中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (29): 4617-4622.doi: 10.3969/j.issn.2095-4344.1809

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

过表达GATA-4骨髓间充质干细胞外泌体中miR-330-3p的时-量变化

贺继刚1，谢巧丽1，王梓豪1，严  丹2，张宏波2   

  1. 云南省第一人民医院，昆明理工大学附属医院，1心脏大血管外科，2重症医学科，云南省昆明市  650032
  • 修回日期:2019-04-12 出版日期:2019-10-18 发布日期:2019-10-18
  • 通讯作者: 张宏波，主治医师，云南省第一人民医院重症医学科，云南省昆明市 650032
  • 作者简介:贺继刚，男，1980年生，河北省抚宁县人，汉族，2013年苏州大学毕业，博士，副主任医师，主要从事干细胞在终末心脏疾病中的应用。
  • 基金资助:

    国家自然科学基金(81460073)，项目负责人：贺继刚；云南省科技厅-昆明医科大学应用基础研究联合专项(2014FB089)，项目负责人：贺继刚；云南省教育厅科学研究基金(2015Z051)，项目负责人：贺继刚；中国博士后科学基金(2015M582764XB)，项目负责人：贺继刚；成都医学院2015年度科研项目(CYZ15-18)，项目负责人：贺继刚；云南省医学后备人才(H-201607)，项目负责人：贺继刚

Time-volume variation in miR-330-3p expression in GATA-4-overexpressing bone marrow-derived mesenchymal stem cell exosomes 

He Jigang1, Xie Qiaoli1, Wang Zihao1, Yan Dan2, Zhang Hongbo2   

  1. 1Department of Cardiovascular Surgery, 2Department of Intensive Care Unit, Affiliated Hospital of Kunming University of Science and Technology, the First People’s Hospital of Yunnan Province, Kunming 650032, Yunnan Province, China
  • Revised:2019-04-12 Online:2019-10-18 Published:2019-10-18
  • Contact: Zhang Hongbo, Attending physician, Department of Intensive Care Unit, Affiliated Hospital of Kunming University of Science and Technology, the First People’s Hospital of Yunnan Province, Kunming 650032, Yunnan Province, China
  • About author:He Jigang, MD, Associate chief physician, Department of Cardiovascular Surgery, Affiliated Hospital of Kunming University of Science and Technology, the First People’s Hospital of Yunnan Province, Kunming 650032, Yunnan Province, China
  • Supported by:

    the National Natural Science Foundation of China, No. 81460073 (to HJG); Yunnan Provincial Department of Science and Technology - Kunming Medical University Joint Research Project, No. 2014FB089 (to HJG); Yunnan Provincial Department of Education Science Research Fund, No. 2015Z051 (to HJG); China Postdoctoral Science Foundation, No. 2015M582764XB (to HJG); 2015 Research Project of Chengdu Medical College, No. CYZ15-18 (to HJG); Yunnan Medical Reserve Talents, No. H-201607 (to HJG)

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摘要：

文章快速阅读：

 

文题释义：
外泌体：是指包含了复杂RNA和蛋白质的小膜泡(30-150 nm)。多种细胞在正常及病理状态下均可分泌外泌体。外泌体可能发挥细胞与细胞之间的信号传导作用，它可以远距离运输并将所携带物质释放入受体细胞。例如，miRNA从一个细胞穿梭到另一个细胞，被称为“外泌体载体miRNA”，能够影响受体细胞蛋白质产生。
microRNA：是指一类由内源基因编码的长度约为22个核苷酸的非编码单链RNA分子，它们在动植物中参与转录后基因表达调控。miRNA在多种调节途径中具有关键作用，包括控制发育、细胞增殖、细胞分化、细胞周期进展、免疫反应、蛋白分泌和许多其他生理和病理过程。该研究中的miR-330-3p可能涉及细胞的抗凋亡作用。

 

