rhFGF4在大肠杆菌中的表达、纯化及其对高糖损伤的人脐静脉内皮细胞的保护作用

doi:10.3969/j.issn.2095-9400.2021.07.001

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摘要目的：制备、鉴定重组人成纤维细胞生长因子4（rhFGF4）蛋白，观察rhFGF4 对高糖损伤人脐静脉内皮细胞（HUVEC）的保护作用及相关分子机制。方法：构建pET3d-hFGF4 重组质粒，利用大肠杆菌表达系统高效表达rhFGF4 蛋白。可溶性目的蛋白大多以包涵体的形式表达，制备方法为建立包涵体清洗、变性及肝素柱柱上复性与纯化。采用MTT法检测rhFGF4 蛋白促细胞增殖的生物学活性，建立细胞损伤模型并用Western blot技术检测NF-κB、JNK以及p38 蛋白的磷酸化表达。结果：成功构建重组质粒pET3d-hFGF4。SDS-PAGE电泳及Western blot鉴定结果表明，rhFGF4 蛋白在大肠杆菌中成功高效表达。细胞实验证实rhFGF4蛋白通过阻止NF-κB、JNK以及p38的磷酸化来修复高糖诱导损伤的HUVEC并抑制其凋亡，降低其MDA、ROS含量。结论：本研究成功得到高纯度并具有生物学活性的rhFGF4蛋白，并证实其对高糖损伤的HUVEC的保护作用与改善氧化应激水平相关。

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	张煜
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	李校堃

关键词 ：重组人成纤维细胞生长因子4, 柱上复性, 人脐静脉内皮细胞, 氧化应激, 细胞凋亡

Abstract：Objective: To prepare and identify recombinant human fibroblast growth factor 4 (rhFGF4) protein and observe the protective effect of rhFGF4 on human umbilical vein endothelial cells (HUVEC) model with high glucose damage. Methods: The recombinant plasmid pET3d-hFGF4 was constructed, and the rhFGF4 protein was efficiently expressed by E.coli expression system. Most soluble objective proteins are expressed in the form of inclusion, including the cleaning, degeneration and restoration and purification of heparin column.The MTT method was used to detect the biological activity of rhFGF4 protein in promoting cell proliferation,the cell damage model was established and the phosphorylation expression of NF-κB, JNK and p38 was detected by Western blot technology. Results: The recombinant plasmid pET3D-HFGF4 was successfully constructed.SDS-PAGE electrophoresis and Western blot identification results showed that rhFGF4 protein was successfully expressed in E.coli. It was also confirmed that rhFGF4 protein repaired HUVEC cells damaged by high glucose and inhibited their apoptosis by preventing the phosphorylation of NF-κB, JNK and p38, and reduced their MDA and ROS content. Conclusion: This study has successfully obtained high purity and biologically active rhFGF4 protein, which provides a useful tool for further research on the role of this protein in vascular diseases caused by high glucose and related mechanisms.

Key words： rhFGF4 on-column refolding human umbilical vein endothelial cells oxidative stress apoptosis

收稿日期: 2020-12-30

基金资助:国家重点研发计划“蛋白质机器与生命过程调控”重点专项（2017YFA0506000）。

通讯作者: 李校堃，教授 E-mail: profxiaokunli@163.com。

作者简介: 张煜，硕士生，Email：1063321062@qq.com。

链接本文:

https://xb.wmu.edu.cn/CN/10.3969/j.issn.2095-9400.2021.07.001 或 https://xb.wmu.edu.cn/CN/Y2021/V51/I7/517

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