褪黑素通过视黄酸相关孤儿受体α可抑制骨髓间充质干细胞的成脂肪分化

背景：前期研究发现，褪黑素能够抑制骨髓间充质干细胞向脂肪细胞诱导分化，但其作用机制有待进一步研究。 目的：探索褪黑素是否通过核受体视黄酸相关孤儿受体α介导抑制骨髓间充质干细胞向脂肪细胞诱导分化的作用机制。 方法：无菌采集骨髓样本，采用密度梯度离心结合贴壁培养分离培养人骨髓间充质干细胞，用流式细胞仪鉴定细胞表面表型。然后将细胞分为褪黑素组、CGP52608组和对照组，分别以含褪黑素、CGP52608脂肪维持液和正常的脂肪诱导液进行诱导，用荧光定量PCR和Western blot法检测核受体视黄酸相关孤儿受体α在骨髓间充质干细胞以及其成脂肪分化过程中的表达特征。用受体特异性激活剂 CGP52608，考察核受体视黄酸相关孤儿受体α激活状态下，骨髓间充质干细胞成脂肪分化的影响。 结果与结论：原代分离的骨髓间充质干细胞生长旺盛，呈纤维梭状，流式鉴定间充质干细胞表型结果示，CD34和CD45表达阴性，CD29，CD44与CD105表达阳性。荧光定量PCR发现骨髓间充质干细胞高表达褪黑素核受体视黄酸相关孤儿受体α，而且褪黑素能够呈浓度依赖性促进骨髓间充质干细胞表达视黄酸相关孤儿受体α；Western blot检测结果也证实褪黑素呈浓度依赖性促进视黄酸相关孤儿受体α在蛋白水平的表达。在成脂肪分化过程中，褪黑素在早期明显上调视黄酸相关孤儿受体α表达，但在中后期就维持在相对低水平。最后，用CGP52608激活视黄酸相关孤儿受体α后，骨髓间充质干细胞成脂肪分化被抑制。因此，褪黑素通过核受体视黄酸相关孤儿受体α抑制骨髓间充质干细胞成脂肪分化，褪黑素在调节脂肪细胞分化过程中发挥重要作用。

Melatonin inhibits adipogenic differentiation of bone marrow mesenchymal stem cells through retinoid-related orphan receptor alpha

BACKGROUND：Our previous studies have demonstrated that melatonin inhibits the adipogenic differentiation of bone marrow mesenchymal stem cels. However, the mechanism underlying the effect of melatonin on adipogenesis is stil unknown.
OBJECTIVE：To determine whether melatonin can inhibit the adipogenesis of bone marrow mesenchymal stem cels through retinoid-related orphan receptor α.
METHODS：Bone marrow mesenchymal stem cels were isolated and purified by density gradient centrifugation combined with cellattachment culture. The phenotype was investigated by flow cytometry. Then, cels were induced for adipogenic differentiation with melatonin, CGP52608 and normal adipose tissue, respectively. The levels of retinoid-related orphan receptor α mRNA and protein were investigated by real-time RT-PCR and western blot assay, respectively. Further, the effect of retinoid-related orphan receptor α on the dipogenic differentiation of bone marrow mesenchymal stem cels was investigated by CGP52608.
RESULTS AND CONCLUSION：The primary isolated bone marrow mesenchymal stem cels were spindle-shaped fibroblast-like cels. These cels did not express hematopoietic stem cels markers： CD34 and CD45; and highly expressed MSC markers： CD29, CD44, and CD10. The result of RT-PCR demonstrated that melatonin nuclear receptor, retinoid-related orphan receptor α, was highly expressed in bone marrow mesenchymal stem cels and the expression of retinoid-related orphan receptor α was further enhanced by melatonin in a dose-dependent manner, which was confirmed at protein level by western blot assay. During adiogenesis, the expression of retinoid-related orphan receptor αmRNA was up-regulated in the early stage, but maintained at a low level in the mild-later stage. While the retinoid-related orphan receptor α was activated by agonist CGP52608, the adipogenic differentiation of bone marrow mesenchymal stem cels was inhibited, which was similar to the inhibitory effect of melatonin. Therefore, melatonin inhibited the adipogenic differentiation of bone marrow mesenchymal stem cels through retinoid-related orphan receptor α, suggesting that melatonin plays an important role in the differentiation of adipocytes.