锶对人骨髓间充质干细胞增殖和成骨分化的影响

Effects of strontium on proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells in vitro

目的观察锶(Sr)对人骨髓间充质干细胞(hMSCs)增殖及成骨分化的影响并探索其最佳作用浓度。方法抽取健康成年志愿者骨髓6ml,体外分离、培养原代hMSCs,流式细胞术鉴定细胞表型。取第4代细胞进行实验,共分为5组,A组为空白对照,培养液中仅加入骨诱导剂(地塞米松10-8mol/L、抗坏血酸50μg/ml、β-甘油磷酸钠10mmol/L,B、C、D、E组培养液在加入骨诱导剂后分别加入3.75×10-3、3.75×10-2、3.75×10-1、3.75mmol/L的SrCl2。定期测定各组hMSCs的增殖情况(MTT法)、碱性磷酸酶(ALP)活性(酶标法)和骨钙素(OCN)含量(放免法),并观察细胞茜素红钙结节染色情况,分析各组钙结节数量及面积。结果随培养时间延长,各组细胞增殖和成骨分化均明显提高(P<0.01)。组间比较显示,第7天时的细胞增殖水平,第7、14天的ALP活性,第14、21天的OCN含量以及第21天的茜素红钙结节数量和面积测定结果,B-E组均明显优于A组(P<0.05);D、E组第7天时的细胞增殖水平及第14天的ALP活性均明显高于A、B、C组(P<0.05),而D组与E组比较无显著差异(P>0.05);第21天时的OCN含量以及茜素红钙结节数量和面积测定结果D组均高于与其他4组(P<0.05)。结论锶可促进hMSCs的增殖及成骨分化,其最佳作用浓度为3.75×10-1mmol/L。

Objective To study the effects of strontium （Sr） on proliferation and osteogenic differentiation of human marrow mesenchymal stem cells （hMSCs） in vitro,and to determine its optimal concentration.Methods Six milliliter of bone marrow was obtained from a healthy adult volunteer,the hMSCs were then isolated and cultured in vitro,and the cell phenotype was identified by flow cytometry.The 4th passage cells were used for experiment,and they were randomly divided into 5 groups：the osteogenic agents （10-8mol/L dexamethasone,50μg/ml ascorbic acid and 10mmol/L beta-sodium glycerophosphate） were used alone in the control group （group A）,and the combination of osteogenic agents and different doses of strontium chloride （3.75×10-3,3.75×10-2,3.75×10-1 and 3.75mmol/L） were used in group B,C,D and E.The proliferation level,alkaline phosphatase （ALP） activity and osteocalcin （OCN） content of MSCs were determined by MTT colorimetric assay,enzyme-labeled method and radioimmunological method,respectively.The amount and area of calcium nodules in each group were observed by alizarin red staining.Results The proliferation and osteogenic differentiation rate of each group increased significantly with the lapse of culture time（P0.01）.The proliferation levels at day 7,both ALP activities and OCN contents at day 7 and 14,and the amount and area of calcium nodules at day 21 in groups B,C,D and E were strikingly higher than those of group A （P0.05）.The proliferation levels at day 7 and day 14 in groups D and E were higher than those of groups A,B and C （P0.05）,while there was no significant difference between group D and E.The content of OCN and amount and area of calcium nodules at day 21 in group D were significantly higher than those of other 4 groups （P0.05）.Conclusion Strontium can promote the proliferation and osteogenic differentiation of hMSCs,and the optimal concentration is 3.75×10-1mmol/L.