脂肪干细胞脂向分化过程中miRNA-27b表达的研究

An Experimental Study of miRNA-27b Expression during Adipogenic Differentiation by Adipose-Derived Stem Cells

目的:探讨与脂肪干细胞脂向分化密切相关的microRNA(miRNA)表达情况,为阐明脂肪干细胞脂向分化的调控机制提供新的线索。方法:采用Ⅰ型胶原酶消化法结合密度梯度离心获得脂肪干细胞,将第3代细胞采用脂向诱导液(α-MEM培养基内加入100mL/L胎牛血清、1μmol/L地塞米松、10μmol/L胰岛素、200μmol/L吲哚美辛和0.5mmol/L3-异丁基-1-甲基黄嘌呤)进行脂向诱导;分别应用miRNA芯片技术及实时定量PCR技术对脂肪干细胞脂向分化中的miRNA-27b表达情况进行检测,分析miRNA-27b的变化趋势,并结合生物信息学方法探讨miRNA-27b与脂肪干细胞脂向分化相关的生物学功能。结果:建立了脂肪干细胞体外脂向分化的模型,miRNA芯片及实时定量PCR结果均表明miRNA-27b在脂肪干细胞体外脂向分化过程中表达显著下调。结论:miRNA-27b可能与脂肪干细胞的脂向分化密切相关,并可能通过调控其靶基因PPARγ的表达而促进脂肪干细胞的脂向分化。

Objective： To investigate miRNA involved in adipogenic differentiations of adipose-derived stem cells （ADSCs）, and the regulation mechanisms of ADSCs＇ osteogenic differentiation thereof. Methods： ADSCs were obtained from the inguinal fat pads of SD rats by type I collagenase digestion and density gradient centrifugation. Adipogenic differentiation of the third generation of ADSCs was induced with the adipogenic medium （α-MEM medium supplemented with 100mL/Lfetal bovine serum, 1μ mol/L dexamethasone, 10 μ mol/L insulin, 200 p,mol/L indomethacin, and 0.5 mmol/L 3-isobuty1-1- methylxanthine） prior to applying miRNA chip technology for examination of miRNA-27b. Real-time quantitative polymerase chain reaction （real-time PCR） was used to detect the expression of miRNA-27b. Moreover, the bioinformatics analysis was utilized to discuss adipogenie-related function of the miRNA-27b. Results： An in vitro model of adipogenic-inductive differentiation of rat ADSCs was successfully constructed. The miRNA-27b was identified by the miRNA chip technology as being down-regulated during adipogenic differentiation of ADSCs. Real-time PCR analysis confirmed the decreased expression of miRNA-27b. Conclusion： The expression of miRNA-27b was significantly down-regulated during adipogenic differentiation of ADSCs, which indicated miRNA-27b may be involved in adipogenic differentiation of ADSCs. The miRNA- 27b may promote adipogenic differentiation of ADSCs through regulating its target gene PPARγ expression.