应用基因表达谱芯片研究骨髓基质细胞诱导分化为成骨细胞中基因表达水平的变化

Research on Differentially Expressed Genes in Process of Marrow Stromal Cells Differentiated into Osteoblasts via Microarray

目的 探讨骨髓基质细胞（MSC）诱导分化为成骨细胞过程中各基因表达水平的变化,同时为合理选择及构建符合临床需求的组织细胞系在基因水平上寻求理论依据.方法 无菌条件下分离SD大鼠MSC进行原代培养,根据培养体系不同,将其分为两组：①诱导分化组（6例）,采用含浓度为1×10-8mol/L地塞米松,浓度为10 mmol/L β-甘油磷酸钠及浓度为50 mg/L维生素C的诱导性DMEM培养基进行诱导分化培养;②对照组（6例）,采用DMEM培养基进行同步培养.通过倒置相差显微镜观察诱导分化组与对照组MSC的形态特征,并采用茜素红染色观察细胞的生长与分化情况.采用基因表达谱芯片杂交技术选出诱导分化组与对照组的表达水平存在差异的基因,并对差异基因进行生物学功能分析.结果 ①分离MSC进行原代培养.培养72 h后,MSC开始增殖,细胞形态多呈梭形、三角形或不规则细胞形态.继续培养6～7 d后,细胞逐渐形成散在的细胞集落,多呈成纤维细胞形态.培养10～12 d后,MSC集落间相互融合成单层细胞.②MSC传代培养,诱导分化组细胞与对照组相比细胞增殖速度缓慢,细胞形态多呈梭形或多角形,且逐渐向成骨细胞转变;培养10～12 d后,密集的细胞间出现较多散在的致密圆形透光性差的钙化结节;钙化结节经茜素红染色呈片状棕染,茜素红染色呈显著阳性.对照组细胞增殖迅速,多呈成纤维细胞形态生长,在常规培养14 d后未出现明显钙化结节.③诱导分化组细胞内表达水平发生改变的基因占总有效基因的27.7％,显著高于对照组MSC传代培养中表达水平发生改变的基因数,差异有统计学意义（P=0.01）.诱导分化组细胞与原代细胞、对照组细胞基因表达水平的相关性较低（R2 =0.524 8,0.495 4）,诱导分化组细胞表达水平发生改变的基因数目增多.诱导分化组与对照组MSC表达水平存在＞3倍差异的部分已知基因,包括参与基因表达或蛋白质合成、修饰、加工,跨膜运输与胞内膜泡运输、细胞外基质与黏附分子、细胞通讯与信号传导、细胞骨架及代谢功能相关的6大类基因,其中表达水平上调的基因为120个,下调的基因为17个.结论 MSC诱导分化为成骨细胞的过程中,细胞形态向成骨细胞转变,且与细胞生长,代谢及骨形成相关的基因表达水平发生改变,为后续构建符合临床需要的成骨细胞系在基因水平上提供理论依据.

Objective To investigate gene expression levels in the process of marrow stromal cell （MSC） differentiation,and to seek theoretical basis for making cell lines which are more suitable for clinical use at genetic level.Methods MSC of SD rats were isolated and cultured.According to cell culture system,cultured MSC were divided into two groups.① Induced differentiation group （6 cases） were cultured in DMEM media adding 1×10 8 mol/L dexamethasone,10 mmol/L beta-glycerin sodium phosphate and 50 mg/L vitamin C.② Control group （6 cases） were cultured in DMEM media synchronously.Morphological characteristics of MSC in two groups were observed by inverted phase contrast microscope,and growth and differentiation of MSC were tested by alizarin red staining.Differential gene of two groups were screened by microarray,and the function of differential gene were analyzed.Results ① MSC were successfully isolated.After 72 h,MSC began to proliferate and cell morphology began changing to fusiform,triangular or irregular shape.After 6 to 7 d,cells gradually formatted dispersed cell colony.Cell colonies fused into a single layer of cells after 10 to 12 d.② MSC proliferation of induced differentiation group was slower than control group.After induction,MSC mainly shaped as spindle and polygon,and gradually transformed into osteoblasts.After 10 to 12 d,scattered dense circular calcified nodules with poor transparency were appeared in dense cells.The alizarin red staining shows calcified nodules appeared flaky brown dyed with a significant positive response.MSC proliferation of control group was rapid and no calcified nodule was appeared after 14 d.③ The number of genes with expression level differences in induced differentiation group were accounted for 27.7% of total effective gene,and were far higher than those in control group,and the difference was statistically significant （P=0.01）.The correlation of gene expression levels between induced differentiation group and primary MSC,control group were low（R2 =0.524 8,0.495 4）.Genes with expression level difference were increased in induced differentiation group.Genes with expression level difference which were greater than 3 times between induced differentiation group and control group were involved in gene expression and protein synthesis,modification,processing,transmembrane transport and intracellular membrane vesicle transport,extracellular matrix and adhesion molecule,cell communication and signal transduction,cytoskeletal and metabolic function.Among those differential genes,expression levels of 120 genes were upregulated,and 17 genes were downregulated.Conclusions MSC transformed into osteoblasts in the process of induced differentiation.Expression levels of genes which were associated with cell growth,metabolism and bone formation were changed.And theoretical basis for making osteoblast cell lines which met clinical needs at genetic level was provided.