| Retinitis pigmentosa, the most common cause of genetically visual impairment and blindness, is a group of sexy photo cell function characterized by the loss of inherited retinal degenerative diseases. Royal College of Surgeons rat is a classic study model of retinitis pigmentosa, in which the retinal pigment epithelium does not play its phagocytic function, and the phagocytic dysfunction will lead to the aging rod outer segment membrane disc can not be swallowed up in time. Therefore a large accumulation is formed in the subretinal space, causing visual impairment, resulting in the loss of their physiological function, and finally the development of RP is increased. Retinal glial cells are functioned as "street sweeper" during retinal development and degeneration from the role clearance of apoptotic cells. Therefore, microglia is enhanced to be easily identified; phagocytosis apoptotic rod cells and apoptotic cells can reduce the toxic effects of the adjacent tissue to maintain the stability of retinal micro-environment so as to effectively clear the accumulation in the subretinal space of apoptotic cells can protect the health of the cones to protect the role of central vision remaining.MFG-E8 was firstly discovered by the phagocytic cells in lymphoid organs, recent studies found in the central nervous system macrophages/microglia cells in MFG-E8 expression. We cultured rat microglia cells were also detected in MFG-E8 expression, suggesting that RP disease process, MFG-E8 is involved in the regulation of retinal phagocytic behavior of microglial cells, but the adjustment mechanism is also through the CrkⅡ-DOCK180-Racl signaling pathway to complete. However, no such pioneer scholars have involved in the issue or the uroimmunomodulation. If the phagocytic function of microglia in regulation does have the participation of MFG-E8, then joined the MFG-E8 exogenous is likely to enhance its phagocytosis, thereby to protect the remnants of photoreceptor cells, which is of positive importance to the RP therepy. Objective:1) To study the law implied in the activation process of the microglia cells in the RCS rat retinal pigment degeneration.2) To study the law of The MFG-E8 in the RCS rat retinal pigment degeneration.3) The MFG-E8 and the relationship between retinal glial cells.4) The CD11b, MFG-E8 and the related cytokine mRNA in retinal development and degenerative changes of the expression of the process. Methods:1) Normal royal college of surgeon (RCS) rats were divided into P14, P35, P60, P90 groups according to their postnatal day. Stain of microglial cells marker (CD11b) was performed by immunofluorescence.2) Normal royal college of surgeon (RCS) rats were divided into P14, P35, P60, P90 groups according to their postnatal day. Stain of MFG-E8 was performed by immunofluorescence.3) Normal royal college of surgeon (RCS) rats were divided into P14, P35, P60, P90 groups according to their postnatal day. Double stain of MFG-E8 and microglial cells marker (CD11b) was performed by immunofluorescence.4) Expressions of MFG-E8, integrin av, integrinβ5, CDllb, TNF-a, interleukin-1β(IL-1β) and MCP-1 mRNA in the neural retina were analyzed by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). Results:1) Retinal immunofluorescence staining showed that CD11b-positive cells of rats aged 14 d of the RCS rat are distributed in the inner retina, mainly of which are in the retinal ganglion cell layer (RGCL) and the inner plexiform layer. In the kernel layer a small amount of CD11b-positive cells is located. CD11b positive cells of Marine age of 35d of the RCS rat mainly found in the retinal inner plexiform layer, a small amount of cell is migrated to the outer nuclear layer, which are clearly visible positive cells. Murine age of 60 d of the RCS rat CD11b-positive cell migration to the outer nuclear layer, outer nuclear layer was activated. Murine age of 90d of the RCS rat CDllb positive cells in outer nuclear layer, which shows obvious activation.2) Retinal immunofluorescence staining showed that MFG-E8-positive cells of rats of the RCS 14d of age in rats are located in the inner retina, mainly of which are in retinal ganglion cell layer (retinal ganglion cells layer, RGCL) and the inner plexiform layer. The age of 35 d mouse RCS rats MFG-E8-positive cells were mainly distributed in the retinal inner plexiform layer and inner nuclear layer, outer nuclear layer shows few positive cells. Murine age of 60d of the RCS rat, the outer nuclear layer shows a large number of MFG-E8-positive cells. Murine age of 90d of the RCS rat, the outer nuclear layer was visible outside the MFG-E8-positive cells.3) Retinal immunofluorescence staining, MFG-E8 in 14,35,60,90d positive cells were stained with CD11b same site.4) Real-time PCR results showed that in normal rats and RCS rat neural layer of retina during embryonic development (PO-14), MFG-E8 mRNA expression levels of the is very low at birth but it would be increased and expanded gradually in later stages. P14 mRNA has the strongest expression, and compare to PO, the difference was of statistical significance (P<0.05). Normal rat retinal development in the mature nerve layer, compare to PO, the difference between the two was of no statistical significance (P> 0.05). The RCS rat retinal degeneration in the nerve layer process (P14-P90), its mRNA expression increased gradually; P30, P45 and P0, the difference was statistically significant (P <0.05); P60 mRNA in the expression reached a peak, compare to P0, the difference was statistically significant (P<0.05); P90 and P0, the difference was statistically significant (P<0.05). Integrin av and P5 with the MFG-E8 in the mRNA have the same expression. 5) Real-time PCR results showed that in normal rats and RCS rat neural layer of retina during embryonic development (P0-P14), CD11b mRNA expression levels of the early low at birth and gradually increase. P7-P14 of mRNA expression is most intense, and PO, the difference was statistically significant (P<0.05). Normal rat retina in the nerve layer mature (P14) after, MFG-E8 mRNA expression level of P0, the difference was not statistically significance (P> 0.05). The RCS rat retinal degeneration in the nerve layer process (P14-P90), its mRNA expression increased gradually. P30, P45 and P0, the difference was statistically significant (P<0.05). P60 mRNA expression reached a peak, and P0, the difference was statistically significant (P<0.05); P90 and P0, the difference was statistically significant (P<0.05). Inflammatory factor TNF-α(tumor necrosis factor), IL-1β(IL-1-β) and the chemokine MCP-1, its mRNA expression changes were consistent with the expression of CD11b mRNA. Conclusion:1) Retina microglia cells in the RCS rat retinal pigment degeneration process was obviously activated, and outwardly migrates to the outer layer.2) In the RCS rat retinal pigment degeneration process,MFG-E8 at the protein level and mRNA expression levels were associated with activation of retinal microglia, which are consistent with law, as well as the affirmed the MFG-E8 expression in the retina, and help to further define the expression in the nerve layer of retina microglia cells.3) In the RCS rat retinal pigment development and degeneration process, integrin av,β5, and MFG-E8 and CD11b (microglia-specific marker) mRNA showed changes of consistency, suggesting that:MFG-E8 retinal nerve layers mediated phagocytosis of microglia, and possibly through CrkⅡ-DOCK180-Rac1 signaling pathway phagocytosis of apoptotic cells.4) Factors Related to Inflammation in TNF-α, IL-1β, MCP-1 of mRNA expression and CD11b (microglia-specific marker) expression changes of the same, suggesting that in the RCS rat retinal pigment degeneration process, leading to small glial cell-mediated inflammatory response.
|
PDF Full Text Request