中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (31): 5014-5019.doi: 10.12307/2022.970

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

松果菊苷延缓人脐静脉内皮细胞衰老的机制

田  甜，欧阳·菊艳，李  瑜，米叶赛·艾尼瓦尔，何娟丽，李振华，王  红   

  1. 新疆医科大学第一附属医院综合内二科，新疆维吾尔自治区乌鲁木齐市   830000
  • 收稿日期:2021-12-06 接受日期:2022-02-08 出版日期:2022-11-08 发布日期:2022-04-24
  • 通讯作者: 王红，博士，教授，硕士研究生导师，新疆医科大学第一附属医院综合内二科，新疆维吾尔自治区乌鲁木齐市 830000
  • 作者简介:田甜，女，1994年生，新疆医科大学第一附属医院在读硕士，主要从事老年相关疾病及机制研究。
  • 基金资助:
    克拉玛依市科技计划项目(2017RC001A-16)，项目负责人：王红；新疆医科大学研究生创新创业项目(CXCY2021008)，项目负责人：田甜

Mechanism by which echinacoside delays the senescence of human umbilical vein endothelial cells

Tian Tian, Ouyang·Juyan, Li Yu, Miyesai·Ainiwaer, He Juanli, Li Zhenhua, Wang Hong   

  1. Second Department of Comprehensive Internal Medicine, The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830000, Xinjiang Uygur Autonomous Region, China
  • Received:2021-12-06 Accepted:2022-02-08 Online:2022-11-08 Published:2022-04-24
  • Contact: Wang Hong, MD, Professor, Master’s supervisor, Second Department of Comprehensive Internal Medicine, The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830000, Xinjiang Uygur Autonomous Region, China
  • About author:Tian Tian, Master candidate, Second Department of Comprehensive Internal Medicine, The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830000, Xinjiang Uygur Autonomous Region, China
  • Supported by:
    Science and Technology Plan Project of Karamay City, No. 2017RC001A-16 (to WH); Postgraduate Innovatian Entrepreneurship Project of Xinjiang Medical University, No. CXCY2021008 (to TT)

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摘要：

文题释义：
胞质多聚腺苷酸化元素结合蛋白1(cytoplasmic polyadenylation element binding protein，CPEB1)：是一种高度保守的RNA结合蛋白，将翻译抑制剂招募到其目的mRNA 3’非翻译区，并与特定序列结合调控mRNA的聚腺苷化。CPEB1广泛参与生物过程，包括细胞周期进程、增殖与肿瘤、细胞衰老和炎症等。
沉默信息调节因子2相关酶类1(silent mating type information regulation 2 homolog 1，SIRT1)：是Sirtuin蛋白家族的一员，是负责细胞调控的组蛋白去乙酰化酶，参与细胞周期调控、DNA修复、凋亡和炎症、自噬和衰老，并在多种疾病中起保护作用，包括神经退行性疾病、心血管疾病、代谢综合征和肿瘤等。

背景：前期工作发现同型半胱氨酸通过活性氧加速脐静脉内皮细胞衰老，此次实验初步研究影响内皮细胞衰老的相关机制。
目的：探讨松果菊苷对人脐静脉内皮细胞中CPEB1、SIRT1、P53、P21基因表达的影响。 
方法：体外培养人脐静脉内皮细胞，加入10 g/L D-半乳糖构建细胞衰老模型，CCK-8法分析松果菊苷(0，10，20，50，100，200，300，400，500 mg/L)预处理12，24 h后的细胞增殖活性，筛选松果菊苷最佳质量浓度及干预时间；然后将人脐静脉内皮细胞分为5组：正常对照组，D-半乳糖组，松果菊苷100，200，300 mg/L组。β-半乳糖苷酶染色法检测松果菊苷预处理12 h后细胞染色阳性率，实时荧光定量-聚合酶链反应、蛋白免疫印迹法检测松果菊苷预处理12 h后人脐静脉内皮细胞中CPEB1、Sirt1、P53、P21 mRNA的表达和CPEB1、Sirt1、P21蛋白的表达。
结果与结论：①CCK-8检测结果表明：松果菊苷预处理12 h的细胞存活率明显高于预处理24 h，与D-半乳糖组相比，松果菊苷100，200，300 mg/L组均不同程度提高细胞活力(P < 0.000 1)，3组间对比差异有显著性意义(P < 0.01)；②与正常对照组相比，D-半乳糖组β-半乳糖苷酶染色阳性率最高(P < 0.000 1)，100，200，300 mg/L松果菊苷预处理12 h后，可一定程度降低β-半乳糖苷酶染色阳性率(P < 0.000 1)；③与正常对照组相比，D-半乳糖组CPEB1、P53、P21 mRNA表达水平均升高(P < 0.000 1)，Sirt1 mRNA表达水平降低(P < 0.000 1)；与D-半乳糖组相比，100，200，300 mg/L松果菊苷预处理12 h后，CPEB1、P53、P21 mRNA表达水平均降低(P < 0.000 1)，Sirt1 mRNA表达水平均升高(P < 0.000 1)；蛋白免疫印迹分析结果与实时荧光定量-聚合酶链反应分析结果一致；④结果表明，松果菊苷可能通过下调CPEB1、P53、P21表达水平，上调Sirt1表达水平，来延缓D-半乳糖诱导的人脐静脉内皮细胞衰老。 

