杜仲诱导大鼠间充质干细胞成骨分化中成骨与成脂相关转录因子的表达

Expression of osteogenesis-and adipogenesis-related transcription factors during osteogenic differentiation of rat mesenchymal stem cells induced by eucommia bark

背景:杜仲对骨质疏松具有明显的干预和治疗作用,前期研究发现杜仲能够显著诱导骨髓间充质干细胞成骨分化和抑制成脂分化,而对于学术界广泛认同的成骨和成脂相关转录因子的表达变化还未见报道。目的:观察杜仲诱导大鼠骨髓间充质干细胞成骨分化过程中成骨和成脂相关转录因子表达的变化。方法:制备杜仲水/醇提取物,诱导培养至第3代的SD大鼠间充质干细胞,提取物按生药2g,定容至15mL体积为原液,诱导时按10-3、10-4倍稀释度稀释。诱导实验分为6组:阴性对照组(加入与诱导物等量的PBS);阳性对照组(诱导物为地塞米松0.01mmol/L,β-甘油磷酸钠10mmol/L,L-抗坏血酸钠50mg/L);杜仲水提取物10-4稀释度诱导组;杜仲水提取物10-3稀释度诱导组;杜仲醇提取物10-4稀释度诱导组;杜仲醇提取物10-3稀释度诱导组,各组于诱导之后的第15天终止培养。RT-PCR和荧光定量PCR方法检测成骨分化转录因子基因Runx2、Osterix和成脂分化转录因子基因过氧化物酶体增殖物激活受体γ、脂肪酸结合蛋白的表达。结果与结论:Runx2除在10-3稀释度水提取物组表达下调,在10-4稀释度醇提取物组上调外,其他各组无显著变化;Osx在各诱导组的表达均明显下调;过氧化物酶体增殖物激活受体γ在各组中无显著变化;脂肪酸结合蛋白在各组均下调。提示杜仲诱导间充质干细胞成骨分化与成脂相关转录因子脂肪酸结合蛋白的表达抑制相关。

BACKGROUND：Eucommia bark has obvious effects of intervention and treatment on osteoporosis.Previous studies have confirmed that eucommia bark can significantly induce differentiation of bone marrow mesenchymal stem cells（BMSCs） into osteoblasts and adipogenic cells.There are no reports concerning changes in osteogenesis-and adipogenesis-related transcription factor expression.OBJECTIVE：To observe changes in expression of osteogenic and adipogenic differentiation related transcription factors during osteogenic differentiation of rat BMSCs induced by eucommia bark.METHODS：Water and MeOH extract of eucommia bark was prepared.BMSCs from Sprague Dawley rat were cultured in vitro for 3 passages.Extract was volume metered into 15 mL as stock solution according to 2 g of crude drug.Extract of eucommia bark were diluted to 10-3 and 10-4 groups.The test contained six groups.Negative control group received the same volume of phosphate buffered saline as inducers.Positive control group induced by dexamethasone 0.01 mmol/L,β-sodium glycerophosphate 10 mmol/L and L-sodium ascorbate 50 mg/L.There were water extract of eucommia bark 10-4 dilution group,water extract of eucommia bark 10-3 dilution group,MeOH extract of eucommia bark 10-4 dilution group and MeOH extract of eucommia bark 10-3 dilution group.The culture was stopped at 15 days following induction in each group.Reverse transcription PCR and fluorescent quantitative PCR were used to detect expression of osteogenic transcription factor Runx2 and Osterix,and adipogenic transcription factor peroxisome proliferator activated receptor γ（PPARg） and fatty acid binding protein.RESULTS AND CONCLUSION：The expression of Runx2 did not change except downregulated at 10-3 dilution of water extract and upregulated at 10-4 dilution of MeOH extract.Osx was downregulated obviously in all induction groups,and the expression of PPARr did not change and fatty acid binding protein was downregulated in all induction groups remarkably.These results suggested that of MSCs induced by eucommia bark was related to inhibition of the expression of fatty acid binding protein.