| BACKGROUNDDiabetic retinopathy (DR) is a serious complication of diabetes, in recent years, with the increasing of diabetic patients year by year, the number of patients suffered from diabetic retinopathy is more and more. As one of the major blinding eye disease, it is very important to have research and study on the pathogenesis. In most studies, the mechanisms of vascular diseases and metabolic factors are more generally accepted, we take more and more attention to the immune factors.AGEs is a special product in diabetic patients. It is formed in the environment of high blood glucose. The proteins have non-enzymatic glycosylation reaction slowly. These proteins which have a longer half life are more affected, such as collagen, lens protein. At the same time the proteins are effected by the blood glucose concentration and the products are difficult to degrade. The formation of AGEs is a slow process. The results show that there may be extensive leakage of blood retinal barrier when the non-diabetic animals were injected AGEs. Eventually the inflammatory immune response were triggered, causing vascular occlusion. The microcirculation disorder were occurred. Animal studies showed that the performance of early diabetic retinopathy similar to that of the low level of chronic immune and inflammatory damage. The anti-inflammatory drugs or inflammatory cytokines, such as corticosteroids, may reduce the inflammatory response. AGEs is the important factor in the diabetic retinopathy, but its role in the immune inflammation remains unclear.The retina is formed of different types of cells. Microglia cell is a kind of glial cell, it is the first line to against pathogens invading to the CNS.It is considered to be the representative immune cells in CNS。Retina is a continuation of CNS, microglial cells also exist in the retina. So retinal microglia is inherent immune cells in the retina. The retinal pigment epithelium (RPE) forms the outer retinal barrier. The current research on the function and immunological characteristics of RPE is unclear。T lymphocytes is not the inherent cells in retina,but it plays a crucial role in the immune response. In recent years, we realize that advanced glycation end products plays a very crucial role in diabetic retinopathy。So how did AGEs affected the retinal microglia, retinal pigment epithelial cell and T lymphocytes, it is not yet clear。We have preliminary study on the immune mechanisms of diabetic retinopathy.1 The microglia cells and retinal pigment epithelial cells stimulated by AGEs may play the role of antigen presenting cells.2 The microglia cells play a stronger role in antigen presentation than retinal pigment epithelial cells.3 At the stimulation of AGEs, the T lymphocyes were cultured with retinal microglia cells or retinal pigment epithelial cells. The activity of T lymphocytes is better, when T lymphocytes were cultured with retinal microglial cells. 4. We found that, Activated T lymphocytes may differetiate to TH1, promoting the immune response.OBJECTIVE1)To investigate the expresstion of major histocompability comlexⅡ(MHC-Ⅱ), co-stimulatory molecule B7.1/CD80, B7.1/CD86 and ICAM-1 on retinal microglial cell after being stimulated by AGEs.2))To investigate the expresstion of major histocompability comlexⅡ(MHC-Ⅱ),co-stimulatory molecule B7.1/CD80, B7.1/CD86 and ICAM-1 on retinal epithelial cell after being stimulated by AGEs.3) To investigate the effect of AGEs on T lymphocyte activation in immune response process. And To investigate the expresstion of major IFN-γ, IL-2, IL-4, IL-10 on T lymphocyte after being stimulated by AGEs.4)To investigate the effect of AGEs on T lymphocyte activation in the co-culture system of RPE cells and T cells.5) To investigate the effect of AGEs on T lymphocyte activation in the co-culture system of retinal microglial cells and T cells.METHODS1) Microglial cells were cultured with AGEs of different concentration (0,10,50,100,500 ug/ml) respectively. The mRNA expression of MHCⅡ, CD80, CD86, ICAM-1 was measured by RT-PCR. And on the protein level, the expression of MHC II,CD80, CD86, ICAM-1 was also measured by Western-Blot.2) RPE cells were cultured with AGEs of different concentration (0,10,50,100,500 ug/ml) respectively. The mRNA expression of MHCⅡ,CD80, CD86,ICAM-1 was measured by RT-PCR. And on the protein level, expression of MHCⅡ,CD80, CD86,ICAM-1 was measured by Western-Blot.3) T lymphocyte cells were cultured with AGEs of different concentration(0,10,50,100,500 ug/ml) respectively in vitro, and then the expression of activated lymphocytes CD69 and CD3 was measured by fluorescence activated cell soter(FACS). T lymphocyte cells were cultured with AGEs of different concentration (0,10,50,100,500 ug/ml) respectively. And on the protein level, expression of IFN-γ, IL-2, IL-4, IL-10 was measured by Western Blot.4) T lymphocyte cells were co-cultured with retinal microglial cells,and with AGEs of different concentration (0,10,50,100,500 ug/ml) respectively in vitro, and then the expression of activated lymphocytes CD69 and CD3 was measured by fluorescence activated cell soter(FACS).5) T lymphocyte cells were co-cultured with RPE cells,and with AGEs of different concentration (0,10,50,100,500 ug/ml) respectively in vitro, and then the expression of activated lymphocytes CD69 and CD3 was measured by fluorescence activated cell soter(FACS).RESULTS1) Compared with control group, AGEs up-regulated the expression MHC-Ⅱ, CD80, CD86, ICAM-1,contrast to control group(P<0.05). On protein level, Western Blot analysis demonstrated that AGEs increased MHC-Ⅱ, CD80, CD86, ICAM-1 proten quantity, contrast to control group.2) Compared with control group, AGEs up-regulated the expression MHC-Ⅱ, ICAM-1 contrast to control group(P<0.05). On protein level, Western Blot analysis demonstrated that AGEs increased MHC-ⅡICAM-1 proten quantity, contrast to control group. However the expression of CD80, CD86 mRNA and protein quantity has no significant difference with that in control group.3) Increasing amount of CD69 and CD3 positive lymphocytes were found in co-cultured with AGEs of different concentration (0,10,50, 100,500 ug/ml), compared with contral group. On protein level, Western Blot analysis demonstrated that AGEs increased IFN-y,IL-2 protein quantity, contrast to control group. However the expression of IL-4,IL-10 has no significant difference with that in control group.3) In the co-cultured system of retinal microglial cells and T cells, AGEs of different concentration (0,10,50,100,500 ug/ml) up regulated the amount of CD69 and CD3 positive lymphocytes.4) In the co-cultured system of RPE cells and T cells, AGEs of different concentration (0,10,50,100,500 ug/ml) up regulated the amount of CD69 and CD3 positive lymphocytes.CONCLUSION1) The property of microglial behaviors depend on the local microenvironment. Appropricate regulation by AGEs may up-regulated the antigen presenting function of microglial cells.2) The property of RPE cells behaviors depend on the local microenvironment. Appropricate regulation by AGEs may up-regulated the antigen presenting function of RPE cells.3) T-lymphocytes were activated by AGEs with appropricate regulation. T-lymphocytes were activated by human MG cells or RPE cells which is antigen presenting cells with immunological characteristics potential. |
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