骨髓间质干细胞向软骨细胞定向分化过程中基因表达谱变化

The changes of gene expression profile during differentiation of marrow mesenchymal stem cells to- wards chondrocytes

目的运用基因芯片分析骨髓间质干细胞（MSCs）向软骨细胞定向分化过程中基因表达谱变化。方法抽取正常骨髓分离出MSCs，诱导向软骨细胞分化。同时提取诱导后0、7、14d细胞RNA，运用Affymetrix全基因组芯片比较不同诱导时间点基因表达差异；通过实时定量聚合酶链反应（Real-timePCR）验证差异表达基因。结果MSCs诱导第7天与第0天比较：表达基因涉及的生物过程主要集中细胞通讯（31．25％）和细胞发育（25．00％）等；而诱导第14天时差异表达基因涉及的生物过程主要与代谢相关：细胞代谢（62．50％），基础代谢（62．50％），大分子代谢（56．25％）。进一步进行基因功能分析显示，MSCs诱导7、14d与第0天比较，差异表达基因主要集中在编码软骨基质蛋白基因、基质金属蛋白酶、生长因子、细胞结构细胞发育、转录因子、信号分子、整合素和趋化因子等相关基因。实验显示诱导早期（0～7d）微环境变化集中在促进细胞增殖、分化和发育；诱导晚期（7～14d）微环境变化主要表现在参与维持代谢和软骨细胞表型稳定。Real—timePCR进一步证实SPARC、CCL25和CCL4基因表达在诱导7d明显上调，基质金属蛋白酶-t1（MMP．11）、金属蛋白酶组织抑制因子-3（TIMP-3）和肿瘤坏死因子相关受体因子5（TRAF5）基因表达在第14天达到高峰，这些基因有可能作为诱导分化过程中早期和晚期分子标志物。结论微环境分子网络改变调控MSCs向软骨细胞定向分化。

Objective To explore the changes of gene expression profile during differentiation of marrow mesenchymal stem cells （ MSCs ） towards chondrocytes. Methods The differentiation of human MSCs towards chondrocytes was induced in vitro and the changes of gene expression profiling were examined by using Affymetrix microarray. The differentially expression genes were confirmed by using real-time poly- merase chain reaction （real-time PCR）. Results Multiple genes showed differential expression on the 7th day and/or 14th day, including the genes encoding extracellular matrix molecule, metalloproteases, cell cy- cle, development, growth factors and chemokines. The changed genes were involved in cells proliferation, differentiation and development in the early stage （0-7 day）, and in cell metabolism and maintenance chon- drocyte phenotype in the late stage （7-14 day）. Real-time PCR identified that SPARC, CCL25 and CCL4 were significantly up-regulated on the 7th day, and matrix metalloproteinase-11 （MMP-11 ）, tissue inhibitor of metalloproteinase-3 （TIMP-3） and tumor necrosis factor receptor-associated factor 5 （TRAFS） reached the peak expression on the 14th day. These differentially expressed gene could be classified as early markers and late markers in differentiation of MSCs towards chondrocytes in vitro. Conclusion The changes in microenvironmental molecular network involved in regulation of the differentiation of MSCs towards chondrocytes.