Background Studies indicated that Mer might be the main contributor to the specific internalization of photoreceptor outer segments （POS） in retinal pigment epithelium （RPE）. It is very important to understand the...Background Studies indicated that Mer might be the main contributor to the specific internalization of photoreceptor outer segments （POS） in retinal pigment epithelium （RPE）. It is very important to understand the mechanism of POS phagocytosis under the pathway of Mer and its ligands. The objective of this study was to identify changes in gene expression profiles caused by Mergene knockout （Mer-/-） during phagocytosis of POS in RPE. Methods RPE from both Mer-/- and wild-type （WT） mice were isolated and cultured to the 3rd passage. POS were subjected to culture medium with 20 nmol/L Gas6 and protein S to activate specific mer-mediated phagocytosis. RPE phagocytosis was evaluated by phagocytosis assays and differential gene expression identified by microarray at 3 and 12 hours; the 0-hour time point served as the control. Three independent samples for each Mer-/- or WT RPE were subjected to the same protocol of microarray. Five genes were confirmed by real-time quantitative PCR （QPCR）. Results The Mer-/- RPE had less internalized POS than WT RPE after both 3 and 12 hours in phagocytosis assay. Compared to WT RPE and the 0-hour control, 38 and 45 different known genes were increased and 68 and 59 known genes were decreased in Mer-/- RPE after 3 and 12 hours, respectively. Abnormal POS phagocytosis in Mer-/- RPE was associated with significant gene expression changes in, for example, signal transduction （WNT, MAPK）, phagocytosis （Vav3, Hsdllb1）, cytoskeleton components （Myo7a）, and metabolism, in a time-specific manner. QPCR results showed Vav3, Hsdllb1, Myo7a, Rtn2 and Itga8 in those independent samples were consistent with microarray. Conclusion Gene expression profiles modulated in a time-specific manner in Mer-/- RPE indicate a possible internalization mechanism for abnormal POS phagocytosis, which gives insight into the mechanism of retinitis pigmentosa caused by the mutation of MerTK in humans.展开更多
文摘Background Studies indicated that Mer might be the main contributor to the specific internalization of photoreceptor outer segments （POS） in retinal pigment epithelium （RPE）. It is very important to understand the mechanism of POS phagocytosis under the pathway of Mer and its ligands. The objective of this study was to identify changes in gene expression profiles caused by Mergene knockout （Mer-/-） during phagocytosis of POS in RPE. Methods RPE from both Mer-/- and wild-type （WT） mice were isolated and cultured to the 3rd passage. POS were subjected to culture medium with 20 nmol/L Gas6 and protein S to activate specific mer-mediated phagocytosis. RPE phagocytosis was evaluated by phagocytosis assays and differential gene expression identified by microarray at 3 and 12 hours; the 0-hour time point served as the control. Three independent samples for each Mer-/- or WT RPE were subjected to the same protocol of microarray. Five genes were confirmed by real-time quantitative PCR （QPCR）. Results The Mer-/- RPE had less internalized POS than WT RPE after both 3 and 12 hours in phagocytosis assay. Compared to WT RPE and the 0-hour control, 38 and 45 different known genes were increased and 68 and 59 known genes were decreased in Mer-/- RPE after 3 and 12 hours, respectively. Abnormal POS phagocytosis in Mer-/- RPE was associated with significant gene expression changes in, for example, signal transduction （WNT, MAPK）, phagocytosis （Vav3, Hsdllb1）, cytoskeleton components （Myo7a）, and metabolism, in a time-specific manner. QPCR results showed Vav3, Hsdllb1, Myo7a, Rtn2 and Itga8 in those independent samples were consistent with microarray. Conclusion Gene expression profiles modulated in a time-specific manner in Mer-/- RPE indicate a possible internalization mechanism for abnormal POS phagocytosis, which gives insight into the mechanism of retinitis pigmentosa caused by the mutation of MerTK in humans.
