中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (13): 2006-2011.doi: 10.12307/2022.325

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

低强度脉冲式超声波上调细胞周期蛋白 D1表达促进骨髓间充质干细胞增殖

李  志1，2，3，华永永4，张剑权4，符  真4   

  1. 湖北医药学院附属太和医院，1普外科三病区，2胚胎干细胞研究湖北省重点实验室，湖北省十堰市  442000；3西安交通大学医学部附属太和医院，湖北省十堰市  442000；4中南大学湘雅医学院附属海口医院肝胆外科，海南省海口市  570000
  • 收稿日期:2020-12-16 修回日期:2020-12-19 接受日期:2021-01-23 出版日期:2022-05-08 发布日期:2021-12-18
  • 通讯作者: 张剑权，主任医师，教授，中南大学湘雅医学院附属海口医院肝胆外科，海南省海口市 570000
  • 作者简介:李志，男，1992年生，湖北省十堰市人，2019年中南大学毕业，硕士，医师，主要从事干细胞转化研究。
  • 基金资助:
    海南省重点科技计划项目(ZDXM2015082)，项目负责人：张剑权；海南省自然科学基金项目(817382)，项目负责人：符真

Low-intensity pulsed ultrasound promotes the proliferation of bone marrow mesenchymal stem cells by inducing cyclin D1 up-regulation

Li Zhi1, 2, 3, Hua Yongyong4, Zhang Jianquan4, Fu Zhen4   

  1. 1Ward Three, Department of General Surgery, 2Hubei Key Laboratory of Embryonic Stem Cell Research, Taihe Hospital, Hubei University of Medicine, Shiyan 442000, Hubei Province, China; 3Taihe Hospital Affiliated to Xi’an Jiaotong University Health Science Center, Shiyan 442000, Hubei Province, China; 4Department of Hepatobiliary Surgery, Haikou Hospital Affiliated to Xiangya School of Medicine, Central South University, Haikou 570000, Hainan Province, China
  • Received:2020-12-16 Revised:2020-12-19 Accepted:2021-01-23 Online:2022-05-08 Published:2021-12-18
  • Contact: Zhang Jianquan, Chief physician, Professor, Department of Hepatobiliary Surgery, Haikou Hospital Affiliated to Xiangya School of Medicine, Central South University, Haikou 570000, Hainan Province, China
  • About author:Li Zhi, Master, Physician, Ward Three, Department of General Surgery, and Hubei Key Laboratory of Embryonic Stem Cell Research, Taihe Hospital, Hubei University of Medicine, Shiyan 442000, Hubei Province, China; Taihe Hospital Affiliated to Xi’an Jiaotong University Health Science Center, Shiyan 442000, Hubei Province, China
  • Supported by:
    Hainan Science and Technology Fund Project, No. ZDXM2015082 (to ZJQ); the Hainan Natural Science Fund Project of China, No. 817382 (to FZ)

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摘要：

文题释义：
低强度脉冲式超声波：是一种强度低至0.03 mW/cm2的机械波，是机械振动能量传输的一种形式，具有低强度和低热效应的脉冲发射，因此对细胞的不良影响很小甚至没有，可以有效促进多种细胞体外增殖，包括成骨细胞、脂肪干细胞、骨髓间充质干细胞、关节软骨细胞等。
细胞周期蛋白D1：是细胞周期信号早期调控机制的重要组成部分，主要通过与CDK-4或CDK-6相互结合形成活性复合物来发挥调控作用，这些复合物可以引起一系列细胞周期信号分子转录激活和表达(主要包括细胞周期蛋白E、细胞周期蛋白A和细胞周期蛋白B)，从而促进细胞周期从G1期向S期过渡来促进细胞增殖。

