人HUT78细胞褪黑素受体的基因与蛋白表达的研究

INVESTIGATION OF MELATONIN RECEPTOR GENE AND PROTEIN EXPRESSION IN HUMAN HUT78 CELL LINE

为了检测人HUT78细胞是否存在褪黑素受体(melatoninreceptor,MR)及其亚细胞分布特点 ,采用异硫氢酸胍 苯酚 氯仿一步法抽提总RNA ,进一步通过RT PCR方法检测MR亚型mt1和MT2 的mRNA ,并将RT PCR的阳性产物通过自动测序仪测序 ;同时应用免疫组化染色方法检测mt1和MT2 受体亚型蛋白在HUT78细胞内的分布特点。RT PCR方法则得到了 3 68bpmt1cDNA片段 ,无 3 2 1bpMT2 cDNA片段 ,同时将mt1阳性条带通过胶回收、测序 ,结果显示扩增产物与人mt1受体亚型的基因序列相吻合 ;免疫组化结果显示 ,人HUT78细胞存在mt1受体蛋白 ,主要分布于细胞膜和细胞浆 ,呈棕褐色阳性颗粒 ,而细胞核上未见阳性颗粒 ,MT2 受体蛋白则未检出。提示褪黑素 (melatonin,Mel)对T淋巴细胞具有直接调节作用 。

To investigate the melatonin receptor(MR) gene and protein expression in human HUT78 cell line, total RNA of HUT78 cells was isolated by single-step method of acid guanidinium-thiocyanate-phenol-chloform, and mt 1 and MT 2 mRNA were determined by reverse transcription-polymerase chain reaction(RT-PCR). The Envision method was applied to immunohistochemistry to identify and localize mt 1 and MT 2 protein. The mt 1 cDNA fragment of the expected size of 370bp was determined, but the MT 2 cDNA fragment of the expected size of 320bp was not determinable by RT-PCR. Sequencing result indicated that the positive product coincided with the cDNA of human mt 1. The mt 1 protein was observed by immunohistochemistry. These buffy positive granules were scattered with some areas stronger than the others, and were primarily located in cytoplasm and membrane, with rare location in nucleus; meanwhile the MT 2 protein was not observed. These results demonstrated the mt 1 mRNA and protein expression in HUT78 cell line. It is indicated that melatonin has directly immune-regulative effects on T lymphocyte, The changes of MR in physiological and pathological stages need to be investigated.