CAJ | 학술논문

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、14 d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-time PCR)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、14 d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(0～7 d)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-11(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.
목적 운용기인심편분석골수간질간세포(MSCs)향연골세포정향분화과정중기인표체보변화.방법 추취정상골수분리출MSCs,유도향연골세포분화.동시제취유도후0、7、14 d세포RNA,운용Affymetrix전기인조심편비교불동유도시간점기인표체차이；통과실시정량취합매련반응(Real-time PCR)험증차이표체기인.결과 MSCs유도제7천여제0천비교:표체기인섭급적생물과정주요집중세포통신(31.25％)화세포발육(25.00％)등；이유도제14천시차이표체기인섭급적생물과정주요여대사상관:세포대사(62.50％),기출대사(62.50％),대분자대사(56.25％).진일보진행기인공능분석현시,MSCs유도7、14 d여제0천비교,차이표체기인주요집중재편마연골기질단백기인、기질금속단백매、생장인자、세포결구세포발육、전록인자、신호분자、정합소화추화인자등상관기인.실험현시유도조기(0～7 d)미배경변화집중재촉진세포증식、분화화발육；유도만기(7 ～14d)미배경변화주요표현재삼여유지대사화연골세포표형은정.Real-timePCR진일보증실SPARC、CCL25화CCL4기인표체재유도7d명현상조,기질금속단백매-11(MMP-11)、금속단백매조직억제인자-3(TIMP-3)화종류배사인자상관수체인자5(TRAF5)기인표체재제14천체도고봉,저사기인유가능작위유도분화과정중조기화만기분자표지물.결론 미배경분자망락개변조공MSCs향연골세포정향분화.
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 polymerase 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 eycle,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 chondrocyte 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 (TRAF5) 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.