摘要
目的探讨多孔块体生物玻璃在体外环境中对成骨细胞黏附、增殖及分化的影响。方法多孔块状生物玻璃浸泡于α-MEM培养基中制备浸提液，采用电感耦合等离子体原子发射光谱仪检测生物玻璃培养基及α-MEM培养基中的离子浓度。通过Giemsa染色、黏着斑蛋白（Venculin）免疫荧光染色检测生物玻璃培养基培养的MC3T3-E1细胞（生物玻璃培养基组）及α—MEM培养基培养的MC3T3-E1细胞（普通培养基组）的细胞个数、核质比、Venculin免疫荧光强度；通过细胞周期检测、MTT检测、Brdu检测比较两组细胞的各个细胞周期数量构成比、OD值、Brdu阳性细胞比；提取两组细胞总RNA并行成骨相关基因碱性磷酸酶、骨钙素、Ⅰ型胶原蛋白分析，经碱性磷酸酶染色和茜素红染色观察两组培养基培养细胞的碱性磷酸酶活性率及细胞矿化率。结果生物玻璃培养基中的Si离子和F离子浓度分别为（40．02±0．67）mg／L、（0．02±0．001）mg／L，α-MEM培养基分别为（2．02±0．01）mg／L、0．00mg／L。Giemsa染色后生物玻璃培养基组和普通培养基组400倍镜下的细胞个数分别为（106．0±6．025）个、（40．20±3．639）个，差异有统计学意义；免疫荧光染色结果显示生物玻璃培养基组细胞的核质比、Venculin免疫荧光强度（分别为40．85±5．720、0．05088±0．02178）较普通培养基组（分别为21．93±4．137、0．02360±0．00318）增高。生物玻璃培养基组处于S期和G2／M期细胞数量构成比较普通培养基组高。生物玻璃培养基组OD值至第3天开始较普通培养基组增高，BrdU阳性细胞比较普通培养基组增高。生物玻璃培养基组细胞碱性磷酸酶、骨钙素、Ⅰ型胶原的表达较普通培养基组增多，碱性磷酸酶活性率（1．328％±0．01536％）高于普通培养基组（0．979％±0．03059％），细胞矿化率（2．953％±0．5363％）高于普通培�
Objective To observe and identify the impact of a type of macro-pore hone block hioactive glass on osteoblast in vitro. Methods Extract fluid of new bioactive glass was prepared with α-MEM culture medium as the bioactive medium group. And the concentrations of different ions were detected with Inductively Coupled Plasma-Atomic Emission Spectrometry in bioactive medium group and α-MEM medium group. MC3T3-E1 cells cultured in bioactive medium group were considered as experimental group and cells cultured in α-MEM medium as control group. Giemsa and immunofluorescence staining was performed to detect the cell numbers, the karyoplasmic ratio and the average fluorescence intensity per cell. Cell proliferation and viability in different groups were detected by cell cycle analysis, MTT assay and BrdU assay, respectively. Total RNAs of cells in different groups were extracted and the expressions of ALP, OCN and collagen I were measured by quantitative real time PCR. ALP staining and alizarin red staining were performed to assess the differentiation and mineralization of MC3TC-E1 ceils in different groups. Results The concentrations of Si and F were 40.02±0.67 mg/L and 0.02±0.001 mg/L in bioactive medium group, higher than 2.02±0.01 mg/L and 0.00 mg/L in α-MEM solution, and the concentration of Ca was lower than that in α-MEM solution. The concentration of P and Na had no difference. In Giemsa staining, the cell numher in 400 times field under a microscope was 106.0±6.025 in bioactive medium group and 40.20±3.639 in α-MEM medium group. In the immunofluorescence of vinculin, the karyoplasmic ratio and tbe expression of vinculin were higher in bioactive medium group （40.85±5.720, 0.050 88±0.021 78） than in α-MEM medium group （21.93±4.137, 0.023 60±0.003 18）. In cell cycle analysis, the proportion of cells retained in S and G2/M phase in the bioactive medium group was more than that in the α-MEM medium group after 72 hours of cell culture. In the BrdU and MTT assay, MC3T3-E1 cells in bioactive medium
出处
《中华骨科杂志》
CAS
CSCD
北大核心
2016年第3期168-176,共9页
Chinese Journal of Orthopaedics
基金
国家自然科学基金（81472043）
关键词
成骨细胞
细胞增殖
细胞分化
Osteoblasts
Cell proliferation
Cell differentiation
