The Research Of The Effection Of Metabotropic Glutamate Receptor 6 On HaCaT Phagocytosis

Objective:To investigate the influence of metabotropic glutamate receptor 6 on Ha Ca T phagocytosis.Methods:1.Culture the human epidermal keratinocyte in vitro.2.Western blot analysis was done to detect the expression mGluR6 in HaCaT Knockdown of m Glu R6 using sh RNA3. Knockdown of m Glu R6 by m Glu R6-sh,and use nc treated Ha Ca T as negative control.4. SEM assessment of cell morphology.5. Immunofluorescence staining, confocal microscopy assess the cytoskeletal proteins of experimental group and control group.6. Use fluorescent microspheres and Ha Ca T to set a model of phagocytosis, and observe by confocal microscopy.7. To determine the fluorescent microspheres Uptake efficiency of Ha CaT by flow cytometer.Results:1.Ha Ca T cells express m Glu R6 richly.2.m Glu R6 has been knockdowned effectively in Ha Ca T cells,and the Lv-shm Glu R6 cell line has been constructed successfully.3.SEM analysis shows an decrease in cell podia number following m Glu R6 knockdown.4.The laser confocal microscope analysis shows m Glu R6 knockdown induces cytoskeletal protein reorganization.5.Confocal microscopy shows an decrease in fluorescent microspheres number in Ha Ca T after m Glu R6 downward.6.Flow cytometry shows an decrease in fluorescent microspheres in Ha Ca T after m Glu R6 downward.Conclusion:1. This is the first demonstration that Ha Ca T cells express m Glu R6.2.The number of lamellipodia decrease after m Glu R6 downward,and the morphology of the Ha Ca T surface alter. SEM analysis shows an decrease in cell podia number and a decrease in podia diameter,and shows a reorganization of cytoskeletal protein.3. Using fluorescent microspheres and Ha Ca T set up a model of phagocytosis,and proved Ha Ca T phagocytosis may be an important mechanism for transfer of melanosomes.4. Flow cytometry shows an decrease in fluorescent microspheres in HaCaT after m Glu R6 downward, and proved m Glu R6 may be an important protein to transfer of melanosomes. This may provide a possible new way to treat pigmentation diseases.