低氧对H2O2预处理MC3T3-E1增殖和成骨分化功能的影响  

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

作　　者：梁静 王君 唐传玲[3] 周琦[1,2] 魏立 胡方琼[1,2] 万荣 Liang Jing Wang Jun Tang Chuan-ling Zhou Qi Wei Li Hu Fang-qiong Wan Rong(Shanghai Key Laboratory for Bone and Joint Diseases,Shanghai Institute of Orthopedics and Traumatology,Shanghai Ruijin Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai First Maternity and Infant Hospita)

机构地区：[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

出　　处：《中国组织工程研究》2017年第4期505-511,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金资助项目(81300505)~~

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H_2O_2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H_2O_2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200μmol/L H_2O_2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400μmol/L H_2O_2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;(3)400μmol/L H_2O_2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400μmol/L H_2O_2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H_2O_2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H_2O_2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。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（H_2O_2） 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 H_2O_2 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 m RNA levels of collage type 1, alkaline phosphatase and Cbfa1. RESULTS AND CONCLUSION： When MC3T3-E1 pretreated with 200 μmol/L H_2O_2 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 H_2O_2 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 H_2O_2 for 6 hours and then cultured in hypoxia, the m RNA expression of Cbfa1 was decreased, but the m RNA expressions of collage type 1 and alkaline phosphatase were significantly increased. These results suggest that MC3T3-E1 pretreated with low concentration of H_2O_2 show a significant decrease in proliferation, while MC3T3-E1 pretreated with a high concentration of H_2O_2 and cultured in hypoxia

关 键 词：细胞低氧 成骨细胞 碱性磷酸酶 缺氧诱导因子1 组织工程 组织构建 缺氧诱导因子 MC3T3-E1 H2O2 Ⅰ型胶原 CBFA1 定量PCR 国家自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]