中华皮肤科杂志 ›› 2022, Vol. 55 ›› Issue (11): 990-995.doi: 10.35541/cjd.20210692

• 论著 • 上一篇    下一篇

白藜芦醇对婴儿血管瘤内皮细胞活性的影响

杨开颖1    邱桐1    龚雪1    兰钰茹1    周江元1    陈思源2    吉毅1   

  1. 1四川大学华西医院小儿外科，成都  610041；2四川大学华西医院重症医学科，成都  610041
  • 收稿日期:2021-09-22 修回日期:2022-04-18 发布日期:2022-11-03
  • 通讯作者: 吉毅 E-mail:jijiyuanyuan@163.com
  • 基金资助:
    国家自然科学基金（81401606、81400862）；四川省科技厅重点研发项目（2019YFS0322）；四川大学优秀青年学者基金（2015SU04A15）；四川大学华西医院学科卓越发展1·3·5工程临床研究孵化项目（2019HXFH056、2020HXFH048、ZYJC21060）

Effects of resveratrol on the activity of infantile hemangioma-derived endothelial cells

Yang Kaiying1, Qiu Tong1, Gong Xue1, Lan Yuru1, Zhou Jiangyuan1, Chen Siyuan2, Ji Yi1   

  1. 1Department of Pediatric Surgery, West China Hospital, Sichuan University, Chengdu 610041, China; 2Department of Critical Care Medicine, West China Hospital, Sichuan University, Chengdu 610041, China
  • Received:2021-09-22 Revised:2022-04-18 Published:2022-11-03
  • Contact: Ji Yi E-mail:jijiyuanyuan@163.com
  • Supported by:
    National Natural Science Foundation of China （81401606, 81400862）; Key Project in the Science & Technology Program of Sichuan Province （2019YFS0322）; Science Foundation for Excellent Youth Scholars of Sichuan University （2015SU04A15）; The 1·3·5 Project for Disciplines of Excellence-Clinical Research Incubation Project, West China Hospital of Sichuan University （2019HXFH056, 2020HXFH048, ZYJC21060）

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摘要： 【摘要】 目的 研究葡萄糖转运体1（Glut-1）抑制剂白藜芦醇（RSV）对婴儿血管瘤内皮细胞（HemEC）活性的影响。方法 收集4例增殖期与4例消退期婴儿血管瘤（IH）组织，采用免疫组化观察Glut-1的表达。从4例增殖期IH组织中提取原代HemEC，采用qPCR与Western印迹检测HemEC与对照脐静脉内皮细胞 （HUVEC）中Glut-1 mRNA与蛋白的表达水平。体外培养HemEC，分别采用0（对照组）、50、100、200、400、800 μmol/L RSV干预24 h，采用CCK-8法检测各组HemEC增殖能力，计算半抑制浓度（IC50）。分别采用Transwell实验、流式细胞仪观察HemEC迁移能力及凋亡水平。组间比较采用t检验或方差分析，组间多重比较采用LSD-t检验。结果 免疫组化显示，增殖期及消退期IH组织中血管内皮细胞Glut-1均阳性，但增殖期表达丰富，消退期表达明显降低。HemEC中Glut-1 mRNA及蛋白表达水平（1.793 ± 0.041、1.959 ± 0.144）均高于HUVEC（0.820 ± 0.073、0.648 ± 0.046，t = 16.35、12.28，均P < 0.001）。采用Glut-1抑制剂RSV干预HemEC后，0 ~ 800 μmol/L组HemEC增殖活性差异有统计学意义（F = 1 043.00，P < 0.001），所有RSV干预组HemEC的增殖活性均低于对照组（P < 0.05）。GraphPad Prism 8计算得出RSV IC50浓度为150 μmol/L，Transwell实验与流式细胞仪检测显示，150 μmol/L RSV干预组HemEC组细胞迁移数（61 ± 5）低于对照组（150 ± 6，t = 15.11，P < 0.001），凋亡率（13.01% ± 0.45%）高于对照组（3.93% ± 0.68%，t = 19.34，P < 0.001）。结论 糖酵解关键酶Glut-1在IH增殖期组织与HemEC中高表达，RSV干预可以抑制HemEC增殖及迁移能力并促进其凋亡。

关键词: 血管瘤, 人脐静脉内皮细胞, 葡萄糖转运体1型, 细胞增殖, 细胞凋亡, 白藜芦醇

Abstract: 【Abstract】 Objective To investigate the effect of the glucose transporter 1 （Glut-1） inhibitor resveratrol on the activity of infantile hemangioma （IH）-derived endothelial cells （HemEC）. Methods IH tissues were collected from 4 cases of proliferating IH and 4 cases of involuting IH, and immunohistochemical study was performed to determine the Glut-1 expression. Primary HemEC were extracted from 4 proliferating IH tissues, real-time fluorescence-based quantitative PCR（qPCR） and Western blot analysis were performed to determine the mRNA and protein expression of Glut-1 in HemEC and human umbilical vein endothelial cells （HUVEC）, respectively. HemEC were cultured in vitro and treated with 0 （control group）, 50, 100, 200, 400 and 800 μmol/L resveratrol for 24 hours, respectively. Cell counting kit-8 （CCK8） assay was performed to evaluate the proliferative ability of HemEC in the above groups, and the 50% inhibitory concentration （IC50） was calculated. The migratory ability and apoptosis level of HemEC were assessed by Transwell assay and flow cytometry, respectively. Intergroup comparisons were performed using t test or analysis of variance, and multiple comparisons were performed using least significant difference-t test. Results Immunohistochemical study showed that Glut-1 was expressed in vascular endothelial cells derived from both proliferating and involuting IH tissues, and the Glut-1 expression was abundant in the proliferating IH but markedly decreased in the involuting IH tissues. The mRNA and protein expression levels of Glut-1 were significantly higher in HemEC （1.793 ± 0.041, 1.959 ± 0.144, respectively） than in HUVEC （0.820 ± 0.073, 0.648 ± 0.046, t = 16.35, 12.28, respectively, both P < 0.001）. After the treatment with Glut-1 inhibitor resveratrol at different concentrations, the proliferative ability of HemEC significantly differed among the control group, 50-, 100-, 200-, 400- and 800-μmol/L resveratrol groups （F = 1 043.00, P < 0.001）, and was significantly lower in all the resveratrol groups than in the control group （all P < 0.05）. The IC50 of resveratrol was calculated to be 150 μmol/L by using GraphPad Prism 8 software. Transwell assay and flow cytometry showed significantly decreased number of migratory HemEC but significantly increased apoptosis rate respectively in the 150 μmol/L resveratrol group （61 ± 5, 13.01% ± 0.45%, respectively） compared with the control group （150 ± 6, 3.93% ± 0.68%, t = 15.11, 19.34, respectively, both P < 0.001）. Conclusion The key glycolytic enzyme Glut-1 was highly expressed in proliferating IH tissues and HemEC, and resveratrol could inhibit the proliferation and migration of HemEC, but promote their apoptosis.

Key words: Hemangioma, Human umbilical vein endothelial cells, Glucose transporter type 1, Cell proliferation, Apoptosis, Resveratrol