Effects of Jiangu Granules on the proliferation of osteoblasts through G_1/S phase cell-cycle regulated proteins

目的观察健骨颗粒对成骨细胞G_1/S期调控的影响,探讨健骨颗粒促进成骨细胞增殖的作用机制。方法制备健骨颗粒血清组、模型血清组和雌二醇血清组。采用酶消化法培养SD大鼠成骨细胞,健骨颗粒含药血清干预,以模型血清和雌二醇血清为对照。运用流式细胞术检测成骨细胞增殖周期,荧光定量PCR法检测成骨细胞G_1/S期调控蛋白Cyclin E、CDK2、p21和转录因子E2F-1 m RNA的表达。结果 15%的雌二醇血清与健骨颗粒血清干预48 h,成骨细胞增殖速度均明显快于模型血清组(P<0.01);G_0/G_1期成骨细胞比例明显降低(P<0.01),S期、G_2/M期细胞比例及增殖指数则明显高于模型血清组(P<0.01);与模型血清组比较,雌二醇血清组与健骨颗粒血清组能提高成骨细胞Cyclin E、CDK2及转录因子E2F-1 m RNA的表达(P<0.01),而降低p21的表达(P<0.01)。结论健骨颗粒通过调节成骨细胞G_1/S期调控机制,推进成骨细胞顺利通过G_1/S检测点,促进成骨细胞增殖。Objective To observe the effects of Jiangu Granules on the G_1/S phase cell-cycle regulated proteins of osteoblasts, so as to reveal the mechanism of Jiangu Granules in promoting osteoblasts proliferation. Methods The Jiangu Granules-serum group, model-serum group and estradiol-serum group were prepared. Osteoblasts of SD rats were cultivated by enzymatic digestion and intervened with Jiangu Granules-serum, model-serum and estradiol-serum(as control) respectively. The cell cycles were analyzed by flow cytometry, while the fluorescence quantitative PCR was applied to measure cyclin E, CDK2, p21 and E2F-1 m RNA. Results The cell proliferation rates of osteoblasts intervened with estradiol serum and Jiangu Granules-serum for 48 hours were faster than that with the same concentration model-serum(P < 0.01). Compared with the model-serum group, the proportion of osteoblasts in the G_0/G_1 phase were significantly reduced after intervention with 15% of estradiol serum and Jiangu Granules-serum for 48 hours(P < 0.01),while the proportion of cells in S phase, G_2/M phase and proliferation index was much higher(P < 0.01). The expression of cycling E, CDK 2 and transcription factor E2F-1 m RNA of osteoblasts in the Jiangu Granules-serum group and the estradiol-serum group was higher than that of model-serum group(P < 0.01), while the expression of p21 m RNA was lower than the model-serum group(P < 0.01). Conclusion Jiangu Granules-serum can adjust the G_1/S phase cellcycle regulated mechanism in osteoblast, so as to push the cells passing G_1/S checkpoint and accelerate the proliferation of osteoblast.国家自然科学基金资助项目(81473706

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