基于Cyclin D1基因沉默探讨健骨颗粒促成骨细胞增殖的机理

CDK-Cyclin can regulate cell proliferation cycle，of which Cyclin D is one of the most important protein. Previous study showed that there were Cyclin D1 and other regulating protein in osteoblast，and Jiangu granule could accelerate the process of osteoblast proliferation cycle to promote the cell proliferation.So we suggest osteoblast proliferation cycle were regulated by CDK-Cyclin，and Jiangu granule promoted the cell proliferation iva CDK-Cyclin. Based on these，the project plans to construct the small interfering RNA(siRNA) expression vector specific to rat osteoblast Cyclin D1 gene. Then the osteoblast will be intervened with Jiangu granula.The expressions of Cyclin D1,Cyclin E,CDK2，CDK4， Rb and E2F in the osteoblasts will be detected by quantitative real time polymerase chain reaction and Western blot methods. Cell proliferation of OB will be measured by CCK-8 Method, while the cell cycle were analyzed by flow cytometry(FCM).So as to observe the effects of Cyclin D1 gene silencing and Jiangu granula intervention to the regulatory of osteoblast proliferation cycle. Thus to further testify the regulating effect of Cyclin D1 on osteoblast proliferation cycle,and investigate the mechanism of Jiangu granula promoting osteoblast proliferation from the perspective of osteoblast proliferation cycle regulating.

细胞增殖周期受CDK-Cyclin系统调控，Cyclin D是其中的关键蛋白。前期研究表明，成骨细胞中存在Cyclin D1等细胞周期蛋白，且补肾健脾中药健骨颗粒能推进成骨细胞增殖周期进程，促进其增殖。因此提出成骨细胞增殖受CDK-Cyclin系统调控，且健骨颗粒通过该系统促进成骨细胞增殖的假说。本项目拟采用siRNA技术沉默体外大鼠成骨细胞Cyclin D1 mRNA表达，健骨颗粒含药血清干预，运用荧光定量PCR、Western-blot检测Cyclin D1及其下游Cyclin E、CDK2、CDK4、Rb、E2F等周期蛋白表达，流式细胞术检测细胞周期分布，CCK-8法检测细胞增殖，观察Cyclin D1沉默与健骨颗粒干预对成骨细胞周期调控的影响，进一步阐明CDK-Cyclin系统对成骨细胞增殖周期的调节作用，并从细胞周期调控的角度探讨健骨颗粒促进成骨细胞增殖的作用机制。

Cyclin D1作为细胞外信号的传感器和整合器，将细胞外的有丝分裂信号连接到细胞内，并与其他周期蛋白一起调控着细胞的增殖周期。前期研究表明，补肾健脾中药健骨颗粒能推进成骨细胞增殖周期进程，促进其增殖。本项目拟采用siRNA基因干扰技术抑制体外大鼠成骨细胞Cyclin D1 mRNA表达，用健骨颗粒含药血清分别干预正常成骨细胞及Cyclin D1基因沉默细胞，运用实时荧光定量PCR、Western-blot检测干预前后成骨细胞中Cyclin D1及其下游Cyclin E、Rb、E2F等周期调节蛋白mRNA与蛋白表达，流式细胞术检测成骨细胞周期分布，CCK-8法检测成骨细胞增殖情况，观察Cyclin D1基因沉默与健骨颗粒干预对成骨细胞增殖周期调控的影响，从而进一步验证Cyclin D1等周期蛋白对成骨细胞增殖周期的调节作用，并从细胞周期调控的角度探讨健骨颗粒促进成骨细胞增殖的作用机制。