https://orcid.org/0000-0002-2453-0802 (田甜) 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 松果菊苷, 人脐静脉内皮细胞, 衰老, CPEB1, Sirt1, P53, P21

Abstract: BACKGROUND: Preliminary work has found that homocysteine accelerates the aging of umbilical vein endothelial cells through reactive oxygen species. This experiment preliminarily studies the relevant mechanisms affecting endothelial cell aging.
OBJECTIVE: To investigate the effects of echinacoside on the expression of cytoplasmic polyadenylate binding protein 1 (CPEB1), sirtuin1 (SIRT1), P53 and P21 genes in human umbilical vein endothelial cells.
METHODS: Human umbilical vein endothelial cells were cultured in vitro. D-galactose 10 g/L was used to construct cell aging model. The proliferation viability of cells treated with echinacoside (0, 10, 20, 50, 100, 200, 300, 400, 500 mg/L) for 12 and 24 hours was analyzed by CCK-8 assay, and the optimal mass concentration and intervention time of echinacoside were screened. Human umbilical vein endothelial cells were divided into normal control group, D-galactose group and echinacoside groups (100, 200, and 300 mg/L). β-Galactosidase staining was used to detect the positive staining rate of cells after echinacoside pre-intervention for 12 hours. Real time RT-PCR and western-blot assay were used to detect the mRNA expression levels of CPEB1, Sirt1, P53 and P21 and the protein expression levels of CPEB1, Sirt1 and P21 in human umbilical vein endothelial cells after echinacoside pretreatment for 12 hours. 
RESULTS AND CONCLUSION: (1) The results of CCK-8 assay showed that the survival rate of echinacoside group at 12 hours was significantly higher than that at 24 hours. Compared with the D-galactose group, cell viability increased in the echinacoside 100, 200, and 300 mg/L groups to varying degrees (P < 0.000 1), and there was a significant difference among the three groups (P < 0.01). (2) Compared with the normal control group, the D-galactose group had the highest positive rate of β-galactosidase staining (P < 0.0001). 100, 200, and 300 mg/L echinacoside pretreatment for 12 hours could reduce the positive rate of β-galactosidase staining to a certain extent (P < 0.000 1). (3) Compared with the normal control group, the mRNA expression levels of CPEB1, P53 and P21 were increased (P < 0.000 1), and the expression level of SIRT1 mRNA was decreased (P < 0.000 1) in the D-galactose group. Compared with the D-galactose group, 100, 200, and 300 mg/L echinacoside pretreatment for 12 hours decreased the mRNA expression levels of CPEB1, P53, and P21 (P < 0.000 1), but increased Sirt1 mRNA expression (P < 0.000 1). Western blot assay results were consistent with real-time fluorescence quantitative polymerase chain reaction results. (4) These findings indicate that echinacoside may delay D-galactose-induced human umbilical vein endothelial cell senescence by down-regulating CPEB1 and P53/P21 expression levels and up-regulating Sirt1 expression levels.

Key words: echinoside, human umbilical vein endothelial cells, aging, CPEB1, Sirt1, P53, P21

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