背景：前期研究发现低强度脉冲式超声波可以促进人骨髓间充质干细胞增殖。
目的：探讨FAK/PI3K/Akt信号通路在低强度脉冲式超声波促进人骨髓间充质干细胞增殖中的作用机制。
方法：将购买的商品化人骨髓间充质干细胞复苏培养至第4代，分为0 mW/cm2超声波组(对照组)、50 mW/cm2超声波组、50 mW/cm2超声波+PF-562271组，其中50 mW/cm2超声波+PF-562271组在超声处理前加入1 μL FAK/PI3K/Akt信号通路抑制剂PF-562271进行预处理。每隔24 h超声刺激1次，每次5 min，刺激3次后采用CCK-8试剂盒检测细胞增殖情况，流式细胞仪检测细胞周期变化，Western blot法检测FAK及其下游靶基因和细胞周期蛋白D1的表达。
结果与结论：①与对照组相比，50 mW/cm2超声波组细胞增殖能力明显增强(P﹤0.05)，使用PF-562271抑制FAK/PI3K/Akt信号通路后，细胞增殖能力明显降低(P﹤0.001)；②与对照组相比，50 mW/cm2超声波组FAK、p-FAK、PI3K、p-PI3K、Akt、p-Akt和细胞周期蛋白D1的表达均上调(P﹤0.05)；与50 mW/cm2超声波组相比，50 mW/cm2超声波+PF-562271组上述指标明显降低(P﹤0.05)；③与对照组相比，50 mW/cm2超声波组G1期细胞比例减少(P=0.026)，S期细胞比例增多(P=0.002)；与50 mW/cm2超声波组相比，50 mW/cm2超声波+ PF-562271组G1期细胞比例升高(P=0.023)，S期细胞比例降低(P=0.035)；④结果表明，低强度脉冲式超声波可能通过FAK/PI3K/Akt信号通路上调细胞周期蛋白D1促进人骨髓间充质干细胞的增殖。

https://orcid.org/0000-0001-6555-7107 (李志)

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, 低强度脉冲式超声波, 增殖, 通路, 细胞周期

Abstract: BACKGROUND: Previous studies found that low-intensity pulsed ultrasound can promote the proliferation of human bone marrow mesenchymal stem cells.
OBJECTIVE: To investigate the mechanism of low-intensity pulsed ultrasound on the proliferation of human bone marrow mesenchymal stem cells via the FAK/PI3K/Akt signaling pathway. 
METHODS: The purchased commercial human bone marrow mesenchymal stem cells were resuscitated and cultured to the fourth generation, and they were divided into 0 mW/cm2 ultrasound group (control group), 50 mW/cm2 ultrasound group, and 50 mW/cm2 ultrasound + PF-562271 group. The 50 mW/cm2 ultrasound + PF-562271 group was pretreated with 1 μL FAK/PI3K/Akt signaling pathway inhibitor PF-562271 before ultrasound treatment, once every 24 hours, 5 minutes each time. After three stimuli, cell proliferation was detected by Cell Counting Kit-8 and the cell cycle was detected by flow cytometry. Western blot assay was used to detect the expression of FAK and its downstream target gene and cyclin D1. 
RESULTS AND CONCLUSION: (1) Compared with the control group, the cell proliferation ability was significantly enhanced in the 50 mW/cm2 ultrasound group (P < 0.05). After inhibiting the FAK/PI3K/Akt signaling pathway using PF-562271, the cell proliferation ability was significantly reduced (P < 0.001). (2) Compared with the control group, the expression levels of FAK, p-FAK, PI3K, p-PI3K, Akt, p-Akt and cyclin D1 were all up-regulated in the 50 mW/cm2 ultrasound group (P < 0.05). Compared with the 50 mW/cm2 ultrasound group, the above indicators were significantly reduced in the 50 mW/cm2 ultrasound + PF-562271 group (P < 0.05). (3) Compared with the control group, the proportion of cells in the G1 phase decreased in the 50 mW/cm2 ultrasound group (P=0.026), and the proportion of cells in the S phase increased (P=0.002). Compared with the 50 mW/cm2 ultrasound group, the proportion of cells in G1 phase increased (P=0.023), and the proportion of cells in S phase decreased in the 50 mW/cm2 ultrasound + PF-562271 group (P=0.035). (4) These results indicate that low-intensity pulsed ultrasound promotes the proliferation of human bone marrow mesenchymal stem cells via FAK/PI3K/Akt signaling pathway-induced cyclin D1 up-regulation.

Key words: stem cells, human bone marrow mesenchymal stem cells, low-intensity pulsed ultrasound, proliferation, pathway, cell cycle

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