中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (6): 821-826.doi: 10.12307/2023.230

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

四氧化三锰纳米颗粒抗氧化损伤保护骨髓间充质干细胞的伸展功能

田沁玉1,2,田兴贵3,田  壮4,眭  翔2,刘舒云2,鲁晓波1,郭全义1,2   

  1. 1西南医科大学附属医院骨与关节外科,四川省泸州市   646000;2中国人民解放军总医院第一医学中心骨科研究所,骨科再生医学北京市重点实验室,全军骨科战创伤重点实验室,北京市   100853;3德累斯顿工业大学附属大学医院骨科,德国德累斯顿   01307;4北京大学第九临床医学院,北京世纪坛医院,北京市   100038
  • 收稿日期:2022-01-14 接受日期:2022-03-17 出版日期:2023-02-28 发布日期:2022-08-04
  • 通讯作者: 郭全义,博士,教授,主任医师,西南医科大学附属医院骨与关节外科,四川省泸州市 646000;中国人民解放军总医院第一医学中心骨科研究所,骨科再生医学北京市重点实验室,全军骨科战创伤重点实验室,北京市100853
  • 作者简介:田沁玉,女,1995年生,四川省泸州市人,西南医科大学在读硕士,主要从事抗氧化纳米材料用于骨及软骨再生方面的研究。
  • 基金资助:
    国家重点研发计划(2019YFA0110600),项目负责人:郭全义

Protection of manganese oxide nanoparticles for bone marrow mesenchymal stem cell spreading against oxidative stress

Tian Qinyu1, 2, Tian Xinggui3, Tian Zhuang4, Sui Xiang2, Liu Shuyun2, Lu Xiaobo1, Guo Quanyi1, 2   

  1. 1Department of Bone and Joint Surgery, Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China; 2Institute of Orthopedics, First Medical Center, Chinese PLA General Hospital, Beijing Key Lab of Regenerative Medicine in Orthopedics, Beijing 100853, China; 3University Center of Orthopaedic, Trauma and Plastic Surgery, University Hospital Carl Gustav Carus at Technische Universität Dresden, Dresden 01307, Germany; 4Ninth Clinical School of Peking University, Beijing Shijitan Hospital, Beijing 100038, China
  • Received:2022-01-14 Accepted:2022-03-17 Online:2023-02-28 Published:2022-08-04
  • Contact: Guo Quanyi, MD, Professor, Chief physician, Department of Bone and Joint Surgery, Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China; Institute of Orthopedics, First Medical Center, Chinese PLA General Hospital, Beijing Key Lab of Regenerative Medicine in Orthopedics, Key Laboratory of Musculoskeletal Trauma & War Injuries, PLA, Beijing 100853, China
  • About author:Tian Qinyu, Master candidate, Department of Bone and Joint Surgery, Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China; Institute of Orthopedics, First Medical Center, Chinese PLA General Hospital, Beijing Key Lab of Regenerative Medicine in Orthopedics, Key Laboratory of Musculoskeletal Trauma & War Injuries, PLA, Beijing 100853, China
  • Supported by:
    the National Key Research and Development Plan Project, No. 2019YFA0110600 (to GQY)

摘要:

文题释义:
四氧化三锰:金属氧化物,为深棕色粉末,不溶于水,溶于强酸并放热。锰作为人体微量元素参与超氧化物歧化酶一种亚型的形成,四氧化三锰在人体内具有可降解性,不会造成体内长期潴留而导致生物毒性或者人体危害。
氧化应激:机体在遭受有害刺激时,体内高活性分子如活性氧自由基或活性氮自由基产生过多,导致细胞、组织和动物机体受到严重损伤,减少氧化应激是保护干细胞功能的手段之一。

