胰岛素受体底物-1的阻断抑制脉络膜内皮细胞的生物学行为。
Blockade of insulin receptor substrate-1 inhibits biological behavior of choroidal endothelial cells.
作者信息
Qian Yi-Yong, Wu Hong-Ya, Liu Gao-Qin, Ren Chi, Lu Pei-Rong, Zhang Xue-Guang
机构信息
Department of Ophthalmology, the First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China.
Department of Ophthalmology, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, China.
出版信息
Int J Ophthalmol. 2019 Sep 18;12(9):1386-1394. doi: 10.18240/ijo.2019.09.03. eCollection 2019.
AIM
To investigate the effects of blockade of insulin receptor substrate-1 (IRS-1) on the bio-function of tube formation of human choroidal endothelial cells (HCECs).
METHODS
Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were performed to determine the expression level of IRS-1 and phospho-IRS-1 in HCECs. Tube formation of HCECs was analyzed using three dimensional Matrigel assay with or without IRS-1 blockage IRS-1 inhibitor (GS-101) and vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor. In addition, cell counting kit (CCK)-8 and Transwell migration assay were exerted to analyze the effects of blockade of IRS-1 on the bio-function of proliferation and migration of HCECs, respectively. The apoptosis of HCECs was examined using flow cytometry (FCM).
RESULTS
RT-PCR and Western blot revealed that IRS-1 phospho-IRS-1 were expressed in HCECs and the expression level was enhanced by stimulation of VEGF-A. The number of tube formation was decreased significantly in GS-101 treated groups compared to phosphate buffered saline (PBS) treated control groups. Furthermore, both cell proliferation and migration of HCECs were decreased in the presence of GS-101. FCM analysis showed that the apoptosis of HCECs was enhanced when the cells were treated with GS-101. Western blot also showed that the expression level of cleaved-caspase 3 in GS-101 treated group was higher than that in control group.
CONCLUSION
Blockade of IRS-1 can inhibit tube formation of HCECs through reducing cell proliferation and migration and promoting cell apoptosis.
目的
研究胰岛素受体底物-1（IRS-1）阻断对人脉络膜内皮细胞（HCECs）管腔形成生物功能的影响。
方法
采用定量逆转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测HCECs中IRS-1和磷酸化IRS-1的表达水平。使用三维基质胶试验分析有无IRS-1阻断剂（IRS-1抑制剂（GS-101））和血管内皮生长因子受体2（VEGFR2）抑制剂时HCECs的管腔形成情况。此外，分别采用细胞计数试剂盒（CCK）-8和Transwell迁移试验分析IRS-1阻断对HCECs增殖和迁移生物功能的影响。采用流式细胞术（FCM）检测HCECs的凋亡情况。
结果
RT-PCR和蛋白质免疫印迹显示，HCECs中表达IRS-1和磷酸化IRS-1，且VEGF-A刺激可提高其表达水平。与磷酸盐缓冲液（PBS）处理的对照组相比，GS-101处理组的管腔形成数量显著减少。此外，GS-101存在时HCECs的细胞增殖和迁移均减少。FCM分析显示，GS-101处理细胞时HCECs的凋亡增加。蛋白质免疫印迹还显示，GS-101处理组中裂解的半胱天冬酶3的表达水平高于对照组。
结论
阻断IRS-1可通过减少细胞增殖和迁移以及促进细胞凋亡来抑制HCECs的管腔形成。
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