中国组织工程研究 ›› 2017, Vol. 21 ›› Issue (4): 505-511.doi: 10.3969/j.issn.2095-4344.2017.04.003

• 骨组织构建 bone tissue construction • 上一篇    下一篇

低氧对H2O2预处理MC3T3-E1增殖和成骨分化功能的影响

梁  静1,2,王  君1,2,唐传玲3,周  琦1,2,魏  立1,2,胡方琼1,2,万  荣2   

  1. 1上海骨与关节病损重点实验室,上海市  200025;2上海市伤骨科研究所,上海瑞金医院,上海交通大学医学院,上海市  200025;3同济大学附属第一妇婴保健院,上海市  201204
  • 收稿日期:2016-12-10 出版日期:2017-02-08 发布日期:2017-03-13
  • 通讯作者: 通讯作者:万荣,副主任医师,上海瑞金医院骨科,上海交通大学医学院,上海市伤骨科研究所,上海市 200025
  • 作者简介:梁静,女,1979年生,汉族,山东省泰安市人,2005年上海第二医科大学毕业,硕士,主管技师,主要从事骨系细胞培养研究。 并列第一作者:王君,主管技师,主要从事分子生物学实验。
  • 基金资助:

    国家自然科学基金资助项目(81300505)

Hypoxia effects on the proliferation and differention of hydrogen peroxide-pretreated  MC3T3-E1

Liang Jing1, 2, Wang Jun1, 2, Tang Chuan-ling3, Zhou Qi1, 2, Wei Li1, 2, Hu Fang-qiong1, 2, Wan Rong2   

  1. 1Shanghai Key Laboratory for Bone and Joint Diseases, Shanghai 200025, China; 2Shanghai Institute of Orthopedics and Traumatology, Shanghai Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 20025, China; 3Shanghai First Maternity and Infant Hospital, Shanghai 201204, China
  • Received:2016-12-10 Online:2017-02-08 Published:2017-03-13
  • Contact: Corresponding author: Wan Rong, Associate chief physician, Shanghai Institute of Orthopedics and Traumatology, Shanghai Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 20025, China
  • About author:Liang Jing, Master, Technologist-in-charge, Shanghai Key Laboratory for Bone and Joint Diseases, Shanghai 200025, China; Shanghai Institute of Orthopedics and Traumatology, Shanghai Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 20025, China Wang Jun,Technologist-in-charge, Shanghai Key Laboratory for Bone and Joint Diseases, Shanghai 200025, China; Shanghai Institute of Orthopedics and Traumatology, Shanghai Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 20025, China Liang Jing and Wang Jun contributed equally to this work.
  • Supported by:

    the National Natural Science Foundation of China, No. 81300505

摘要:

文章快速阅读:
 

文题释义:
氧化应激:细胞内活性氧簇增加导致的氧化应激(OS),活性氧高度活跃,过量或过长时间的活性氧形成可以损伤细胞成分,活性氧引起蛋白质、脂类、染色体和线粒体DNA氧化损伤的积聚。H2O2(活性氧成分之一)可以抑制MC3T3-E1细胞的增殖和成骨分化,并且呈浓度和时间依赖性。实验用小鼠成骨细胞样细胞MC3T3- E1在不同浓度的H2O2干预下,MC3T3-E1的细胞活性随着氧化应激损伤的加重(H2O2浓度增加,干预时间延长),细胞活性下降明显,显微镜下看到有大量细胞死亡。
低氧环境:低氧并不仅仅定义为通常的氧分压,它是和组织或细胞的代谢需求相关的。低氧表示的是没有满足组织需要的病理性低氧分压。低氧诱发的活性氧簇可引起所有细胞成分的损伤,并且参与调控成骨细胞的活性和分化。将氧化应激损伤的MC3T3-E1再置于低氧(1%O2)环境下培养,结果MC3T3-E1的增殖和分化都会受影响,这和氧化应激损伤的程度及低氧时间都有关系,首先是细胞增殖受到抑制,随着氧化应激损伤的加重,低氧培养时间的延长,会进一步影响细胞的分化和功能,以至于出现不可逆的损伤和细胞死亡。

