| Diabetic retinopathy is the common and severe microvascular complication of diabetes. It has been suggested that the functional loss and the death of neurons in the diabetic retina can be attributed to reactive changes in Müller cells. Retina Müller cell is the specialized glial cell, it spans the entire thickness of retina, ensheaths all retinal neurons, maintains retinal structure and function, and participates in many retinal pathology processes. Calcium ions distribute in intracellular and extracellular, play important physiological function, [Ca2+]i is important regulation factor of cell growth and signal transmission, it is important material foundation of cell physiological function, and center step of many signal transmission processes after acceptor excited, it has been suggested that retinal calcium ions concentration is highly related with diabetic course. At present, oral antidiabetic drugs and insulin are main methods for treating diabetes. High concentration glucose is one of the important risk factors of DR, the development of DR is correlated with blood glucose. Insulin is a kind of ectogenic treatment, it promotes diabetes develop at early time of application, and it has been suggested that insulin can stimulate DNA synthesis of microvascular endothelial cells, accelerate proliferation of endothelial cells, promote formation of vascular bud, induce neovascularization, and high concentration insulin can induce VEGF expression in Müller cells, then accelerate neovascularization in diabetes. Consequently, this experiment observed effects of high concentration glucose and high concentration insulin on [Ca2+]i in cultured retina Müller cells in vitro, expected to illuminate their effects in diabetes, accordingly provide new ideas forits prevention and cure.Objective:To observe the effects of high concentration glucose and high concentration insulin on [Ca2+]i in cultured rabbit retinal Müller cells in vitro.Methods:Tissue suspension culture method was used to establish the primary culture system. Müller cells were cultured with high concentration glucose and high concentration insulin for different time (1day, 3days, 5days). Fluorescent probe and laser confocal microscopy were conducted to quantitatively assay the changes of [Ca2+]i in Müller cells cultured with normal, high concentration glucose, high concentration insulin, high concentration glucose and insulin in vitro on the different conditions of high calcium and Ca2+ channel blocker outside the cells.Results:1. According to the results of phase contrast microscope, HE stain, immunohistochemistry stain, immunofluorescence stain and transmission electron microscope, the purity of Müller cells was more than 95%. Müller cells were fusiform, in radiation arrangement, growing slowly. By GFAP immunohistochemistry stain, There were buffy silk screen structures in kytoplasm. By GFAP immunofluorescence stain with FITC, they were almost positive. By transmission electron microscope, cell nucleus was big and anomalous with two or more nucleoli, there were glycogen granule and diagnostic intermediate filament(8~10nm) in kytoplasm. 2. According to the results of fluo-3 fluorescent probe, there were calcium ions with green fluorescence in retina Müller cells, the calcium ion concentration depended on its intensity.①The [Ca2+]i of Müller cells cultured with high concentration glucose, high concentration insulin, high concentration glucose and insulin were higher than normal (P<0.01). At 1st day and 3rd day the [Ca2+]i of Müller cells cultured with high concentration glucose and insulin was higher than that with high glucose, but at 5th day it was lower than that with high concentration glucose.②On the condition of high calciumoutside the cells, the [Ca2+]i of Müller cells cultured with normal nutrient solution decreased (P<0.01), the [Ca2+]i of Müller cells cultured with high concentration glucose, high concentration insulin, high concentration glucose and insulin increased (P<0.01) but still higher than normal. At 1st day and 3rd day the [Ca2+]i of Müller cells cultured with high concentration glucose and insulin was higher than that with high concentration glucose, but at 5th day it was lower than that with high concentration glucose.③On the condition of Ca2+ channel blocker outside the cells, all of them decreased (P<0.01), the [Ca2+]i of Müller cells cultured with high concentration glucose, high concentration insulin, high concentration glucose and insulin were still higher than normal. At 1st day and 3rd day the [Ca2+]i of Müller cells cultured with high concentration glucose and insulin was higher than that with high concentration glucose, but at 5th day it was lower than that with high concentration glucose.Conclusions : 1. The effects of high concentration glucose, high concentration insulin on [Ca2+]i in cultured retinal Müller cells in vitro are obviously, [Ca2+]i of Müller cells increased. 2. At 1st day and 3rd day, the [Ca2+]i of Müller cells cultured with high concentration glucose and insulin was higher than that with high concentration glucose, but at 5th day it was lower than that with high concentration glucose, the decrease showed the efficacy of insulin for long-term application. 3. Ca2+ channel blocker made the [Ca2+]i of Müller cells cultured with normal, high concentration glucose, high concentration insulin, high concentration glucose and insulin all obviously decreased,it effectively controlled [Ca2+]i by interfering extracellular calcium ions enter cells, and it maybe researched and applicated in retinopathy in the future.
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