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目的探讨信号选择性甲状旁腺素(parathyroid hormone,PTH)模拟肽在促进小鼠成骨细胞成骨过程中对Wnt信号传导通路相关因子的影响,研究其成骨的作用机制。方法取2～3日龄C57BL乳鼠颅盖骨分离培养成骨细胞,倒置相差显微镜观察形态变化,ALP染色和茜素红染色对成骨细胞进行鉴定。取贴壁生长的第1代细胞,随机分为4组:PTH(1-34)组、G1R19(1-34)组和G1R19(1-28)组分别加入10 nmol/L PTH(1-34)、10 nmol/L G1R19(1-34)、100 nmol/L G1R19(1-28),空白对照组加入等体积的0.1%三氟乙酸(trifluoroacetic acid,TFA)。实时定量PCR法检测Wnt信号通路相关基因β-catenin、骨钙素(osteocalcin,OC)、Wnt2、Wnt5b、Wnt7b基因的表达。结果倒置相差显微镜观察示,培养7 d可见细胞排列紧密,为三角形或多边形,呈铺路石样;细胞培养14 d ALP染色可见细胞质中出现蓝染颗粒,成骨诱导培养28 d茜素红染色可见细胞外基质中红色矿化结节形成。实时定量PCR检测发现,各处理组OC、Wnt5b mRNA相对表达量均高于空白对照组,Wnt2 mRNA相对表达量均低于空白对照组;PTH(1-34)组、G1R19(1-34)组Wnt7b mRNA相对表达量高于空白对照组,G1R1(91-34)组高于PTH(1-34)组和G1R1(91-28)组;PTH(1-34)组β-cateninmRNA相对表达量显著高于空白对照组;以上差异均有统计学意义(P<0.05)。其余各因子mRNA表达各组间比较差异均无统计学意义(P>0.05)。结论在所检测的Wnt相关因子中,PTH(1-34)和G1R19(1-34)对经典Wnt信号因子作用明显,G1R19(1-28)对非经典Wnt信号因子作用明显。
Objective To investigate the effect of signal selective parathyroid hormone (PTH) peptidomimetic on the Wnt signaling pathway-related factors in promoting the osteoblast osteoblast formation in mice and to investigate its osteogenic mechanism. Methods Osteoblasts were isolated and cultured from calvaria of C57BL neonatal rats aged 2-3 days. The morphological changes were observed under inverted phase contrast microscope. The osteoblasts were identified by ALP staining and alizarin red staining. The first generation of adherent cells were randomly divided into 4 groups: PTH (1-34) group, G1R19 (1-34) group and G1R19 (1-28) group were treated with 10 nmol / L PTH ), 10 nmol / L G1R19 (1-34) and 100 nmol / L G1R19 (1-28). The blank control group was added with 0.1% trifluoroacetic acid (TFA). Real-time quantitative PCR was used to detect the expression of β-catenin, osteocalcin (OC), Wnt2, Wnt5b and Wnt7b genes in Wnt signaling pathway. Results Inverted phase contrast microscopy showed that cells were arranged in a triangular or polygonal shape on the 7th day after culturing. Parasitic samples were found on the 7th day after culture. Cytoplasm of blue staining granules appeared on day 14 after ALP staining. Alizarin red staining of osteoblasts induced 28 days Red mineralized nodules in the extracellular matrix. Real-time quantitative PCR showed that the relative expression of OC and Wnt5b mRNA in each treatment group was higher than that in the blank control group, and the relative expression of Wnt2 mRNA was lower than that in the blank control group; PTH (1-34), G1R19 (1-34) The relative expression of Wnt7b mRNA in the PTH (1-34) group was higher than that in the blank control group, which was higher in the G1R1 (91-34) group than in the PTH (1-34) group and the G1R1 (91-28) group Higher than the blank control group; The above differences were statistically significant (P <0.05). There were no significant differences among the other groups (P> 0.05). Conclusions Among the Wnt-related factors detected, PTH (1-34) and G1R19 (1-34) exert obvious effects on the canonical Wnt signaling factor, whereas G1R19 (1-28) has a significant effect on the non-canonical Wnt signaling factor.