强骨饮对CKIP-1过表达成骨细胞的影响研究

Effect of Qiangguyin on Rat Osteoblasts in CKIP-1 Overexpressing

目的:观察强骨饮对酪蛋白激酶2相互作用蛋白1(CKIP-1)过表达慢病毒感染的体外小鼠成骨细胞的调控情况,研究强骨饮治疗骨质疏松的分子作用机制。方法:从2d龄SD乳大鼠分离成骨细胞,构建CKIP-1过表达慢病毒,将成骨细胞分成4组:a组为空白成骨细胞对照组,b组为空载慢病毒感染的成骨细胞,c组为CKIP-1过表达慢病毒感染的成骨细胞,d组为CKIP-1过表达慢病毒感染成骨细胞+强骨饮处理。qRT-PCR检测成骨抑制基因CKIP-1和成骨相关基因ALP及RUNX2的表达,CKK-8检测细胞相对数目,茜素红染色鉴定成骨细胞,低倍光镜下观察钙化结节计数。结果:qRT-PCR检测:相比a组、b组,c组的CKIP-1有较高表达,差异有统计学意义(P<0.01),RUNX2和ALPL表达水平极低,差异有统计学意义(P<0.01);c组与d组比较,d组的CKIP-1表达水平显著降低,差异有统计学意义(P<0.01),RUNX2和ALPL表达显著升高,差异有统计学意义(P<0.01)。CKK-8检测:相比a组、b组,c组的细胞增殖水平显著降低,差异有统计学意义(P<0.01);相比c组,d组强骨饮处理后,细胞增殖比例有升高的趋势,且差异有统计学意义(P<0.01)。茜素红染色:相比a组、b组,c组钙化结节明显减少;相比c组,d组钙化结节显著增多。结论:CKIP-1过表达对成骨细胞的增殖、成骨分化及矿化有明显抑制作用,强骨饮能提高CKIP-1过表达病理状态下成骨细胞的活性和成骨分化及矿化能力。

Objective：To observe the effect of Qiangguyin on osteoblasts of mice infected by lentivirus overexpressing casein kinase 2 interaction protein 1（CKIP-1）,and to study the molecular mechanism of Qiangguyin for treating osteoporosis.Methods：Osteoblasts were isolated from 2-day-old SD lactating rats to construct CKIP-1 over-expressed lentivirus.Osteoblasts were divided into 4 groups.a group,blank osteoblasts control group;b group,empty-loaded osteoblasts infected by lentivirus;c group,osteoblasts infected by lentivirus overexpression CKIP-1;d group,CKIP-1 overexpression osteoblasts infected by lentivirus combined with Qiangguyin treatment.The expression of CKIP-1,ALP and RUNX2 were detected by qRT-PCR,the relative number of osteoblasts was detected by CKK-8,the osteoblasts were identified by alizarin red staining,and the number of calcified nodules was observed under low power microscope.Results：The qRT-PCR assay showed：compared with group a and group b,the expression of CKIP-1 in group c was significantly higher（P〈0.01）,the expression of RUNX2 and ALPL was extremely low（P〈0.01）;compared with group d,the expression of CKIP-1 in group c was significantly lower（P〈0.01）,and the expression of RUNX2 and ALPL was significantly higher（P〈0.01）.The CKK-8 assay showed：compared with group a and group b,the level of cell proliferation in group c was significantly decreased（P〈0.01））;compared with group c,the percentage of cell proliferation in group d after Qiangguyin treatment was increased,and the difference was significant（P〈0.01）.Alizarin red staining showed：compared with group a and group b,calcified nodules in group c decreased significantly;compared with group c,calcified nodules in group d increased significantly.Conclusion：The CKIP-1 overexpression is significantly inhibited the proliferation,osteogenic differentiation and mineralization of osteoblasts.Qiangguyin can increase osteoblast activity,osteogenic differentiation and mineralization in the pathological state of CKIP-1 overexpression ability.