甾体类激素影响骨内微血管内皮细胞表达谱与转录组的初探

Preliminary study of impact of steroids on expression profile and transcriptome of bone microvascular endothelial cells

目的 探讨对甾体类激素导致股骨头坏死的分子机制.方法 使用Ⅰ型胶原酶和胰蛋白酶消化法分离人股骨头松质骨内的微血管内皮细胞,通过血管假性血友病因子、血小板-内皮细胞黏附分子-1、血管内皮钙黏蛋白和血管细胞黏附分子-1等血管内皮细胞标志物抗体进行免疫荧光鉴定,用双标的lncRNAs＋mRNAs表达谱芯片和单标microRNAs芯片进行差异性转录本检测,通过生物信息学软件并参考数据库信息,分析出lncRNAs-microRNAs-mRNAs的空间位置和表达水平的相关性.结果 体外培养的股骨头内微血管内皮细胞在甾体类激素损伤情况下出现mRNAs、microRNAs（其中miR-339上调,miR-100、miR-222、miR-23、miR-933下调）和lncRNAs（其中73种表达上调,166种表达下调）等分子特异性的变化,利用靶基因预测等生物信息学工具,构建出了转录本模块间的共表达调控网络模型.结论 甾体类激素导致骨内微血管内皮细胞miR-100、miR-222、miR-933、miR-339和miR-23等转录本发生明显变化,它们直接攻击过氧化氢酶、成纤维细胞生长因子和神经生长因子等关键酶和细胞因子,对松质骨内的骨组织和循环系统产生重要影响.

Objective To explore the differential expression profiles and transcriptomes of human bone microvascular endothelial cells via microarray and elucidate the molecular mechanisms underlying steroids-induced osteonecrosis.Methods Bone microvascular endothelial cells in cancellous bone of femoral head were harvested by type Ⅰ collagenase and trypsin digestion and identified by immunofluorescent labeling with primary antibodies specific for vascular endothelial cells＇ biomarkers such as yon Willebrand factor,platelet endothelial cell adhesion molecule 1,vascular endothelial cadherin and vascular cell adhesion molecule 1.Their differential expression profiles and transcriptomes were tested by double-labeled lncRNAs ＋ mRNAs microarrays and single-labeledd microRNAs microarrays.The data were analyzed by bioinformatical software with database checking.The authors spatial correlations and expression relevance were established among lncRNAs,microRNAs and mRNAs.Results Steroids incurred specific changes in mRNAs,microRNAs and lncRNAs transcribed in human bone microvascular endothelial cells of femoral head in vitro cultured.Thus a model of coexpression network was constructed among transcript modules with bioinformatical tools such as target gene prediction.Conclusion Steroids induced significant changes in transcripts of bone microvascular endothelial cells such as miR-100,miR-222,miR-933,miR-339 and miR-23.They exert considerable influences on osseous tissues and circulatory systems in cancellous bone by targeting cytokines and enzymes such catalase,fibroblast growth factor and nerve growth factor.Through further explorations of these transcripts,we can not only elucidate the underlying pathogenesis,but also discover specific biomarkers and therapeutic targets for corresponding diseases.