| Velvet antler is a periodically regenerated organ which has a precious medical value.In China,deer antlers usually refer to the unguided tender angle of Cervus nippon and Cervus elaphus.Melatonin(MLT),playing an important role in regulating the physiological cycle of seasonal breeding animals,is an indole endocrine hormone and mainly secreted by the pineal gland.Previous studies have shown that MLT can affect the growth cycle of velvet,and its receptor MT1 has a high expression in the top proliferation area of velvet and is closely related to the antler production.However,the mechanism of melatonin promoting the proliferation of velvet anter mesenchymal cells is not clear.At pressent,we use velvet anter mesenchymal cells as a cell model and employ RNAi method to uncover these questions.The results will give us a deep understanding of MLT’s function on velvet mesenchymal cells proliferation and provide new idea to improve the deer antler production.The main contents and results are as follows:(1)Velvet mesenchymal cells were transfected GR-2 interference plasmid for 48 h,and MT1 mRNA expression was decreased by 60.1%(p <0.05)compared with the control group.And the expression of MT1 protein was decreased by 62.9%(p <0.05)after transfection for 72 h,suggesting that shiRNA interference was efficiency.(2)MTT assay was applied to illustrated velvet mesenchymal cells proliferation.Result showed that the cell viability was significantly decreased in the MT1 knockdown group than the control group(48h,p <0.001).To further demostreat the function of melatonin on velvet mesenchymal cells proliferation,we treated cells with melatonin(800pg / mL)for 48 h,the result showed as melatonin can significantly increased the cell survival rate(p < 0.05),indicating that MT1 can drive cell proliferation.(3)We employed flow cytometry after MLT treatment to demostreat the cell cycle.Result showed that compared with control group,the cell proportion in G2/M phase was significantly decreased(p <0.05),however,cells proportion in S phase was significantly increased(p <0.05);(4)qRT-PCR result showed that IGF-1 and IGF-1R was significantly decreased(p <0.001)after kncokdown MT1.While melatonin(800pg/mL)significantly increaseda IGF-1 expression(0.001 <p <0.01),and IGF-1R expression(p <0.001).The results of ELISA showed that IGF-1 secretion was significantly decreased(p <0.05)after MT1 interference.(5)MTT assay demostreated that compared with MT1 knockdown group,the cell viability was significantly increased by treated with MLT(0.001 <p <0.01).The results showed that melatonin played a pivotal part in proliferation of velvet mesenchymal cells,including regulatng the cell viabilt,the cell cycle of mesenchymal cells and the secretion of IGF-1. |
