Deer Antler Split Layer Cell Isolation And Culture, Effect Of Melatonin On Deer Antler Mesenchymal Cells In Vitro

Sika deer antler production is an important indicator of deer breeding work, which determines the economic effects of deer. Melatonin is a kind of photoperiodic signal hormone, can affect antler growth in many ways, improve the production of antler. Melatonin(MLT) can play a biological effect role only to combine their corresponding receptors, Therefore studying melatonin and its receptor (MTNR) has an important economic and biological breeding efficiency. In this study, We cultivate sika deer antler ectomesenchymal cells and chondrocytes cells individually in vitro conditions, through the different concentration levels of melatonin acts on the antler mesenchymal cells, to investigate the influence of different concentration levels melatonin on antler mesenchymal cells under the culture condition in vitro, and signaling pathway that makes melatonin functional.The sample of this institute was gathered from53(Shuangyang species) deer farm. In this study We use cell culture, RT-PCR, Western blot, confocal microscopy positioning, flow technology, MTT technology etc, by comparing several different concentration levels of melatonin on deer antler ectomesenchymal cell growth and proliferation, and its receptor expression in the cells, is designed to:(1) screen the concentration level of melatonin which has the best proliferation effect on deer ectomesenchymal cells, and can offer grouns for increasing deer antler production;(2) Investigate and discuss the signaling pathway between melatonin and its receptors, to lay the further foundation for further improve antler production capacity. The main research contents and results are as follows:1. Adopted the technique of rt-PCR and Western blot techniques tested the melatonin receptor MT1mRNA and protein expression of ectomesenchymal cells in pilose antler, electrophoresis target band after reverse transcription detected, showing that MT1mRNA in antler mesenchymal cells can transcriped normally;2. Using cell immunofluorescence technology localization subcellular distribution of MT1protein in antler mesenchymal cells, the results show that, MT1protein properly translated in antler mesenchymal cells. Melatonin receptor MT1protein located in the cytoplasm and the inner nuclear membrane.3. The effect of various concentrions of exogenous melatonin on deer antler mesenchymal cellsThe experiment set up six different doses of exogenous MLT groups (0pg/mL,400pg/mL,800pg/mL,1200pg/mL,1600pg/mL,2000pg/ml), detect the impact the exogenous MLT on the growth and development and function of antler mesenchymal cell, The proliferative capability of these cells were estimated by MTT method, and the cell cycle, distribution was detected by flow cytometry. At the same time, using enzyme-linked immunoassay determination of testosterone and estradiol in the nutrient solution concentration. The results show that, MLT can promote proliferation of deer antler mesenchymal cells, the concentration of800pg/ml of MLT promoting effect is obvious; MLT influence to the DAN synthesis phase of the cell and increase the number of interstitial cell cycle. Different concentrations of the MLT which is added to the cell culture medium and cultured to the antler mesenchymal cells, can promote the secretion of testosterone in the mesenchymal cells, but has no significant effect on estradiol.