microRNA-16在脂肪基质细胞脂向分化过程中的表达变化

Changes in microRNA-16 expression during adipogenic differentiation of adipose tissue-derived stromal cells

背景:触发和控制脂肪基质细胞脂向分化的调控研究将有助于人为控制脂肪基质细胞的分化方向,促进对细胞分化的深入了解。目的:了解脂肪基质细胞脂向分化过程中microRNA-16的表达变化情况。设计、时间及地点:观察对比试验,2007-10/2008-03在四川大学口腔疾病研究国家重点实验室完成。材料:出生30d雄性体健SD幼鼠8只。成脂诱导培养液[DMEM培养液中加入地塞米松(1μmol/L)、3-异丁基-1-甲基黄嘌呤(0.5mmol/L)、胰岛素(10mg/L)]。方法:从SD幼鼠体内取出脂肪,采用密度梯度离心结合贴壁培养法获得了纯度高的脂肪基质细胞,采用脂向诱导液对第3代的脂肪基质细胞进行诱导培养,并以未诱导的细胞为对照。主要观察指标:应用microRNA芯片技术检测脂肪基质细胞脂向诱导后7d和未诱导microRNA-16的表达差异。采用实时定量PCR检测microRNA-16在脂肪基质细胞脂向分化前后表达量变化。结果:①成功获得纯度较高的脂肪基质细胞,成功诱导其脂向分化,诱导培养后7d观察到细胞形态学上表现出典型的脂肪细胞改变:细胞相互融合,成片生长,变为细长纺锤形;PPARγ免疫细胞化学染色呈阳性;油红O染色脂滴被染成橙红色。②成脂诱导前后microRNA-16表达明显下调,诱导前microRNA-16表达量为90.25。诱导后microRNA-16表达量为56.75,③实时定量PCR结果显示microRNA-16表达显著下调,与microRNA芯片结果一致。结论:PCR及芯片结果一致支持microRNA-16在脂肪基质细胞脂向分化过程中表达显著下调,可能在脂肪基质细胞脂向分化过程参与调控。

BACKGROUND： The study on regulation adipogenic differentiation of adipose tissue-derived stromal cells （ADSCs） is benefit for artificial control differentiation direction of adipose stromal cells, and promote further understanding of cell differentiation. OBJECTIVE： To explore the differential expression of microRNA-16 during adipogenic differentiation of ADSCs. DESIGN, TIME AND SETTING： The comparison observation was performed at the State Key Laboratory of Oral Diseases in October 2007 to March 2008. MATERIALS： Eight 30-days-old Sprague-Dawley （SD） rats. Adipogenic-differentiated culture medium [DMEM including dexamethasone （1 μ mol/L）, 3-isobutyl-l-methyl xanthine （0.5 mmol/L） and insulin （10 mg/L）. METHODS： Removed the adipose from yonge SD rats, and treated with density gradient centrifugation and adherent culture to obtained ADSCs. Induced and cultured the third generation cells into adipogenic differentiation with the inductive medium, and the non-induced cells were served as the control group. MAIN OUTCOME MEASURES： The differential expression of microRNA-16 in experimental group at 7 days after adipogenic-differentiation and in the control group were detected by microRNA chip technology. Realtime quantitative PCR was carried out to explore the differentially expression of microRNA-16 during adipogenic differentiation of ADSCs. RESULTS： High-purity ADSCs were acquired successfully. The ADSCs were induced into osteoplastic differentiation, and displayed typical adipocytes changes 7 days after induction, the cells turned to slender fusiform and mixed together, which showed positive PPARy immunohistochemical staining and the lipid droplet was stained orange-red by oil red staining. The expression of microRNA-16 was significant down regulated during adipogenic differentiation, which changed from 56.75 to 90.25. Results of realtime quantitative PCR was concordance with that of microRNA chip technology. CONCLUSION： The expression of microRNA-16 is significant down regulated during the adipogenic differentiation of ADSCs. Maybe it takes part in adipogenic differentiation of ADSCs.