摘要
背景:
活性氧簇增加可以导致氧化应激,体内低氧环境在生理和病理状态下都存在,但处于氧化应激状态的骨组织或者已经有氧化应激损伤的骨系细胞在低氧环境下增殖和分化功能的变化未见相关报道。
目的:拟观察小鼠成骨细胞样细胞(MC3T3-E1)在不同浓度H2O2干预后处于低氧环境下的成骨细胞生物学特性的改变,以期揭示老年骨质疏松和糖尿病患者骨折愈合过程延长的细胞学机制。
方法:用不同浓度H2O2干预的小鼠成骨细胞样细胞置于不同的氧浓度下培养,用细胞计数试剂盒(CCK8)检测细胞增殖活性的变化,用碱性磷酸酶染色和钙结节染色检测细胞向成骨方向分化能力的改变,并收集RNA检测成骨分化早期特异性基因信使RNA表达的变化。
结果与结论:①小鼠成骨细胞样细胞用200 μmol/L H2O2预处理6 h,细胞的增殖活性随着低氧培养时间延长而增加,但仍然低于正常对照组;成骨分化早期的碱性磷酸酶染色减弱,钙结节形成有明显的减少;②用400 μmol/L H2O2预处理6 h,则细胞的增殖活性下降明显,没有受氧浓度的影响,成骨分化早期的碱性磷酸酶染色减弱,低氧使碱性磷酸酶表达进一步降低,钙结节形成明显减少,但氧浓度改变对其影响不大;③400 μmol/L H2O2预处理6 h和再低氧干预时小鼠成骨细胞样细胞的Cbfa1的信使RNA表达降低,常氧下400 μmol/L H2O2影响小鼠成骨细胞样细胞的Ⅰ型胶原和碱性磷酸酶的表达,低氧培养以及氧化应激损伤后的小鼠成骨细胞样细胞成骨分化早期基因Ⅰ型胶原和碱性磷酸酶的信使RNA表达增加,并且差异有显著性意义;④结果证实,低浓度的H2O2预处理小鼠成骨细胞样细胞再低氧更容易影响细胞的增殖,而高浓度的H2O2预处理小鼠成骨细胞样细胞再低氧更容易影响细胞向成骨方向的分化能力,尤其是早期碱性磷酸酶的形成。

中国组织工程研究杂志出版内容重点:组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松组织工程
ORCID: 0000-0001-5443-9898(梁静)

关键词: 组织构建, 成骨细胞, 缺氧诱导因子, MC3T3-E1, H2O2, 碱性磷酸酶, Ⅰ型胶原, cbfa1, 定量PCR, 国家自然科学基金

Abstract:

Abstract
BACKGROUND
: The intracellular accumulation of reactive oxygen species leads to oxidative stress. Hypoxia is widespread in physiological and pathological condition. Variation of bone proliferation and differentiation when bone tissues cultured or bone cells induced toxicity by reactive oxygen species under hypoxia have not yet been reported.
OBJECTIVE: To observe the biological characteristics of MC3T3-E1 pretreated with different concentrations of hydrogen peroxide (H2O2) in hypoxia, thus understanding the cell mechanism underlying prolonged bone healing in the elderly with osteoporosis and diabetes.
METHODS: The MC3T3-E1 cells pretreated with different concentrations of H2O2 were cultured in different oxygen concentrations. The proliferation of MC3T3-E1 was detected by cell counting kit-8. The cell differentiation was detected through alkaline phosphatase staining and alizarin red staining. Total RNAs were extracted and used for analyzing the mRNA levels of collage type 1, alkaline phosphatase and Cbfa1.
RESULTS AND CONCLUSION: When MC3T3-E1 pretreated with 200 μmol/L H2O2 for 6 hours, the cell proliferation was increased with time, but lower than that in the control group. The alkaline phosphatase activity was weakened, and the number of mineralized nodes was decreased at the early stage of differentiation. When MC3T3-E1 pretreated with   400 μmol/L H2O2 for 6 hours, the cell proliferation was decreased obviously. The alkaline phosphatase activity was still weakened, and the number of mineralized nodes was decreased further, but not affected by hypoxia. When MC3T3-E1 pretreated with 400 μmol/L H2O2 for 6 hours and then cultured in hypoxia, the mRNA expression of Cbfa1 was decreased, but the mRNA expressions of collage type 1 and alkaline phosphatase were significantly increased. These results suggest that MC3T3-E1 pretreated with low concentration of H2O2 show a significant decrease in proliferation, while MC3T3-E1 pretreated with a high concentration of H2O2 and cultured in hypoxia show a decrease in osteogenic differentiation, especially at the early stage of alkaline phosphatase formation.

中国组织工程研究杂志出版内容重点:组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松组织工程

Key words: Cell Hypoxia, Osteoblasts, Alkaline Phosphatase, Hypoxia-Inducible Factor 1, Tissue Engineering

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