碱性成纤维细胞生长因子对生长板软骨细胞增殖与分化的影响

摘要

背景: 体外培养软骨细胞经历多次传代后,其增殖能力将逐渐退化.实验表明,碱性成纤维细胞生长因子可促进软骨细胞增殖,并可能影响软骨细胞的表型与分化.目的:观察碱性成纤维细胞生长因子对生长板软骨细胞增殖和分化的影响,并筛选其用于体外软骨细胞培养的最佳剂量.设计、时间及地点:以细胞为观察对象的观察对比实验,于2004-10/2005-02在暨南大学医学院生化教研室组织工程实验室完成.材料: 选用12只新西兰白兔用于分离软骨细胞,碱性成纤维细胞生长因子由英国Peoro Tech公司生产.方法: 分离并在低血清条件下培养兔生长板软骨细胞.根据实验需要加入0.1,2.5,5,10,25,50及100 μg/L 8个剂量的碱性成纤维细胞生长因子,进行各项指标观察.主要观察指标: 应用改良四甲基偶氮唑比色法检测细胞增殖倍数: 应用羟脯氨酸法测定软骨细胞胶原产量:应用酶动力学方法测定碱性磷酸酶活性.结果: ①碱性成纤维细胞生长因子剂量在5-100 μg/L,范围内可以促进软骨细胞增殖,并以25 μg/L时刺激效果最为显著.②当碱性成纤维细胞生长因子剂量高?5 μg/L时,软骨细胞的胶原合成被抑制.③当碱性成纤维细胞生长因子剂量高于1 μg/L时,软骨细胞的碱性磷酸酶活性被抑制.结论: 碱性成纤维细胞生长因子可以刺激生长板软骨细胞增殖.并在剂量高于25μg/L时抑制生长板软骨细胞的分化.%BACKGROUND: The proliferative capacity of in vitro cultured chondrocytes is gradually reduced following several passages. Many studies have demonstrated that basic fibrublast growth factor (bFGF) can promote chondrocyte proliferation, and influence its phenotype and differentiation. OBJECTIVE: To observe the influence of bFGF on the proliferation and differentiation of growth plate chondrocytes and to screen the best dosage for in vitro culture of chondrocytes. DESIGN, TIME AND SETTING: For observation and comparison of cells, the experiment was performed at Tissue Engineering Laboratory, Department of Biochemistry, Jinan University Medical School between October 2004 and February 2005. MATERIALS: Twelve New Zealand rabbits were selected for chondrocyte isolation; bFGF was purchased from PeproTech, UK. METHODS: Rabbit growth plate chondrocytes were isolated and cultured in low serum. Different doses of bFGF (0, 1, 2.5, 5, 10, 25, 50 and 100 μg/L) were added. MAIN OUTCOME MEASURES: Cell proliferative multiple was determined using modified MTT method; chondrocyte collagen content was determined using hydroxyproline method; alkaline phosphatase activities were detected by enzyme kinetics. RESULTS: bFGF (5-100 μg/L) could promote chondrocyte proliferation, especially at dose of 25 μg/L. bFGF at dose of larger than 25 μg/L would inhibit collagen production; bFGF at dose of larger than 1 μg/L would inhibit alkaline phosphatase activities. CONCLUSION: bFGF can stimulate growth plate chondrocyte proliferation, and inhibit growth plate chondrocyte differentiation at dose25 μg/L.