovascular homeostasis and cardiovascular disease.
【学位授予单位】：大连海事大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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