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目的:研究外源性纤维黏连蛋白(fibronectin,FN)对大鼠成骨细胞增殖、分化及凋亡的影响,探讨应用FN治疗骨缺损性疾病的可行性。方法:酶消化法分离成骨细胞,通过细胞对染料Alamar蓝摄取检测细胞增殖,ELISA方法检测碱性磷酸酶活性,流式细胞术检测FN对成骨细胞细胞周期及凋亡的影响,Western印迹法检测FN作用后成骨细胞Bcl-2及Bax基因的蛋白表达变化。采用SPSS12.0软件包对实验数据进行单因素方差分析。结果:FN浓度为40μg/mL以上时,能促进大鼠成骨细胞的增殖,与对照组相比有显著性差异(P<0.05);FN能增加成骨细胞ALP活性,各组与对照组相比均有显著性差异(P<0.05),并有明显的剂量依赖性;不同浓度的FN作用成骨细胞后,可使处于DNA合成期(S期)的细胞百分率增加;增殖指数(proliferation index,PI)增加,细胞凋亡百分率减少,与对照组相比有显著性差异(P<0.05);FN作用后的细胞,Bcl-2基因的蛋白表达量增加,Bax基因的蛋白表达量减少,有剂量依赖性。结论:外源性FN能促进大鼠成骨细胞增殖、分化,使大鼠成骨细胞的DNA合成期(S期)的细胞百分比增加,同时抑制大鼠成骨细胞的凋亡,使大鼠成骨细胞胞质内Bcl-2基因的蛋白表达量增加,同时减少Bax基因的蛋白表达量。
Objective: To investigate the effects of fibronectin (FN) on proliferation, differentiation and apoptosis of rat osteoblasts and to explore the feasibility of using FN to treat bone defects. METHODS: Osteoblasts were isolated by enzymatic digestion. Cell proliferation was detected by Alamar blue dye uptake. Alkaline phosphatase activity was detected by ELISA. Flow cytometry was used to detect the effect of FN on cell cycle and apoptosis of osteoblasts. Western blotting Assay was used to detect the protein expression of Bcl-2 and Bax in osteoblasts after FN treatment. One-way ANOVA was used to analyze the experimental data using SPSS12.0 software package. Results: The FN concentration of 40μg / mL could promote the proliferation of rat osteoblasts, which was significantly different from the control group (P <0.05). FN increased the activity of ALP in osteoblasts, (P <0.05), and there was a dose-dependent manner. After treated with different concentrations of FN, osteoblasts could increase the percentage of cells in the DNA synthesis phase (S phase); proliferation index (P <0.05). The protein expression of Bcl-2 and Bcl-2 in the cells after FN treatment were increased and the protein expression of Bax decreased , In a dose-dependent manner. Conclusion: Exogenous FN can promote the proliferation and differentiation of rat osteoblasts, increase the percentage of cells in the osteoblast DNA synthesis phase (S phase), and inhibit the apoptosis of rat osteoblasts at the same time. Rats The protein expression of Bcl-2 in the cytoplasm of osteoblasts increased, while the protein expression of Bax decreased.