摘要
目的:探讨Hedgehog/Gli1信号通路在雷奈酸锶(strontium ranelate,Sr)促进骨髓间充质干细胞(BMSCs)向成骨细胞分化中的作用。方法:体外分离培养大鼠BMSCs,诱导其成骨分化,根据实验目的加入不同浓度的Sr、Hedgehog受体拮抗剂cyclopamine(Cy)及Gli1小干扰RNA(Gli1-siRNA)。用Western blotting法检测Gli1及Runx2的表达,酶标法检测碱性磷酸酶(ALP)活性,茜素红染色法检测钙结节水平。结果:应用不同浓度的Sr(0.1-5 mmol/L)处理BMSCs细胞7 d后,细胞内Gli1蛋白表达增高,Sr的浓度为3 mmol/L时,Gli1表达达到高峰;使用Cy与Sr共处理BMSCs 7 d,能拮抗Sr对Gli1蛋白表达的上调作用;应用Gli1-siRNA转染细胞后,能下调Gli1蛋白的表达,并抑制Sr对Gli1下游Runx2蛋白表达的上调作用,还可拮抗Sr对ALP活性及钙化结节形成的促进作用。结论:Hedgehog/Gli1通路参与了Sr促进骨髓间充质干细胞向成骨分化的过程。
AIM: To explore whether strontium ranelate( Sr) promotes osteogenic differentiation of rat bone mesenchymal stem cells( BMSCs) through the Hedgehog / Gli1 signaling pathway. METHODS: BMSCs were isolated from4-week-old rats by adherent culture and induced to differentiate into osteoblasts. According to the experimental purposes,the cells were exposed to different concentrations of Sr,cyclopamine( Cy,an inhibitor of Hedgehog receptor) or Gli1-siRNA. The expression of Gli1 and Runx2 in the cells was detected by Western blotting. The activity of alkaline phosphatase( ALP) was measured by the method of colorimetry,and the mineralized nodules were observed under microscope with alizarin red staining. RESULTS: Exposure to Sr at concentrations of 0. 1 to 5 mmol / L for 7 d markedly increased the expression of Gli1 in the BMSCs,and the increase in Gli1 expression was the most obvious following Sr exposure at concentration of 3 mmol / L. Cy at concentration of 10 μmol / L inhibited Sr-induced up-regulation of Gli1 expression. Transfection of the BMSCs with Gli1-siRNA not only obviously inhibited Sr-induced up-regulation of Gli1 and Runx2( a downstream protein of Gli1) expression,but also antagonized Sr-induced enhancement of ALP activity and the formation of mineralized nodules.CONCLUSION: The Hedgehog / Gli1 pathway is involved in Sr-induced osteogenic differentiation of rat BMSCs.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2015年第2期234-238,共5页
Chinese Journal of Pathophysiology
基金
广东省自然科学基金资助项目(No.S2012010009403)
广东省科技计划(No.2011B031800002)
广州市科技计划(No.2012J4300082)