中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (1): 1-6.doi: 10.12307/2022.001

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

低氧预处理增强老年人骨髓间充质干细胞条件培养基对H9C2细胞氧化应激损伤的保护作用

宋慧芳1，2，谈佳音2，康  毅3，李  斌2，毕志斐2，龙  女2，夏仲年2，郭  蕊4   

  1. 山西医科大学，1第一医院，2人体解剖学教研室，3山西省肿瘤医院，4形态学实验室，山西省太原市   030001
  • 收稿日期:2021-01-13 修回日期:2021-01-20 接受日期:2021-02-23 出版日期:2022-01-08 发布日期:2021-10-23
  • 通讯作者: 宋慧芳，博士，副教授，山西医科大学，山西省太原市 030001 郭蕊，博士，高级实验师，山西医科大学形态学实验室，山西省太原市 030001
  • 作者简介:宋慧芳，女，1981年生，山西省壶关县人，博士，副教授，主要从事干细胞衰老及心脏、肝脏修复方向研究工作。
  • 基金资助:
    国家自然科学基金项目(81702239)，项目负责人：宋慧芳；山西省自然科学基金项目(201701D221252)，项目负责人：宋慧芳；山西省自然科学基金项目(201801D221397)，项目负责人：郭蕊

Hypoxic pretreatment enhances the protective effect of aged human bone marrow mesenchymal stem cells conditioned medium against H9C2 oxidative stress damage

Song Huifang1, 2, Tan Jiayin2, Kang Yi3, Li Bin2, Bi Zhifei2, Long Nü2, Xia Zhongnian2, Guo Rui4   

  1. 1First Hospital, 2Department of Human Anatomy, 3Shanxi Provincial Cancer Hospital, 4Morphology Laboratory, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
  • Received:2021-01-13 Revised:2021-01-20 Accepted:2021-02-23 Online:2022-01-08 Published:2021-10-23
  • Contact: Song Huifang, PhD, Associate professor, First Hospital, and Department of Human Anatomy, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China Guo Rui, PhD, Senior experimentalist, Morphology Laboratory, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
  • About author:Song Huifang, PhD, Associate professor, First Hospital, and Department of Human Anatomy, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81702239 (to SHF); the Natural Science Foundation of Shanxi Province, No. 201701D221252 (to SHF), No. 201801D221397 (to GR)

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摘要：

文题释义：
丝氨酸/苏氨酸蛋白激酶：在细胞代谢、细胞存活、凋亡、细胞周期、转录调控等多种生物学过程中发挥重要作用。
人骨髓间充质干细胞：是成体干细胞的一种，因其具有易于体外分离培养、低免疫原性等特点，是机体损伤修复和干细胞移植治疗的常用种子细胞，此种细胞已获批多项干细胞临床试验项目。

背景：老年缺血性心脏损伤患者自体干细胞移植治疗效果欠佳，需要探索提高老年干细胞修复再生能力的有效途径。
目的：探讨低氧预处理老年人骨髓间充质干细胞条件培养基对心肌的保护作用，为改善老年患者自体干细胞移植疗效提供实验支持。 
方法：将青年人骨髓间充质干细胞置于常氧培养箱培养，老年人骨髓间充质干细胞分别置于常氧和低氧培养箱培养，收集条件培养基分为青年常氧培养基组、老年常氧培养基组和老年低氧培养基组。H9C2细胞用3种条件培养基在常氧培养箱中培养24 h后，加入300 μmol/L 过氧化氢继续培养30 min诱导氧化应激，采用CCK-8法检测细胞活性，活性氧试剂盒检测活性氧水平反映氧化应激损伤程度，Western blot检测Bax、Bcl-2表达量反映细胞凋亡水平，Western blot检测p-AKT表达量反映AKT活化水平。另外，老年低氧培养基处理及过氧化氢诱导细胞氧化应激前，用含50 μmol/L LY294002的DMEM培养液培养2 h，以阻断PI3K/AKT通路的激活，然后采用CCK-8法检测细胞活性。
结果与结论：①与老年常氧培养基组比较，老年低氧培养基组H9C2细胞活力显著增加(P < 0.01)；凋亡相关蛋白Bax表达显著降低(P < 0.05)，凋亡抑制蛋白Bcl-2表达显著增高(P < 0.05)；活性氧水平显著降低(P < 0.05)；②与老年常氧培养基组比较，老年低氧培养基组H9C2细胞中p-AKT蛋白表达显著增高(P < 0.05)；③与老年低氧培养基组比较，应用LY294002抑制PI3K/AKT信号通路后，H9C2细胞活力下降(P < 0.05)；④结果提示，低氧预处理老年人骨髓间充质干细胞条件培养基通过AKT通路减弱H9C2细胞氧化应激损伤，减少细胞凋亡，提高细胞活性。
https://orcid.org/0000-0003-3471-9497(宋慧芳)；https://orcid.org/0000-0001-8684-1414(郭蕊)

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: 干细胞, 人骨髓间充质干细胞, 老年, 条件培养基, 低氧, 预处理, H9C2细胞, AKT, 氧化应激

Abstract: BACKGROUND: The therapeutic effect of autologous stem cell transplantation in aged patients suffered with ischemic heart injury is not good as young. It is important to explore effective methods to improve the repair and regenerate ability of aged stem cells. 
OBJECTIVE: To investigate the protective effect of hypoxia preconditioning aged human bone marrow mesenchymal stem cells conditioned medium on myocardium and to provide experimental support for more effective autologous stem cell transplantation in aged patients. 
METHODS: The young human bone marrow mesenchymal stem cells were placed in a normoxic incubator, and the aged human bone marrow mesenchymal stem cells were placed in a normoxic or hypoxic incubator respectively. The culture media were collected and divided into three groups: norCM-young, norCM-old and hypoCM-old. H9C2 cells were treated with three groups of conditioned media, and then treated with 300 μmol/L H2O2 for 30 minutes to establish an oxidative stress model. The cell survival rate of H9C2 was detected by the CCK-8 assay. Reactive oxygen species level detection by reactive oxygen species assay kit reflected the degree of oxidative stress damage. Bax and Bcl-2 expression levels by western blotting reflected the apoptosis level. p-AKT expression level detected by western blot assay reflected AKT activation level. In addition, H9C2 was cultured with Dulbecco’s Modified Eagle Medium containing 50 μmol/L LY294002 for 2 hours before hypoCM-old and H2O2 treatment to block the PI3K/AKT pathway and cell viability was detected by the CCK-8 assay.  
RESULTS AND CONCLUSION: (1) Compared with norCM-old group, hypoCM-old significantly improved the survival rate of H9C2 cells under oxidative stress damage (P < 0.01); significantly reduced the expression of apoptosis-related protein Bax (P < 0.05), increased the expression of apoptosis inhibitor protein Bcl-2 (P < 0.05); and reduced the ROS level in H9C2 cells (P < 0.05). (2) Compared with the norCM-old group, the p-AKT protein level in H9C2 cells treated with hypoCM-old was significantly increased (P < 0.05). (3) Compared with the hypoCM-old group, when the PI3K/AKT pathway of H9C2 was inhibited, the H9C2 cell survival was reduced (P < 0.05). (4) The results indicate that the conditioned media of aged human bone marrow mesenchymal stem cells pretreated with hypoxia could attenuate the oxidative stress damage of H9C2 cells, reduce apoptosis and enhance survival through the activation of AKT pathway. 

Key words: stem cells, human bone marrow mesenchymal stem cells, aged, conditioned medium, hypoxia, preconditioning, H9C2 cells, AKT, oxidative stress 

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