C-Mpl基因对骨骼代谢的影响及其机制

发布时间：2018-06-03 10:37

本文选题：C-Mpl + 骨小梁　；参考：《第三军医大学学报》2017年23期

【摘要】：目的观察C-Mpl对骨骼代谢平衡的影响,并对其机制进行初步研究。方法以6月龄野生型(WT)小鼠和C-Mpl敲除小鼠(C-Mpl-/-)作为研究对象。采用Micro-CT扫描,进行股骨远端骨小梁参数分析,包括骨密度(BMD)、骨体积与组织体积比(BV/TV)、骨小梁厚度(Tb.Th)、骨小梁数目(Tb.N)、骨小梁分离度(Tb.Sp)和结构模型指数(SMI)。通过三点弯曲试验记录股骨生物力学强度,Von Kossa及茜素红矿化染色观察骨矿化水平,微板法检测血清钙、磷水平变化。TRAP染色观察破骨细胞数量,体外流式细胞仪检测破骨细胞凋亡。流式细胞仪检测破骨细胞的ROS含量,q PCR和Western blot分析p38、p-p38、Fox O1和Nrf2表达变化。结果 Micro-CT扫描结果提示,与WT小鼠比较,C-Mpl-/-小鼠股骨干骺端松质骨明显增多,BMD、BV/TV、Tb.N显著升高,而Tb.Sp和SMI明显降低,差异均有统计学意义(P0.05);Tb.Th两组差异无统计学意义。生物力学分析结果显示,C-Mpl-/-小鼠断裂负荷和刚度明显低于WT小鼠(P0.05)。C-Mpl-/-小鼠股骨Von Kossa染色和成骨细胞茜素红染色均弱于WT小鼠,且血清中钙、磷含量亦明显低于WT小鼠(P0.05)。TRAP染色结果显示,与WT小鼠相比较,C-Mpl-/-小鼠破骨细胞数量显著减少(8.667±1.202 vs 18.000±1.732,P0.05)。流式细胞仪检测发现,C-Mpl-/-小鼠破骨细胞凋亡率较WT小鼠明显增加[(14.54±1.17)%vs(5.38±0.56)%,P0.05],且C-Mpl-/-小鼠破骨细胞的ROS水平明显低于WT小鼠破骨细胞(6 833.0±176.4 vs 12 933.0±202.8,P0.05)。q PCR结果显示C-Mpl-/-小鼠破骨细胞中Fox O1和Nrf2表达水平较WT小鼠破骨细胞明显降低(P0.05),Western blot结果也证实C-Mpl-/-小鼠破骨细胞中p-p38、Fox O1和Nrf2表达均显著下降(P0.05)。结论 C-Mpl基因对骨骼代谢存在一定影响,C-Mpl敲除显著加速小鼠体内破骨细胞的凋亡,抑制破骨细胞生成,最终可能导致C-Mpl-/-小鼠的骨骼衰老速度和骨质疏松程度均明显减慢。
[Abstract]:Objective to observe the effect of C-Mpl on bone metabolic balance and its mechanism. Methods six months old wild type WTM mice and C-Mpl knockout mice (C Mpl- r -) were used as the study subjects. The trabecular parameters of distal femur were analyzed by Micro-CT scanning, including bone density, ratio of bone volume to tissue volume, trabecular thickness, trabecular number, trabecular separation and structural model index. Biomechanical strength of femur was recorded by three point bending test. Bone mineralization level was observed by Von Kossa and alizarin red mineralization staining. Serum calcium and phosphorus levels were detected by microplate method. The number of osteoclasts was observed by trap staining. Apoptosis of osteoclasts was detected by flow cytometry in vitro. The ROS content of osteoclasts was detected by flow cytometry (FCM) and Western blot was used to analyze the expression of p38, p-p38, Fox O1 and Nrf2 in osteoclasts. Results the results of Micro-CT scan showed that compared with WT mice, the number of bone in metaphyseal cancellous bone of C-Mpl-R mice was significantly increased, while Tb.Sp and SMI were significantly decreased. There was no significant difference between the two groups. The results of biomechanical analysis showed that the breaking load and stiffness of C-Mpl-r / -mice were significantly lower than those of WT mice (P 0.05N. C-Mpl-r / -mice), the Von Kossa staining of femur and alizarin red staining of osteoblasts were weaker than those of WT mice, and the calcium in serum was lower than that in WT mice. Compared with WT mice, phosphorus content was significantly lower than that of WT mice (P 0.05N. Trap staining). Compared with WT mice, the number of osteoclasts in C-Mpl-r / -mice was significantly decreased by 8.667 卤1.202 vs 18.000 卤1.732P 0.05. Flow cytometry showed that the apoptosis rate of osteoclasts in C-Mpl-r-mice was significantly higher than that in WT mice [14.54 卤1.17)%vs(5.38 卤0.56], and the ROS level of osteoclasts in C-Mpl-r-mice was significantly lower than that in WT mice (6 833.0 卤176.4 vs 12 933.0 卤202.8% P0.05. Q PCR). The results showed that Fox in C-Mpl-r-mouse osteoclasts was significantly lower than that in WT mice. The expression levels of O1 and Nrf2 in osteoclasts of WT mice were significantly lower than those in osteoclasts of WT mice. The results of Western blot also confirmed that the expressions of p-p38, Fox O1 and Nrf2 in osteoclasts of C-Mpl-r-mice were significantly lower than those of osteoclasts of WT mice (P 0.05). Conclusion C-Mpl gene has a certain effect on bone metabolism. C-Mpl knockout can accelerate the apoptosis of osteoclasts and inhibit the formation of osteoclasts in mice, which may result in the reduction of bone senescence and osteoporosis in C-Mpl-r-mice.
【作者单位】：第三军医大学军事预防医学院全军复合伤研究所创伤、烧伤与复合伤国家重点实验室;解放军第九八医院骨科;解放军第九八医院神经外科;
【基金】：国家自然科学基金面上项目(81573084) 重庆市科技创新领军人才支持计划(CSTCKJCXLJRC06)~~
【分类号】：R580

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