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目的探讨高浓度全反式视黄酸(all-trans retinoic acid,atRA)抑制大鼠骨髓间充质干细胞(bone marrowmesenchymal stem cells,BMSCs)向脂肪细胞分化的机制。方法体外分离、培养和诱导大鼠BMSCs;在成脂诱导液中加入0.1、0.5、1.0、5.0μmol/L atRA,光学显微镜下观察细胞形态及油红O染色变化;油红O染色提取法分析不同浓度atRA对BMSCs成脂分化的影响;real-time PCR测定细胞视黄酸受体(RARα、RARγ)、CCAAT增强子结合蛋白α(CEBPα)及过氧化物酶体增殖激活受体γ2(PPARγ2)mRNA水平的变化。结果细胞形态及油红O染色观察发现,在0.1、0.5、1.0、5.0μmol/L atRA作用下大鼠BMSCs向脂肪细胞分化受到抑制,且呈浓度依赖性;油红O染色提取法半定量测定结果显示,随着atRA浓度升高,分化细胞内脂肪含量降低(P<0.01);real-time PCR检测显示与血清组(未加atRA)相比,加入atRA后,大鼠BMSCs的CEBPα和PPARγ2表达显著降低(P<0.05),RARα表达无显著差异[血清组vs 1.0μmol/L atRA组:(0.008 4±0.001 9)vs(0.009 1±0.001 3),P>0.05],但RARγ表达显著增加[血清组vs 1.0μmol/L atRA组:(0.022 6±0.001 2)vs(0.053 1±0.002 3),P<0.01]。结论 0.1、0.5、1.0、5.0μmol/L atRA可抑制大鼠BMSCs向脂肪细胞分化,其机制可能与RARγ的上调及CEBPα、PPARγ2的下调有关。
Objective To investigate the mechanism of high-concentration all-trans retinoic acid (atRA) on adipocyte differentiation of rat bone marrow mesenchymal stem cells (BMSCs). Methods BMSCs were isolated, cultured and induced in vitro; 0.1, 0.5, 1.0, 5.0 micromol / L atRA was added into adipogenic medium, and the cell morphology and oil red O staining were observed under optical microscope. Oil red O staining The effects of different concentrations of atRA on the adipogenic differentiation of BMSCs were detected by real-time PCR. The expressions of retinoic acid receptor (RARα, RARγ), CCAAT enhancer binding protein α (CEBPα) and peroxisome proliferator-activated receptor γ2 (PPARγ2 ) mRNA level changes. Results Cell morphology and oil red O staining showed that the differentiation of rat BMSCs into adipocytes was inhibited in a concentration-dependent manner under the action of 0.1, 0.5, 1.0 and 5.0 μmol / L atRA. Semi-quantitative determination by oil red O staining The results showed that the fat content of differentiated cells decreased with the increase of atRA concentration (P <0.01). The real-time PCR results showed that compared with the serum group (without atRA), the expression of CEBPα and PPARγ2 (P <0.05), but there was no significant difference in the expression of RARα between the two groups [serum group vs 1.0μmol / L atRA group: (0.008 4 ± 0.001 9) vs (0.009 1 ± 0.001 3), P> 0.05] Increased [serum group vs 1.0 μmol / L atRA group: (0.022 6 ± 0.001 2) vs (0.053 1 ± 0.002 3), P <0.01]. Conclusions 0.1, 0.5, 1.0 and 5.0 μmol / L atRA can inhibit the differentiation of rat BMSCs into adipocytes, which may be related to the upregulation of RARγ and the downregulation of CEBPα and PPARγ2.