摘要
背景：前期实验已经证明过表达GATA-4骨髓间充质干细胞(bone marrow mesenchymal stem cells，BMSCs)分泌的外泌体(exosomes)可以通过抑制心肌细胞凋亡，有效修复心肌梗死引起的心肌损伤，进一步发现在其外泌体中miRNA-330-3p明显高表达且功能涉及抗凋亡，提示miRNA-330-3p可能是外泌体修复心肌损伤的关键分子。
目的：探讨过表达GATA-4小鼠BMSCs分泌的外泌体(BMSCsGATA-4-exosomes)中miR-330-3p的时-量变化。
方法：在过表达GATA-4小鼠BMSCs培养体系内加入miR-330-3p模拟剂(miR-330-3p-mimic)及抑制剂(miR-330-3p-inhibitor)作为BMSCsGATA-4-miR-330-3p-mimic组及BMSCsGATA-4-miR-330-3p-inhibitor组，将BMSCsGATA-4组、BMSCsGATA-4-空载体组、BMSCs组、BMSCsGATA-4-miR-330-3p-mimic-空载体组及BMSCsGATA-4-miR-330-3p-inhibitor-空载体组作为混杂因素对照组。上述各组与基因开启剂强力霉素共培养24，48 h，提取各组细胞分泌的外泌体。采用RT-PCR检测各组细胞及其外泌体内miR-330-3p的表达量。采用光镜评估BMSCsGATA-4-miR-330-3p-mimic组、BMSCsGATA-4-miR-330-3p-inhibitor组与BMSCs组细胞在24，48 h的形态变化。
结果与结论：①BMSCsGATA-4-miR-330-3p-mimic组细胞与外泌体内miR-330-3p的表达率最高，且随着时间推移其miR-330-3p的表达逐渐增多；②BMSCsGATA-4-miR-330-3p-inhibitor组细胞与外泌体内miR-330-3p的表达率最低，且随着时间推移其miR-330-3p的表达逐渐减少；③BMSCsGATA-4-miR-330-3p-mimic组、BMSCsGATA-4-miR-330-3p-inhibitor组与BMSCs组在24，48 h的形态无变化；④结果表明，BMSCs内miR-330-3p表达与外泌体内miR-330-3p表达正相关，BMSCs过表达miR-330-3p可以有效提高外泌体中miR-330-3p的表达，而静默miR-330-3p可以有效减少外泌体中miR-330-3p的表达。


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
ORCID: 0000-0002-1877-1757(张宏波)

关键词: 心肌梗死, 心肌损伤, 骨髓间充质干细胞, GATA-4, 外泌体, miR-330-3p

Abstract:

BACKGROUND: Previous experiments have demonstrated that exosomes secreted by bone marrow mesenchymal stem cells overexpressing GATA-4 (BMSCsGATA-4) can effectively repair myocardial injury induced by myocardial infarction through inhibiting cardiomyocyte apoptosis. We further discovered that miRNA-330-3p is highly expressed in BMSCsGATA-4 exosomes and involved in anti-apoptosis, suggesting that miRNA-330-3p may be a key molecule for exosomes to repair myocardial injury.
OBJECTIVE: To investigate the time-volume variation in miR-330-3p expression in BMSCsGATA-4 exosomes. 
METHODS: Experimental group 1 (BMSCsGATA-4–miR-330-3p-mimic) and experimental group 2 (BMSCsGATA-4–miR-330-3p-inhibitor) were established by adding miR-330-3p-mimic or miR-330-3p-inhibitor to the culture systems of BMSCsGATA-4 exosomes, respectively. The BMSCsGATA-4, BMSCsGATA-4–empty vector, BMSCs, BMSCsGATA-4–miR-330-3p-mimic–empty vector, and BMSCsGATA-4–miR-330-3p-inhibitor–empty vector groups were taken as confounding factors and incubated for 24 or 48 hours with doxycycline, a gene opener. Exosomes secreted by each group were extracted. RT-PCR was used to detect the expression level of miR-330-3p in each group of cells and secreted exosomes. Morphological changes in the BMSCsGATA-4-microRNA-330-3p-mimic, BMSCsGATA-4-microRNA-330-3p-inhibitor, and BMSCs groups at 24 and 48 hours of incubation with doxycycline were observed under light microscopy. 
RESULTS AND CONCLUSION: The expression of miR-330-3p was highest in the cells and exosomes of the BMSCsGATA-4–miR-330-3p-mimic group (P < 0.05), and increased gradually with time. Conversely, the expression of miR-330-3p was lowest in the cells exosomes of the BMSCsGATA-4-miR-330-3p-inhibitor group, and moreover, it decreased gradually with time. There were no morphological changes in the BMSCsGATA-4–miR-330-3p-mimic, BMSCsGATA-4–miR-330-3p-inhibitor, and BMSC group at 24 and 48 hours of incubation with doxycycline. Therefore, miR-330-3p expression in BMSCs is positively correlated with that in exosomes. Expression of miR-330-3p in exosomes can be effectively increased through miR-330-3p overexpression while reduced by silencing.

Key words: myocardial infarction, myocardial injury, bone marrow mesenchymal stem cells, GATA-4, exosomes, miR-330-3p, National Natural Science Foundation of China

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