大鼠BMSCs自发钙化过程中成骨相关基因表达谱的分析

发布时间：2018-04-23 00:25

本文选题：BMSCs + 自发钙化基因芯片　；参考：《中国修复重建外科杂志》2014年02期

【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。
[Abstract]:Objective to analyze the changes of osteoblast-associated gene expression profile during spontaneous calcification of rat BMSCs. Methods BMSCs was isolated from the bone marrow of 3 day old SD rats and amplified to the fourth passage. The spontaneous calcification model was established in vitro. Cell growth and spontaneous calcification were observed by inverted phase contrast microscope and observed by alizarin red staining. The cells were collected for microarray analysis and bioinformatics analysis of differentially expressed genes. Real-time fluorescence quantitative PCR(real-time quantitative PCR RT-qPCR) was used to verify the results of chip analysis. Results spontaneous calcification of rat BMSCs occurred in vitro and the cells began to pile up after 7 days of culture. Alizarin red staining was weakly positive, and after 14 days of culture, the cells accumulated obviously and formed a typical calcium nodular structure with alizarin red staining positive. A total of 576 differentially expressed gene probes were detected during spontaneous calcification, corresponding to 378 rat genes. There were 359 differentially expressed gene probes on day 0 and day 7, and only 13 probes on day 7 and day 14. The differentially expressed genes can be divided into 6 categories according to their expression patterns, and the differentially expressed genes which are closely related to bone cell biology can be divided into seven categories, namely angiogenesis, apoptosis, bone related genes. Cell cycle, development, cell communication and osteogenic signaling pathway. Functional correlation analysis showed that 12 functionally related cell cycle class genes were down-regulated during spontaneous calcification. 13(matrix metalloproteinase 13 Mmp13, secreted phosphoric acid 1(secreted phosphoprotein 1, Cxcl12Mmp2mmp3mmp3Apoe and Itga7 had functional connections with more differentially expressed genes. The results of RT-qPCR verification of MMP13 Wnt inhibitor 1Cxcl12 and cyclin A2 were in agreement with the results of gene microarray. Conclusion the first 7 days after inoculation is the critical period for determining the spontaneous calcification of rat BMSCs. The process of spontaneous calcification of BMSCs is mediated by cellular communication genes, bone related genes, and cell cycle genes, including TGF- 尾 signaling pathway. Mitogen-activated protein kinase signaling pathway and Wnt signal pathway are regulated in many ways.
【作者单位】：四川大学华西药学院药理系;香港大学李嘉诚医学院矫形与创伤外科;四川大学华西医院再生医学研究中心;四川大学华西医院生物治疗国家重点实验室·干细胞与组织工程研究室;
【基金】：国家自然科学基金资助项目(31070872、31170948)~~
【分类号】：R329

【共引文献】