The Mechanism Of Metabotropic Glutamate Receptor 6 In The Morphology Of Melanocytes And Melanosome Transport

Objective:To investigate the mechanism of metabotropic glutamate receptor 6 in the the morphology of melanocytes and the melanosome transportMethods:1. Separation and purification of human primary melanocytes; synthesis of lentivirus vector, transfection assay after transfection efficiency.The experiment was divided into knockdown group, blank control group and negative control group. Transfection assay after transfection efficiency.2. Western blotting to detect the lentivirus knockdown efficiency.3. Scanning electron microscope imaged the change of melanocyte morphology, after the melanocyte were treated with lentivirus to knockdown the metabotropic glutamate receptor 6.4. Melanocyte and keratinocyte were co-cultured, then the morphology changes were imaged by scanning microscopy to observe changes in melanocytes and keratinocytes cocultured system after melanocyte transfected, using NaOH method to determine the keratinocytes melanin content in the co-cultured system; using double immunofluorescence staining, and flow cytometry to evaluate melanosome transfer efficiency.5. Immunofluorescence staining to observe the changes of β-tublin and the melanin distribution in co-cultured keratinocyte by laser confocal microscope.Results:1. A scanning electron microscope found knockdown the mGluR6 will enable melanocyte morphology change. The end of the cell dendrites was significantly wider and shorter, the number of surface filopodias was less and shorter in the knockdown group.2. In co-cultured, we found there are the contact between melanocytes and keratinocytes in the system, and filopodias of the area close to the direction of keratinocytes on melanocytes than in the opposite direction. In the knockdown group, the filopodias decreased.3. After co cultured for 48 h, the knockdown group keratinocytes melanin content was significantly lower than the control group.4. Confocal microscopy of fluorescently labeled melanosomes、pan-keratin, found there are melanosomes in the keratinocytes, the number decreased in the knockdown group than the control group. Flow cytometry analysis showed the melanosome in keratinocytes content increased with time, compared with the control group, knockdown group transfer efficiency decreased obviously.5. Confocal microscopy of fluorescently labeled β-tublin, found the control group of β-tublin into a tube bundle uniform distribution. But the knockdown group changed into a punctate distribution.Conclusion:1. Metabotropic glutamate receptor 6 can affect morphology and the β-tublin in melanocytes.2. Metabotropic glutamate receptor 6 can affect the filodias between melanocyte and keratinocyte.3. Metabotropic glutamate receptor 6 can affect the transportation of melanosome.4. Metabotropic glutamate receptor 6 can affect the melanosome transport via the ?lopodias.