Identification And Location Of Melatonin Receptors In Leucocytes Of Human And The Mechanism Of Effects Of Melatonin On The Leucocytes

Identification and Location of Melatonin Receptors in Leucocytes of Human and the Mechanism and Signification of Melatonin on the Leucocytes As one of the important neuro-endocrinology hormone, melatonin was usually thinked of as a hormone which mainly regulate the physiology rhythm in living body. With the proceeding of melatonin study these years, it has been found gradually that melatonin can also regulate the function of reproductive system, immune system and have effect of antioxidation and antitumor. Because of the importance of all these function and the characteristic of small molecular, hydrophobic and wide-existing, melatonin and its further function is being paid more and more attention by many scientists and laboratories. The pineal hormone melatonin, due to its lipophilic nature, has access to every cell and every part of a cell in the body, suggesting that it could exert effects on blood immune cells. Considering this, a study on the effect of melatonin on immune cells was carried out.In this study, using several study technics and methods we managed to resolve some questions as these: 1. In order to further investigate the possible implication of melatonin-mediated immune regulative role, we combined reverse transcription- polymerase chain reaction(RT-PCR) techniques and immunohistochemistry staining to identify the mRNA expression of the melatonin receptors and localize them in the human peripheral blood lymphocytes, granulocytes and HUT78 cells. 2. To study the effects of melatonin on peripheral blood lymphocytes(PBL) and HUT78 cells line proliferation. 3. To study the effects of melatonin on PBL proliferation and apoptosis from patients with type2 diabetes mellitus(DM). 4. To study on T-lymphocytes early responses to melatonin by genomic-scale gene expression analysis of immunity. The final destination is to supply some new data for the study of the mechanism how the melatonin regulate the immune function.Main methods and results: The first part: Methods: Human peripheral venous blood was obtained from healthy volunteers, lymphocytes and granulocytes were isolated by centrifugation over gradient. The HUT78 cells line was obtained from Shanghai Institute of Biochemistry and Cell Biology, Sciences Chinese Academy. The total RNA of cells that we investigated were extracted, reversibly transcripted, amplified. Shanghai Genecore Biotechnologies made the sequence analysis. The mt1 antisense premier is 5'-AGAAGATGACTATGATCATG-3', the mt1 sense premier 5'-GGTGCTGATGT CGATATTTA-3'. The MT2 antisense premier is 5'-TCCTGGTGATCCTCTCCGTG CTCA-3', the MT2 sense premier 5'-AGCCAGATGAGGCAGATGTGCAGA-3'.The Envision method was applied to immunohistochemistry staining, with primary antibody against human mt1 receptor subtype and MT2 receptor subtype kindly donated by Prof. Pang S.F. at physiology department of Medical College in Hongkong University. Results: 1. RT-PCR: RT-PCR analysis demonstrated mRNA expression of the mt1 receptor subtype in human peripheral blood lymphocytes, granulocytes, and HUT78 cells, the mRNA expression of the MT2 receptor subtype in these cells were not found. Electrophoresis showed positive production of RT-PCR with the mt1 receptor subtype in above cells, but did not show positive production of RT-PCR with the MT2 receptor subtype in these same cells. Sequencing of RT-PCR production showed that the DNA sequence of production coincided with the sequence of the mt1 cDNA from Genebank. 2.Immunohistochemistry staining: the mt1 receptor subtype was observed in these cells investigated. These buffy positive granules were scattered with some areas stronger than the others, and primarily located to cytoplasm and membrane, with rare location in nucleus. It was also suggested that the mt1 receptor subtype exist in these cells. The second part: Methods: Human peripheral blood lymphocytes(PBL) and HUT78 cells were cultured with melatonin at different concentrations and different time. MTT colorimetric assay was applied to observe the proli...