大鼠骨髓间充质干细胞向成骨与成脂分化过程中相关基因的表达变化

Gene expression changes during osteogenic and adipogenic differentiations of rat bone marrow mesenchymal stem cells

目的探讨大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)向成骨与成脂分化过程中相关基因表达的变化。方法采用全贴壁法分离培养大鼠BMSCs,并观察其形态学特征的变化,MTT法检测其生长状况,并绘制生长曲线。分别采用成骨和成脂诱导剂对第4代BMSCs进行诱导分化,应用碱性磷酸酶试剂盒、茜素红和油红O染色液检测其ALP活性、成骨和成脂分化能力;RT-QPCR检测诱导0、7、14和21 d的成骨分化相关基因Runt相关转录因子2(Runx2)、骨钙素(osteocalcin,OCN)、碱性磷酸酶(ALP)及成脂分化相关基因过氧化物酶体增殖物激活受体γ(peroxidase proliferator activated receptor gamma,PPARγ)和脂肪酸结合蛋白(FABP4)的表达变化。结果全骨髓贴壁法能成功分离培养BMSCs,传代细胞生长增殖迅速,以长梭形细胞生长为主,细胞生长曲线呈S形。第4代BMSCs分别经成骨和成脂诱导剂诱导后,ALP、茜素红和油红O染色均呈阳性;诱导7、14和21 d后,Runx2、OCN、ALP、PPARγ和FABP4基因mRNA的表达量均显著高于0 d(P<0.05);成骨分化过程中,Runx2和ALP在第7天时表达量最高,之后呈下降趋势,OCN的表达量呈稳定上升趋势;成脂分化过程中,PPARγ在第7天时表达量最高,FABP4始终高表达。结论 BMSCs具有易于体外分离培养、扩增和经诱导后具有多向分化潜能等特点,成骨和成脂分化相关基因的表达量随诱导时间延长而变化,呈明显的时序性表达差异,提示分别在成骨与成脂分化过程中起重要调控作用,为BMSCs在骨、细胞和基因等工程中的机制研究提供了实验依据。

Objective To investigate the change of expression of relevant genes during osteogenic and adipogenic differentiations of rat bone marrow mesenchymal stem cells.Methods BMSCs from rats were isolated and cultured by adherent culture,observed for morphological change and determined for growth by MTT assay,based on which the growth curve was plotted.The osteogenic and adipogenic differentiations of BMSCs of passage 4 were induced with the corresponding inducers,and the alkaline phosphatase（ALP）activity,osteogenic and adipogenic differentiation abilities of the cells were determined by ALP kit,alizarin red staining and oil red staining respectively.The mRNA expressions of Runx2,osteocalcin（OCN）,ALP,peroxidase proliferator activated receptor gamma（PPARγ）and fatty acid binding protein 4（FABP4）after induction for 0,7,14 and 21 d were determined by RT-QPCR.Results BMSCs were successfully isolated and cultured.Passaged cells were proliferated rapidly,most of which were in a long spindle form.The cell growth curve was in an S shape.After induction with osteogenic and adipogenic inducers,the results of ATP activity test,alizarin red staining and oil red staining of BMSCs of passage 4 were positive.The expression levels of mRNA of osteogenic genes（Runx2,OCN and ALP）and adipogenic genes（PPARγ and FABP4）after induction for 7,14 and 21 d were significantly higher than those before induction（0 d）（P〈0.05）.During osteogenic differentiation,the expression levels of Runx2 and ALP reached the maximums after induction for 7 d then showed decreasing trends,while that of OCN showed a steady increasing trend.However,during adipogenic differentiation,the expression level of PPARγreached the maximum after induction for 7 d,while that of FABP4 was higher throughout.Conclusion BMSCs are easy to be isolated,cultured and amplified in vitro,with potential of multi-differentiation after induction.The expression levels of genes related to osteogenic and adipogenic differentiations changed with the increasing time for induction,indicating the regulatory roles of the genes.It provides an experimental basis for study on the mechanism of BMSCs in bone,cell and gene engineering.