连续应用特立帕肽对正常和去势小鼠骨代谢的影响

Prolonged continuous infusion of teriparatide promotes bone metabolism in normal but not in castrated mice

目的探讨连续泵入特立帕肽(TPTD)对去势和正常小鼠骨代谢的影响,为研究TPTD及其相关肽破骨作用的动物模型选择提供实验依据。方法6周龄雌性C57BL小鼠24只,分别接受去势(OVX)或者假手术(SHAM),1周后用微量泵泵入TPTD或者等量的溶剂(VEH),分成SHAM-VEH,SHAM-TPTD,OVX-VEH,OVX-TPTD四组。2周后,获取胫骨组织经显微CT测量胫骨和皮质骨相关参数。并对胫骨组织行进行组织切片HE染色和TRAP染色,统计破骨细胞数目及生长板厚度。测量小鼠血清中的钙离子浓度。取部分颅骨原代成骨细胞加雌二醇(E2)和TPTD刺激培养48h后提取细胞总蛋白,分析β-catenin和RANKL蛋白的表达。结果OVX小鼠的骨小梁骨量较SHAM小鼠的骨量显著减少(P<0.05)。TPTD泵入显著减低SHAM小鼠松质骨骨量股骨中段皮质骨面积,与此相反,TPTD泵入增加OVX小鼠松质骨骨量,不改变皮质骨面积。TRAP染色结果发现,OVX较SHAM小鼠胫骨骨松质破骨细胞明显增多,SHAM-TPTD较SHAM小鼠增多(P<0.05)。细胞学蛋白分析结果显示,E2和TPTD持续刺激48h后,β-catenin表达量较TPTD单纯刺激降低,RANKL表达相对升高。结论TPTD的连续应用促进正常小鼠的骨吸收,但对去势小鼠不具有明显的促骨吸收作用,去势小鼠不适用于研究TPTD及相关肽的促破骨作用。

Objective To investigate the effects of continuous pumping of teriparatide (TPTD) on bone metabolism in ovariectomized and normal mice and provide experimental evidence for the selection of animal models for studying the effects of TPTD and its related peptides on osteoclasts.Methods Twenty-four female C57BL mice (6-weeks old) were subjected to ovariectomy (OVX) or sham operation followed 7 days later by continuous pumping of TPTD or the solvent vehicle (VEH) via a micropump (SHAM-VEH,SHAM-TPTD,OVX-VEH,and OVX-TPTD groups;n=6).Two weeks later,the tibial and femoral bones were harvested for micro-CT scanning to measure the parameters of the tibia and the femoral cortical bone. Histopathological examinations of the tibial tissue were conducted using HE staining and TRAP staining and the number of osteoclasts and the growth plate thickness were determined.The serum Ca2 + levels of the mice were measured.The primary osteoblasts from the cranial bone were treated with estradiol (E2) and TPTD for 48 h,and the expressions of β-catenin and RANKL protein in the cells were analyzed.Results The trabecular bone mass of OVX mice was significantly lower than that of sham-operated mice (P<0.05).Continuous TPTD pumping significantly reduced tibial cancellous bone mass and femoral cortical bone area in the sham-operated mice,while in the castrated mice,TPTD pumping increased the cancellous bone mass without changing the cortical bone area.TRAP staining showed that cancellous osteoblasts in the tibia increased significantly in the castrated mice as compared with the sham-operated mice,and TPTD pumping significantly increased the number of cancellous osteoblasts in the sham-operated mice (P<0.05).In the primary cultured osteoblasts,treatment with both E2 and TPTD obviously lowered the expression of β-catenin and increased the expression of RANKL as compared with TPTD treatment alone.Conclusion Continuous pumping of TPTD promotes bone resorption in normal mice but does not produce obvious bone resorption effect in the ovariectomized mice,suggesting that castrated mice are not suitable models for studying the effect of TPTD and the related peptides on the osteoclasts.