背景:由于干细胞移植微环境原位产生的大量自由基造成移植干细胞伸展功能受损,进而降低细胞的移植存活率。近10年来,大量抗氧化应激纳米酶研究的出现为保护干细胞伸展功能提供了新的方法。四氧化三锰(Mn3O4)作为一种新型纳米酶,含有的锰作为人体微量元素之一,具有抗氧化应激性能以及生物可降解性,可作为一种保护干细胞伸展功能的新型纳米材料。
目的:制备纳米酶四氧化三锰颗粒,检测其在氧化应激环境中对骨髓间充质干细胞抗氧化作用和伸展功能的影响。
方法:水热法制备四氧化三锰纳米颗粒,用扫描电子显微镜、X射线衍射分别表征材料形貌和结构;动态光散射仪检测模拟人体环境下的颗粒粒径和Zeta电位;在中性环境下检测四氧化三锰的抗氧化能力,使用CCK-8和活-死染色法检测四氧化三锰对骨髓间充质干细胞的生物毒性;过氧化氢诱导骨髓间充质干细胞氧化应激,检测四氧化三锰对骨髓间充质干细胞在氧化应激状态下抗氧化能力及伸展能力的影响。
结果与结论:①扫描电子显微镜和X射线衍射测试结果显示该材料为平均直径70-80 nm 的四氧化三锰纳米颗粒;动态光散射仪检测显示,在模拟人体缓冲环境下的四氧化三锰的粒径约为100 nm,Zeta电位约为+20 mV,抗氧化性能实验显示四氧化三锰具有一定的抗氧化能力;②CCK-8、活-死染色、活性氧清除实验显示四氧化三锰在40 mg/L的质量浓度下对骨髓间充质干细胞无生物毒性且有一定的活性氧清除能力;③细胞伸展实验显示过氧化氢对骨髓间充质干细胞的伸展功能具有明显抑制作用,而四氧化三锰加入细胞培养微环境后,细胞伸展面积较对照组无明显统计学差异(P > 0.05),且单独使用四氧化三锰不会抑制骨髓间充质干细胞的伸展功能(P > 0.05);④结果表明,在细胞微环境中加入四氧化三锰纳米颗粒能够抵抗氧化应激,并且对骨髓间充质干细胞的伸展功能具有一定的保护作用。

https://orcid.org/0000-0001-7572-6736 (田沁玉) 

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: 四氧化三锰, 纳米颗粒, 纳米酶, 抗氧化应激, 活性氧清除, 干细胞移植, 干细胞伸展, 干细胞保护

Abstract: BACKGROUND: During stem cell transplantation, the generation of a large number of free radicals damages the cells in situ, and then reducing the cell survival. In the past decade, the emergence of nanozymes provides a new method for scavenging excess free radicals. Among them, manganese oxide (Mn3O4) is one of the promising nanomaterials, because it could provide Mn trace elements in human body as well as its well antioxidative stress properties and biodegradability. Mn3O4 can be used as a new type of nanomaterial to protect the extension function of stem cells.
OBJECTIVE: To produce Mn3O4 nanoparticles for detecting its effect on scavenging and spreading function of bone marrow mesenchymal stem cells during oxidative stress. 
METHODS:  Mn3O4 nanoparticles were prepared by hydrothermal method. Scanning electron microscope and X-ray diffraction were used to characterize the morphology and structure of Mn3O4, respectively. The particle size and zetapotential data of the nanoparticle in human-simulated environment were detected by the dynamic light scattering. The antioxidant capacity of Mn3O4 was tested in neutral environment. The CCK-8 and live-dead staining assays were utilized to verify the biological toxicity of Mn3O4 to bone marrow mesenchymal stem cells. H2O2 was used to induce oxidative stress of bone marrow mesenchymal stem cells. The effect of Mn3O4 on antioxidant ability and spreading ability of bone marrow mesenchymal stem cells was detected under oxidative stress. 
RESULTS AND CONCLUSION: (1) The results of scanning electron microscope and X-ray diffraction exhibited that the material was Mn3O4 with average particle size about 70-80 nm. The dynamic light scattering showed that the particle size of the nanoparticles in PBS solution was about 100 nm, and the Zeta potential was about +20 mV. Antioxidant performance experiments showed that Mn3O4 had a certain antioxidant capacity. (2) The CCK-8 assay, live-death staining and reactive oxygen scavenging experiment demonstrated that Mn3O4 did not show biotoxicity and exhibited a certain level of reactive oxygen scavenging ability under 40 mg/L. (3) The cell spreading analysis showed that H2O2 had a significant inhibitory effect on the spreading function of bone marrow mesenchymal stem cells. After adding Mn3O4 into the cell culture microenvironment, the cell spreading area had no significant difference compared with the control group (P > 0.05). Moreover, the single use of Mn3O4 did not inhibit the spreading function of bone marrow mesenchymal stem cells (P > 0.05). (4) The results have shown that the addition of Mn3O4 nanoparticles in the cell microenvironment can resist oxidative stress and have a certain protective effect on the spreading function of bone marrow mesenchymal stem cells.

Key words: manganese oxide, nanoparticle, nanozyme, anti-oxidative stress, reactive oxygen species scavenging, stem cell transplantation, stem cell spreading, stem cell protection

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