上海交通大学学报(医学版) ›› 2019, Vol. 39 ›› Issue (8): 866-.doi: 10.3969/j.issn.1674-8115.2019.08.010

• 论著·基础研究 • 上一篇    下一篇

血管内皮生长因子对体外培养的人 Tenon s囊成纤维细胞的 纤维化及其富含半胱氨酸的酸性分泌蛋白表达的作用

项潇琼,罗丽颖,傅扬,顾青,唐敏   

  1. 上海交通大学附属第一人民医院眼科,上海市眼底病重点实验室,上海 200080
  • 出版日期:2019-08-28 发布日期:2019-09-23
  • 通讯作者: 唐敏,电子信箱:tmsmile@sina.com。
  • 作者简介:项潇琼(1993—),女,硕士生;电子信箱: 1505817876@qq.com。

Effects of vascular endothelial growth factor on the fibrosis and the of secreted protein acidic and rich in cysteine in cultured human Tenons fibroblast

XIANG Xiao-qiong, LUO Li-ying, FU Yang, GU Qing, TANG Min   

  1. Shanghai Key Laboratory of Ocular Fundus Diseases; Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China
  • Online:2019-08-28 Published:2019-09-23

摘要: 目的 ·探讨血管内皮生长因子( vascular endothelial growth factor,VEGF)对体外培养的人 Tenons囊成纤维细胞( human Tenons fibroblast,HTF)的纤维化及其富含半胱氨酸的酸性分泌蛋白( secreted protein acidic and rich in cysteine,SPARC)表达的作用。方法 ·取斜视手术患者的 Tenons囊组织进行培养,并采用免疫荧光技术对获得的 HTF进行鉴定。采用不同浓度的 VEGF刺激培养 HTF,并依据刺激条件将 HTF分为 4组,即 0 ng/mL组、25 ng/mL组、50 ng/mL组和 100 ng/mL组。采用蛋白质印迹和实时定量 PCR分析 SPARC、Ⅰ型胶原蛋白( collagen-Ⅰ)和基质金属蛋白酶 -9(matrix metalloprotein 9,MMP-9)的表达以及胞外信号调节激酶(extracellular signal-regulated kinase,ERK)通路磷酸化活性的改变。采用细胞活力测定实验( MTS法)和划痕实验检测 HTF的增殖和迁移能力。结果 ·通过免疫荧光技术及倒置相差显微观察可以鉴定,所培养的原代细胞为 HTF。在 VEGF刺激作用下,与 0 ng/mL组相比,其他 3组 HTF中的 SPARC、collagen-Ⅰ和 MMP-9蛋白及其 mRNA表达均有所增加,HTF中 ERK通路的磷酸化活性均有所上调, HTF细胞的增殖及迁移能力亦均有所增加,且在 50 ng/mL组中影响最大。结论 · VEGF在促进 HTF纤维化的过程中伴随着细胞基质蛋白 SPARC的上调,提示 SPARC可能是这一过程的潜在调控位点。

关键词: 人 Tenons囊成纤维细胞, 富含半胱氨酸的酸性分泌蛋白, 血管内皮生长因子, 纤维化

Abstract: Objective · To investigate the effects of vascular endothelial growth factor (VEGF) on the of secreted protein acidic and rich in cysteine (SPARC) andthefibrosisinculturedhumanTenonsfibroblast(HTF) in vitro. Methods · HTF cells were obtained Tenons capsule tissues of patients undergoing strabismus surgery. Immunofluorescence was used to identify the HTF cells. HTF cells were cultured with different concentrations of VEGF, and which were divided into four groups, i.e., 0 ng/mL group, 25 ng/mL group, 50 ng/mL group and 100 ng/mL group. The of SPARC, collagen-Ⅰ, and matrix metalloprotein 9 (MMP-9) and the activity of extracellular signal-regulated kinase (ERK) pathway were analyzedWestern blotting and real-time quantitative PCR (qPCR). The abilities of proliferation and migration of HTF cells were detectedMTS assay and scratch test, respectively. Results · HTF cells were observed and identifiedinverted phase contrast microscope and immunofluorescence. Under the stimulation of VEGF, the of protein and mRNA of SPARC, collagen-I and MMP-9 of HTF cells in other three groups were increased compared with 0 ng/mL group; the phosphorylation activities of ERK pathway were up-regulated, and the proliferation and migration abilities of HTF cells were up-regulated. And the effect was the most obvious in the 50 ng/mL group. Conclusion · VEGF is involved in promoting the fibrosis of HTF cells accompaniedthe up-regulation of the SPARC, which suggests SPARC may become a potential regulatory site.

Key words: human Tenonsfibroblast(HTF), secretedprotein acidic and rich in cysteine(SPARC), vascular endothelialgrowth factor(VEGF), fibrosis

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