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目的:检测体外培养的人骨髓间充质干细胞(bone mesenchymals stromal cells,BMSCs)褪黑素受体的表达,并探讨其意义。方法:10例12～18岁志愿者,分别从髂前上棘处穿刺抽取10ml骨髓,肝素抗凝。采用密度梯度离心法分离BMSCs,体外培养并传至P3代进行表型鉴定。抽提细胞总RNA,采用逆转录-聚合酶链式反应(RT-PCR)方法检测褪黑素受体MT1、MT2 mRNA的表达;同时应用免疫组化染色方法检测细胞MT1、MT2蛋白的表达。结果:分离的单个核细胞经培养至P3代,使用流式细胞仪进行细胞表型鉴定,体外培养细胞的表型与BMSCs的表面标志相符。细胞抽提总RNA以MT1和MT2两种受体的RT-PCR扩增产物电泳呈阳性条带;免疫组化结果显示细胞存在MT1和MT2蛋白的表达,主要分布在细胞膜和细胞浆,呈棕褐色阳性颗粒,而细胞核上未见阳性颗粒。结论:人BMSCs中存在褪黑素受体MT1、MT2的表达,它是褪黑素作用的靶细胞。这为进一步研究BMSCs褪黑素信号通路提供了实验基础。
Objective: To detect the expression of melatonin receptor in human mesenchymal stromal cells (BMSCs) cultured in vitro and to explore its significance. Methods: Ten volunteers aged 12 to 18 years old were enrolled in this study. 10ml bone marrow was obtained from the anterior superior iliac spine and heparin was anticoagulated. BMSCs were isolated by density gradient centrifugation, cultured in vitro and passaged to P3 for phenotypic identification. Total RNA was extracted and the expression of MT1 and MT2 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). The expressions of MT1 and MT2 protein were detected by immunohistochemical staining. Results: The isolated mononuclear cells were cultured to P3 generation, and the phenotypes of cells were identified by flow cytometry. The phenotypes of cultured cells were in accordance with the surface markers of BMSCs. Cell total RNA was extracted by RT-PCR amplification products of MT1 and MT2 were positive bands; Immunohistochemistry showed the presence of MT1 and MT2 protein expression in cells, mainly in the cell membrane and cytoplasm, brown Brown-positive particles, while no positive particles on the nucleus. Conclusion: The expression of melatonin receptors MT1 and MT2 is present in human BMSCs, which is the target cell of melatonin. This provided the experimental basis for further study of melatonin signaling pathway in BMSCs.