本文選題：硫丹 + 細胞骨架��； 參考：《大連海事大學》2017年碩士論文

【摘要】：硫丹屬于持久性有機污染物,具有難降解性、生物蓄積性、長距離遷移性以及高毒性。本課題前期研究發(fā)現(xiàn)硫丹具有內皮細胞毒性,但硫丹對內皮細胞屏障功能的影響尚不清楚。本研究以人臍靜脈內皮細胞(HUVEC-C)為實驗對象,主要探討了硫丹對血管內皮細胞骨架的影響及其信號通路,并著重分析了由于細胞骨架重構而引起的血管內皮通透性改變的分子機制。本研究利用免疫染色法分析了硫丹暴露對HUVEC-C細胞骨架的微絲結構(F-actin)和粘著斑激酶(FAK)分布的影響,用qRT-PCR方法測定了骨架相關基因ROCK2和PXN的mRNA表達水平。用蛋白印記法(Western blot)分析了基質金屬蛋白酶(MMP-3)、層粘連蛋白(LAMC1)、FAK和pFAK的表達。用RNAi干擾的方法探討了 LAMC1對HUVEC-C細胞存活率、細胞骨架和內皮細胞通透性的影響。用Western blot和qRT-PCR方法分別檢測了細胞間粘附連接蛋白E-cadherin和β-catenin及縫隙連接關鍵因子Cx43的變化。最后利用Coaster transwell system探討了硫丹暴露影響HUVEC-C細胞通透性的時間和劑量效應。結果發(fā)現(xiàn),硫丹暴露能夠引起HUVEC-C細胞骨架破壞,ROCK2和PXN的mRNA表達降低,MMP-3蛋白表達升高而降解LAMC1,引起FAK和pFAK的表達降低,這些提示硫丹暴露可能通過影響MMP3/LAMC1/pFAK信號通路而破壞內皮細胞骨架。LAMC1 siRNAs顯著降低HUVEC-C細胞生存率,引起細胞骨架破壞和內皮細胞通透性升高,說明LAMC1在維持血管內皮細胞結構與功能中起重要作用。硫丹通過降低E-cadherin和β-catenin表達而影響內皮細胞間連接。硫丹使縫隙連接功能減弱,與Cx43的表達降低有關。硫丹暴露48h導致內皮細胞通透性的升高,具有劑量效應關系。本研究揭示了硫丹能夠通過破壞血管內皮細胞骨架的微絲結構,減弱細胞間粘附連接和縫隙連接功能,從而引起了內皮細胞的通透性增加,影響血管內皮細胞的屏障作用。首次發(fā)現(xiàn)LAMC1參與了硫丹暴露引起的血管內皮細胞骨架重構和通透性變化。本研究為硫丹是否作為危險因素導致血管穩(wěn)態(tài)失衡而引起心血管疾病提供了重要的理論依據(jù)。
[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.
【學位授予單位】：大連海事大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R114

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