摘要
目的探讨骨形态发生蛋白-2(BMP-2)/Smad通路在雷奈酸锶(Sr)促进大鼠骨髓间充质干细胞(BMSCs)分化为成骨细胞过程中的作用。方法体外分离培养大鼠BMSCs,根据实验目的加入不同浓度Sr、BMP-2的拮抗剂noggin及Smad1小干扰RNA(SiRNA)。酶标法检测碱性磷酸酶(ALP)活性,茜素红染色检测钙结节,Western blotting法检测磷酸化Smad1/5/8及Runt相关转录因子-2(Runx2)蛋白的表达。结果应用0.1~10 mmol/L Sr处理BMSCs细胞1 h后,细胞内磷酸化Smad1/5/8表达增高,其中浓度为1 mmol/L时,表达达到高峰;在Sr处理BMSCs前,应用BMP-2拮抗剂noggin预处理细胞2 h能抑制Sr对磷酸化Smad1/5/8表达的上调作用;应用0.1~5 mmol/L Sr处理细胞6 h后,细胞内Runx2表达增高,其中浓度为1 mmol/L时,表达达到高峰;在Sr处理BMSCs前,应用Smad1 SiRNA转染细胞后能下调Smad1/5/8、磷酸化Smad1/5/8的表达,并抑制Sr对Runx2表达的上调作用,还拮抗Sr对ALP活性及钙化结节形成的促进作用。结论 BMP-2/Smad通路参与了Sr促进BMSCs成骨分化。
Objective To explore whether strontium ranelate (Sr) promotes osteoblast lineage differentiation of rat bone mesenchymal stem cells (BMSCs) through the bone morphogenetic protein-2 (BMP-2)/Smad signaling pathway. Methods Cultured rat BMSCs were exposed to different concentrations of Sr, noggin (an inhibitor of BMP-2) or Smadl siRNA. The activity of alkaline phosphatase (ALP) in the exposed cells was detected by colorimetry, and the formation of mineralized nodules was observed with alizarin red staining. The expressions of phosphorylated (p) Smadl/5/8 and Runt-related transcription factor 2 (Runx2) in the cells were detected by Western blotting. Results Exposure to Sr at 0.1 to 10 mmol/L for 1 h markedly increased the expression of p-Smadl/5/8 in the BMSCs, and the increment was the most obvious following I mmol/L Sr exposure. Preconditioning with 100 ng/ml noggin for 2 h inhibited Sr-induced up-regulation of p-Smadl/5/8 expressions. Exposure of the cells to 0.1 to 5 mmol/L Sr for 6 h significantly enhanced Runx2 expression, and the peak enhancement occurred following I mmol/L Sr exposure. Transfection of the BMSCs with Smadl siRNA decreased the basal level of Smadl/5/ 8 protein expression, and also inhibited Sr-induced up-regulation of p-Smadl/5/8 and Runx2 expressions as well as Sr-induced enhancement of ALP activity and formation of mineralized nodules. Conclusion The BMP-2/Smad pathway is involved in St-induced osteoblast differentiation of rat BMSCs;
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2013年第3期376-381,共6页
Journal of Southern Medical University
基金
广东省科技计划项目(2011B031800002)
广州市科技计划项目(2012J4300082)
广东省自然科学基金(S2012010009403)