摘要目的 寻找青年人与老年人的骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)中表达差异明显的高度保守microRNA(miRNA),分析该miRNA在年轻和老年小鼠骨组织中的表达差异,探讨该miRNA在原发性骨质疏松中发挥的作用.方法 通过基因表达数据库(Gene Expression Omnibus,GEO)查找青年人与老年人BMSCs中表达差异明显且人鼠高度保守的miRNA;在体外培养的人骨髓间充质干细胞(human bone mesenchymal stem cells,hBMSCs)中转染该miRNA,检测转染后第7天的碱性磷酸酶(alkaline phosphatase,ALP)活性;微计算机断层扫描技术(微CT)检测年轻小鼠与老年小鼠皮质骨与松质骨参数,验证自然衰老骨质疏松模型的构建;定量检测2组小鼠骨组织中该miRNA的表达差异;分析骨参数与miRNA的相关性.结果 通过基因表达数据库(GEO数据库)发现28个上调(下调)大于2倍且P＜0.05的miRNA,hBMSC分别转染28个miRNA后发现可明显下调ALP活性的miRNA为mir-124-3p、mir-126-3p、mir-128-3p、mir-424-5p(P＜0.05);微CT检测年轻小鼠与老年小鼠皮质骨和松质骨参数提示自然衰老骨质疏松模型构建成功;与年轻小鼠相比,mir-124-3p在老年小鼠骨组织内高表达;在皮质骨中,mir-124-3p与骨小梁分离度呈正相关,与骨体积分数、骨小梁数量和骨密度呈负相关.在小梁骨中,mir-124-3p与骨小梁分离度呈正相关,与骨小梁模式因子呈负相关(P＜0.05).结论 青年人与老年人BMSCs表达差异明显的mir-124-3p可抑制hBMSCs向成骨分化,该miRNA在老年小鼠骨组织中表达上调,可能与骨质疏松的发生发展有关.

abstractsObjective To investigate different expression levels between young and old bone marrow mesenchymal stem cells in microRNAs (miRNAs) that are significantly conserved between humans and mice.Additional studies have been conducted to discover changes in miRNA expression in old mice relative to that in young adults and discussed the roles of miRNAs in primary osteoporosis.Methods MiRNAs that are highly conserved between human and mice,and are expressed at significantly different levels in the bone marrow mesenchymal stem cells of young and old people were identified by searching the Gene Expression Omnibus (GEO) database.Human bone mesenchymal stem cells (hBMSCs) were transfected with miRNA mimics,and their relative alkaline phosphatase (ALP) activity levels were then determined.Micro-CT scanning was employed to quantitatively characterize cortical and cancellous bones of young and old mice,and to confirm that these mice accurately modeled natural aging osteoporosis.Simultaneously,we investigated differences in expression levels of miRNAs that influence ALP activity in hBMSCs in the two groups of mice.Correlations between miRNA expression levels,and parameters of bone mass and bone strength were studied.Results 28 miRNAs were found to be more than 2 fold up-regulated (down-regulated) with statistical significance (P＜0.05) in the GEO database.We also found that ALP activity was lower in hBMSCs transfected with 4 miRNAs (mir-124-3p,mir-126-3p,mir-128-3p,mir-424-5p,P＜0.05 or P＜ 0.01).The micro-CT scans indicated that the mice are accurately modeled natural aging osteoporosis.Expression of mir-124-3p increased significantly in older mice.This upregulation correlated positively with trabecular separation,and negatively with trabecular pattern factor in trabecular bone.However,in cortical bone,its expression correlated positively with trabecular separation,and negatively with bone volume fraction,trabecular number,and bone mineral density (P＜ 0.05).Conclusion Hsa-mir-124-3p,which is expressed differently in young and old bone marrow stromal cells,inhibited the osteogenic differentiation of hBMSCs.Upregulation of this miRNA in the bone tissue of aged mice may be related to the development of osteoporosis.