摘要
目的研究不同强度和时间的机械牵张应力刺激对成骨细胞的分化及Wnt信号转导通路的影响。方法采用FlexcellFX5000细胞加力系统,分别采用3%,6%,12%的形变幅度的正弦波对MC3T3细胞进行牵张应力干预,0.5 Hz的频率,时间分别为2,4,8 h。干预结束后分别检测细胞的ALP活性;提取细胞总RNA后采用Real time-PCR分别检测骨钙蛋白(Osteocalcin,OC)、Runx^2、Osterix、Wnt1、β-catenin、DKK-1 m RNA的表达;提取细胞总蛋白后采用Werstern blot法分别检测Wnt1、β-catenin和Phosphor-33/37-β-catenin的蛋白表达量。结果 3%和6%强度牵张干预后,成骨细胞ALP活性升高,OC、Runx^2、Osterix、Wnt1、β-catenin m RNA表达升高,而DKK-1 m RNA表达下降。6%的效应高于3%,且干预4 h后升高幅度最大。12%强度牵张干预后,成骨细胞的ALP活性和OC m RNA表达水平下降。Wnt1、β-catenin和Phosphor-33/37-β-catenin蛋白表达水平与上述m RNA表达水平相符。结论中小强度的牵张刺激可以上调Wnt信号转导通路,促进成骨细胞分化,而大强度及长时间连续干预则无成骨分化作用,甚至有抑制作用。
Objective To study the effect of different magnitude and duration of mechanical strain on osteoblast differentiation and the Wnt signaling pathway. Methods Osteoblast cell line MC3T3 was used in this study. The cells were subjected to strain loading of 0.5Hz, magnitude of 3% , 6% , and 12% , and duration of 2, 4, 8h, respectively. Alkaline phosphatase （ALP） activity was measured immediately after all the mechanical loading. Real-time PCR was used to detect osteocalcin （OC）, Runx2, osterix, Wntl, β-catenin, and DKK-1 mRNA. Western blotting was used to detect protein level of Wntl, β-catenin, and phosphor-33/37- β-catenin. Results ALP activity and expression of OC, Runx2, Osterix, Wntl, and β-catenin mRNA increased after 3% and 6% strain, but the DKK-1 mRNA decreased. The efficacy of 6% was greater than that of 3% , and 4h mechanical strain was greater than other duration. However, 12% strain reduced the ALP activity and OC mRNA expression. The protein expression of Wntl and the ratio ofβ-catenin/P-β-catenin was in accordant with mRNA expression. Conclusion Moderate and low mechanical strain increases osteoblast differentiation mediated by the Wnt signal pathway, while unsuitable mechanical stimulus such as high magnitude or long-term duration has no effect on or even inhibits the differentiation of osteoblasts.
出处
《中国骨质疏松杂志》
CAS
CSCD
北大核心
2016年第1期9-13,共5页
Chinese Journal of Osteoporosis
基金
国家自然科学基金项目(81170323)
上海体育学院研究生教育创新计划(yjscx2015007)
上海市人类运动能力开发与保障重点实验室(上海体育学院)(11DZ2261100)
