釉基质蛋白对猪骨髓基质细胞黏附、伸展及增殖的影响

目的：研究釉基质蛋白（enamel matrix proteins,EMPs）对体外培养的猪骨髓基质细胞（bone marrow stromal cells,BMSCs）黏附、伸展和增殖活性的影响。方法：抽取猪髂骨骨髓．全血培养法获得骨髓基质细胞。培养液中EMPs的浓度分别为25、50、100、200μg／ml,以不加EMPs为对照。用比色法检测不同浓度EMPs对BMSCs黏附的影响。通过计数预定视野中伸展的细胞数,计算BMSCs在培养1h、3．5h、6、5h后的伸展率。MTT法测定各组细胞的增殖活性。对实验数据行单因素方差分析和SNK法组间比较。结果：猪BMSCs在含有EMPs的培养液中生长良好。对照组以及不同浓度EMPs实验组对细胞黏附的影响无统计学差异。在1h、3．5h、6．5h．各组细胞的伸展率无显著不同。EMPs对BMSCs的促增殖作用呈浓度和时间依赖性．200μg／ml浓度的EMPs从实验的第3天开始．显著促进猪BMSCs的增殖。结论：EMPs对体外培养的猪BMSCs的黏附和伸展无显著影响．200μg／ml浓度的EMPs可显著促进猪BMSCs增殖,为联合应用EMPs和BMSCs修复牙周组织缺损提供了理论依据。

Effects of enamel matrix proteins on the attachment,spreading and proliferation of porcine bone marrow stromal cells in vitro

PURPOSE: The purpose of this study is to investigate the influence of enamel matrix proteins(EMPs) on the attachment, spreading and proliferation of cultured porcine bone marrow stromal cells (BMSCs). METHODS: BMSCs were obtained from porcine bone marrow aspiration and cultured in DMEM medium with 10% FBS.The third passage cells were exposed to various concentrations of EMPs (25,50,100 and 200microg /ml).Controls were BMSCs cultured in DMEM medium without EMPs. The cell attachment was analyzed by a colorimetric assay and cell spreading rates were performed at culture time of 1h, 3.5h and 6.5h by analysis of micrographs taken at predetermined sites of each wells. BMSCs proliferation rates were carried out over a 9-day period and assessed by an MTT assay. A parametric one-way analysis of variance (ANOVA) and multiple comparisons were used to test the treatments based on the Stument-Newman- Keuls test. RESULTS: It was shown that BMSCs well attached and spread on EMPs-coated substrata,however, there were no significant differences between the control group and experimental groups with various concentrations of EMPs. The proliferation of BMSCs was significantly stimulated by EMPs in a dose- and time- dependent manner,and EMPs at a concentration of 200microg/ml significantly enhanced BMSCs proliferation from day 3 over the experiment. CONCLUSIONS: The results demonstrate that EMPs stimulate BMSCs proliferation, whereas this factor does not affect the attachment and spreading of these cells. These findings lend theoretical support to the combined application of EMPs and BMSCs in repairing periodontal defects. Supported by National "863" Project(Grant No.2002AA205011) and Educational Development Foundation of Shanghai Municipality.