中文摘要：

目的：在经典wnt信号通路抑制剂大鼠Dickkopf相关蛋白1（DKK1）的干预下,观察益骨汤含药血清对成骨细胞的经典Wnt信号通路的影响,探究益骨汤治疗骨质疏松的作用机制,为临床使用补肾活血中药防治骨质疏松症提供实验依据。方法：将40只雌性SD大鼠随机分成2组,益骨汤水提液组和空白对照组,连续灌胃10天,获取含药血清。从乳鼠颅盖骨中分离培养成骨细胞,实验分为4组,即A组（益骨汤含药血清组）、B组（DKK1组）、C组（DKK1＋益骨汤含药血清组）、D组（空白血清对照组）。进行细胞增殖MTT检测、碱性磷酸酶活性检测,RT-PCR检测经典Wnt/β-catenin通路中β-链蛋白（β-catenin）、低密度脂蛋白受体相关蛋白5（LRP-5）、DKK1基因的表达情况。结果：根据MTT法动态观察各组成骨细胞增殖,发现成骨细胞增殖呈规律性：48 h至72 h成骨细胞呈对数增长期,96 h后细胞增殖逐渐受到抑制。同一时间点与B组比较,A、C、D各组成骨细胞的增殖和ALP活性显著较高,差异均有统计学意义（P〈0.05）。同一时间点与D组比较,A组成骨细胞的增殖和ALP活性显著较高,差异均有统计学意义（P〈0.05）。与B组比较,A、C、D组β-catenin mRNA、LRP-5 mRNA、TCF mRNA的表达均明显升高,DKK1 mRNA的表达明显降低,差异均有统计学意义（P〈0.05）。与D组比较,A组β-catenin mRNA、LRP-5 mRNA、TCF mRNA的表达均明显升高,DKK1 mRNA的表达明显降低,差异均有统计学意义（P〈0.05）。结论：益骨汤含药血清可能是通过抑制DKK1的高表达,从而上调经典Wnt/β-catenin信号中β-catenin、LRP-5、TCF基因的表达,达到促进成骨细胞增殖,起到抗骨质疏松作用。

英文摘要：

Objective：From the intervention of classic Wnt signaling pathway inhibitor Dickkopf associated protein 1（DKK1）,to observe the effect of Yigu tang containing medicated serum on canonical Wnt signaling pathway,and to discuss the mechanism of Yigu tang for osteoporosis,so as to provide experimental basis for the clinical application of kidney-nourishing and blood-activating Chinese medicine for osteoporosis.Methods：Divided 40 female SD rats into Yigu tang extract group and blank control group randomly.Both groups were administrated by gavage for 10 days successively,and medicated serum was obtained.Osteoblasts were isolated and cultured from calvaria bones of neonatal rats.The experiment was carried out in four groups：group A（Yigu tang containing medicated serum group）,group B（DKK1 group）,group C（DKK1 and Yigu tang containing medicated serum group）,group D（blank serum control group）.MTT assay of cell proliferation and alkaline phosphatase（ALP） activity assay were carried out.The expression of p- catenin,low density lipoprotein receptor related protein 5（LRP- 5） and DKK1 gene in classic Wnt/p- catenin signaling pathway were determined by reverse transcription-polymerase chain reaction（RT- PCR）.Results：According to MTT assay,observed the proliferation of osteoblasts in all groups dynamically,and discovered the proliferation of osteoblasts exhibiting regularity：from 48 h to 72 h,osteoblasts showed logarithmic growth phase,and the proliferation was inhibited gradually after 96 h.Comparing with those in group B at the same time point,the proliferation of osteoblasts and ALP activity in group A,C and D were obviously higher（P〈0.05）.Comparing with those in group D at the same time point,the proliferation of osteoblasts and ALP activity in group A were evidently higher（P〈0.05）.Comparing with those in group B,the expression of β-catenin mRNA,LRP- 5 mRNA and TCF mRNA in group A,C and D were increased significantly,while the expression of DKK1 mRNA was decreased obviously?