大鼠BMSCs自发钙化过程中成骨相关基因表达谱的分析  

EXPRESSION PROFILE OF OSTEOGENIC-RELATED GENES DURING SPONTANEOUS CALCIFICATION OF RAT BONE MARROW MESENCHYMAL STEM CELLS

作　　者：焦雪峰[1] 黄永灿[2] 黄益洲 吴诚光 邓力[4] 

机构地区：[1]四川大学华西药学院药理系,成都610041 [2]香港大学李嘉诚医学院矫形与创伤外科 四川大学华西医院 [3]再生医学研究中心 [4]生物治疗国家重点实验室·干细胞与组织工程研究室

出　　处：《中国修复重建外科杂志》2014年第2期133-141,共9页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(31070872、31170948)~~

摘　　要：目的分析大鼠BMSCs自发钙化过程中成骨相关基因表达谱的改变。方法取健康3日龄SD大鼠骨髓分离培养BMSCs并扩增至第4代,体外构建自发钙化模型。于培养7、14 d,倒置相差显微镜观察细胞生长状况及自发钙化进程,并行茜素红染色观察。分别于培养0、7、14 d时收集细胞进行基因芯片分析,并对差异表达基因进行生物信息学分析。实时荧光定量PCR(real-time quantitative PCR,RT-qPCR)验证芯片分析结果。结果大鼠BMSCs在体外发生了自发钙化,并于培养7 d后细胞开始堆集,茜素红染色为弱阳性;培养14 d后细胞堆积明显并形成典型的茜素红染色阳性钙结节样结构。自发钙化过程中共检测到576个差异表达的基因探针,对应378个大鼠基因。其中0 d和7 d相比有359个差异表达的基因探针,而7 d和14 d相比只有13个差异表达的基因探针。差异表达基因依据表达模式不同可分为6类;依据生物学功能亲和关系,与骨细胞生物学密切关联的差异表达基因又可分为7大类,即血管生成、细胞凋亡、骨相关基因、细胞周期、发育、细胞通讯和成骨信号通路。基因功能关联性分析显示有12个在功能上联系密切的细胞周期类基因在自发钙化过程中发生了下调;此外,基质金属蛋白酶13(matrix metalloproteinase 13,Mmp13)、分泌型磷酸蛋白1(secreted phosphoprotein 1,Spp1)、Cxcl12、Mmp2、Mmp3、Apoe和Itga7与较多的差异表达基因存在功能上的联系。Spp1、Mgp、Mmp13、Wnt抑制因子1、Cxcl12和细胞周期素A2的RT-qPCR验证结果与基因芯片结果一致。结论细胞接种后的前7 d是决定大鼠BMSCs自发钙化的关键期。BMSCs自发钙化过程受细胞通讯类基因、骨相关基因、细胞周期类基因、TGF-β信号通路、丝裂原活化蛋白激酶信号通路和Wnt信号通路等多方面的共同调控。Objective To analyze the expression profile changes of osteogenlc-related genes aurmg spontaneous calcification of rat bone marrow mesenchymal stem cells （BMSCs）. Methods BMSCs were isolated from 3-day-old healthy Sprague Dawley rats; cells at the 4th generation were used to establish the spontaneous calcification model in vitro. Spontaneous calcification process was recorded by inverted phase contrast microscope observation and alizarin red staining after 7 and 14 days of culture. For gene microarray analysis, cell samples were collected at 0, 7, and 14 days after culture; the differentially expressed genes were analyzed by bioinformatics methods and validated by real-time quantitative PCR （RT-qPCR） assay. Results Rat BMSCs calcified spontaneously in vitro. When cultured for 7 days, the cells began to aggregate and were weakly positive for alizarin red staining. After 14 days of culture, obvious cellular aggregation and typical mineralized nodules were observed, the mineralized nodules were brightly positive for alizarin red staining. A total of 576 gene probe-sets expressed differentially during spontaneous calcification, corresponding 378 rat genes. Among them, 359 gene probe-sets expressed differentially between at 0 and 7 days, while only 13 gene probe-sets expressed differentially between at 7 and 14 days. The 378 differentially expressed genes were divided into 6 modes according to their expression profiles. Moreover, according to their biological functions, differentially expressed genes related to bone cell biology could be classified into 7 major groups： angiogenesis, apoptosis, bone-related genes, cell cycle, development, cell communication, and signal pathways related to osteogenic differentiation. Incell cycle group, 12 down-regulated genes were linked with each other functionally. Matrix metalloproteinase 13 （Mmp13）, secreted phosphoprotein 1 （Sppl）, Cxcll2, Mmp2, Mmp3, Apoe, and Itga7 had more functional connections with other genes. The results of genes Sppl, Mgp, Mmpl3, Wnt inhi

关 键 词：BMSCS 自发钙化 基因芯片 基因表达谱 生物信息学 大鼠 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]