贵州医科大学学报

2008, (04) 335-339

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细胞骨架在高迁移率族蛋白B1诱导人脐静脉内皮细胞表达分泌IL-6中的作用
Effects of Cytoskeleton on the Level of IL-6 Secreted by HUVEC after The Induction of Human HMGB1

徐佳;杨莉;李志杰;刘靖华;邓鹏;姜勇;
XU Jia,YANG Li,LI Zhijie,LIU Jinghua,DENG Peng,JIANG Yong (Department of Pathophysiology and Key Laboratory of Functional Proteomics of Guangdong Province,The Southern Medical University,Guangzhou 510515,Guangdong,China)

摘要(Abstract):

目的:观察体外重组高迁移率族蛋白B1(HMGB1)诱导人脐静脉内皮细胞(HUVEC)分泌释放白介素-6(IL-6)的量效关系,探讨细胞骨架在HMGB1诱导HUVEC释放IL-6中的作用。方法:构建原核表达质粒并表达纯化H is-EGFP-HMGB1及H is-EGFP融合蛋白,刺激体外培养的HUVEC;利用液相芯片分析系统观察HMGB1及相关细胞骨架抑制剂作用或低温培养作用下细胞分泌IL-6因子的情况。结果:酶切和DNA测序证明所构建原核表达载体正确,表达纯化的重组蛋白经SDS-PAGE电泳鉴定大小正确,刺激体外培养HUVEC后IL-6高表达且具有明显量效关系;Nocodazole、Cytochalasin D等细胞骨架抑制剂作用及低温培养后可明显抑制IL-6表达。结论:体外重组的人HMGB1蛋白可以有效诱导HUVEC分泌和释放IL-6,而细胞骨架结构在此过程中起重要作用,并具有明显的能量依赖性。
Objective: To understand the role of cytoskeleton in the expression of interleukin-6(IL-6) in human umbilical vein endothelial cells(HUVECs) under the stimulation of the fusion HMGB1 protein.Methods: The coding sequence of HMGB1 was amplified and subcloned into pET14b-MCS-EGFP plasmid for the construction of fusion protein expressing vector.The recombinant vector was transformed and the expression of fusion protein was induced and purified.The fusion proteins and different cytoskeleton inhibitors were added to the cell culture media of HUVECs and IL-6 level in the supernatant of the cell culture media was measured with LiquiChip system.Results: The recombinant vector was identified by PCR,endonucleases digestion and DNA sequencing.After protein was expressed and purified,the molecular mass and purity were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).High level expression of IL-6 was detected in cell culture media dose-dependently after stimulation of HMGB1.IL-6 level was significantly decreased if be pretreated with Cytochalasin D and nocodazole or under the low temperature incubation.Conclusions: The fusion protein HMGB1 can induce the expression of IL-6 in HUVECs effectively and cytoskeleton may play an important role in this process.

关键词(KeyWords): 高迁移率族蛋白质类;细胞骨架;内皮,血管;脐静脉;人;白细胞介素6
high mobility group proteins;cytoskeleton;endothelium,vascular;umbilical veins;persons;interleukin-6

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基金项目(Foundation): 国家自然科学基金(NO.306708280NO.U0632004);; 广东省科技计划项目(NO.A1090202)

作者(Author): 徐佳;杨莉;李志杰;刘靖华;邓鹏;姜勇;
XU Jia,YANG Li,LI Zhijie,LIU Jinghua,DENG Peng,JIANG Yong (Department of Pathophysiology and Key Laboratory of Functional Proteomics of Guangdong Province,The Southern Medical University,Guangzhou 510515,Guangdong,China)

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