腐胺促进人骨髓间充质干细胞成骨分化

Putrescine Promotes Human Marrow Mesenchymal Stem Cells to Differentiate along Osteogenic Pathway

目的:探索腐胺对人骨髓间充质干细胞(MSC)增殖分化的影响,以期建立一种新的MSC成骨分化诱导体系。方法:采集3份健康人骨髓MSC,应用MTT实验检测腐胺对细胞增殖的影响。实验分为:1腐胺组(100μmol/L腐胺),2阳性对照组(加入以地塞米松、抗坏酸磷酸盐和β-磷酸甘油组成的标准诱导体系),3阴性对照组(未加腐胺体系),MSC培养1周后应用PCR法检测细胞Runx-2表达水平,2周后进行碱性磷酸酶原位组织化学染色,并将部分细胞裂解后测定细胞碱性磷酸酶活性及蛋白质浓度。此外,以标准成脂分化诱导体系为阳性对照,MSC培养体系中加入腐胺,培养2周后油红O染色,观察腐胺诱导MSC成脂细胞分化作用。结果:在一定范围内腐胺促进人骨髓MSC增殖,且效应呈浓度依赖性。腐胺(100μmol/L)培养MSC 1周后,细胞Runx-2表达水平显著增加;2周后,组织化学染色显示,细胞呈现明显的碱性磷酸酶活性,单位细胞蛋白质浓度内酶活性显著高于阴性对照组(0.87±0.012 vs 0.52±0.010)(P<0.01),也明显高于阳性对照组(0.83±0.029)(P=0.02)。在该浓度下,油红O染色显示腐胺组MSC未发生脂肪细胞分化。结论:腐胺可促进MSC增殖并促使其向成骨细胞分化,可作为MSC体外成骨细胞分化新诱导成分之一。

Objective：To investigate the effects of putrescine on the growth and differentiation of human bone marrow mesenchymal stem ceils （MSC） to develop a new inductive medium mixture for their osteogenic differentiation. Methods：Human bone marrow MSC were collected from three healthy donors and were used to observe the growthpromoting activity of putrescine with MTT test. Experiments were divided into 3 goroups： （ 1 ） putrescine group, （ 2 ） positive control group（ presence of dexamethasone, ascorbate, and glycerol phosphate） and negative group （ d-alpha with 5% FCS）. The cellular expression level of Runx-2 was detected by PCR assay after the culture was maintained for 1 week. After 2 weeks, the intracellular activity of alkaline phosphatase was revealed by histochemistry staining, the phosphatase activity, and the protein concentration in the cell lysates were also detected. Furthermore, MSC were cultured in the presence of putrescine for 2 weeks and Oil-red O staining was performed to reveal the differentiated adipocytes; the cells induced by the standard agent cocktail were used as the positive control. Results： Putrescine promoted the proliferation of human marrow MSC in a dose-dependent manner. MSC exposed to putrescine at a concentration of 100 μmol/L for 1 week expressed greatly higher level of Runx-2, compared with the negative control. Alkaline phosphatase activity was evidently observed after MSC were maintained in the presence of putrescine for 2 weeks. The phosphatase activity contrasted to the protein content in putrescine-treated MSC was significantly higher than that of the control cells （0.87 ±0.012 vs 0. 52 ±0.010） （ P 〈 0.01 ）, and also greatly higher than that of the positive control （0.83 ± 0.029） （ P = 0.02）. Oil red O staining showed that MSC treated by putrescine did not differentiate into adipoblasts. Conclusion： Putrescine can promote the proliferation and osteogenic differentiation of MSC, suggesting the potential application of putrescine as a novel inductive agent for in vitro osteogenesis of MSC.