健骨颗粒通过G1/S期调控蛋白对成骨细胞增殖的影响

贾晓康 黄云梅 李超雄 林煜 林向全 黄美雅 吴银生

[摘要] 目的 观察健骨颗粒对成骨细胞G1/S期调控的影響，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G1/S期调控蛋白Cyclin E、CDK2、p21和转录因子E2F-1 mRNA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）；G0/G1期成骨细胞比例明显降低（P < 0.01），S期、G2/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 mRNA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G1/S期调控机制，推进成骨细胞顺利通过G1/S检测点，促进成骨细胞增殖。

[关键词] 绝经后骨质疏松症；成骨细胞；细胞周期；G1/S期；健骨颗粒

[中图分类号] R285.5；R681 [文献标识码] A [文章编号] 1673-7210（2016）03（c）-0009-05

[Abstract] Objective To observe the effects of Jiangu Granules on the G1/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 mRNA. 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 G0/G1 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， G2/M phase and proliferation index was much higher （P < 0.01）. The expression of cycling E， CDK 2 and transcription factor E2F-1 mRNA 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 mRNA was lower than the model-serum group （P < 0.01）. Conclusion Jiangu Granules-serum can adjust the G1/S phase cell-cycle regulated mechanism in osteoblast， so as to push the cells passing G1/S checkpoint and accelerate the proliferation of osteoblast.

[Key words] Postmenopausal osteoporosis； Osteoblast； Cell cycle； G1/S phase； Jiangu Granules

骨质疏松症形成的主要原因是骨形成、骨吸收偶联的破坏，使骨形成减少、骨量降低。成骨细胞在骨代谢、骨形成和维持成年骨骼系统正常中有着主要作用[1]，而成骨细胞增殖周期的进程受周细胞周期蛋白的调控，其中G1/S期检测点是细胞周期进程中最重要的检测点[2]，主要受Cyclin E、CDK2、p21和转录因子E2F等G1/S期调节蛋白的调控。前期研究表明，健骨颗粒可以促进成骨细胞增殖，对防治骨质疏松症有积极的作用[3-4]。本研究以成骨细胞G1/S期调控为切入点，观察补肾健脾中药健骨颗粒对体外培养成骨细胞G1/S期调控蛋白Cyclin E、CDK2、p21和转录因子E2F mRNA的影响，探讨健骨颗粒促进成骨细胞增殖的作用机制，对于进一步认识成骨细胞增殖的机制，治疗和预防骨质疏松症有着重要意义。

1 材料与方法

1.1 实验药物