| Background and Objective:With the development of science and technology to raise the level of diagnosis and treatment for diabetes, patients with diabetes prolonged and suffuered with complications of diabetes increased accordingly. The level of eye disease caused by diabetes is up and up, and the incidence of blind eyes in DR is the first, from the 1960s and continue to today's retinal laser photocoagulation is as inhibiting angiogenesis and macular edema which is the most commonly and effective method, but its damage to retina is irreversible. So deeply understand the DR angiogenesis mechanism to prevent its development has become the focus of research on DR. DR happened because diabetes is caused by the body's blood anoxic environment, causing increased oxidative stress, oxygen free radicals, oxidation product accumulation, damage the retinal tissue, and vascular lesions in capillaries, which cause the formation of new blood vessels. The angiogenesis is closely related to various growth factors ,such as VEGF, bFGF PDGF, etc, the VEGF is the most valued, at home and abroad research, many studies shows that the VEGF is the main pathogenic factor. The study showed that cells secreted VEGF in retina include Müller cells and endothelial cells, etc, and in which Müller cells play a dominant role. And it is also useful in the cell two-way signaling between neurons, participating in the retina information processing, and they are the only cells in retina which can synthesize glutamine synthase(GS) to eliminate the toxicity to neurons caused by high concentration glutamic acid. Once the GS was inhibited, corresponding neurons functions were lost. In the early of DR, Müller cells will occur a series of changes from ultrastructure to physiological function, and this abnormal structure and function affect early abnormal function of nerve cells, patients with diabetes, in the early Müller cells will show increasig expression of GFAP and VEGF which affect the whole progression of DR. So slow or reverse its structure and function abnormal become the focus in prevention and treatment of DR.The experiment will use Müller cells to charify whether zinc supplement can inhibit abnormal secretion of VEGF in Müller cells, in order to prevent and cure DR.Research contents and methods:(1) The cultured of Müller cells.(2) The appraisal of Müller cells: The cells has been identified by electron laboratory to be rats retinal Müller cells by Song E director laboratory.(3) Experimental group: Design of normal, high glucose, high glucose with zinc and normal glucose with zinc groups, normal concentration of glucose is 5mmol/L, high glucose concentration of glucose is 25mmol/L, high glucose with zinc contain zinc sulfate in the medium are 2.5μmol/L, 5μmol/L, 7.5μmol/L and 10μmol/L, normal glucose with zinc group containing zinc sulfate in the medium is 5μmol/L.(4)Time effect: Under the conditions of normal and high glucose concentration (25mmol/L), cultivate retinal Müller cells in a 24 hole boards, altogether divides five groups, and five holes, respectively in the 1d, 2d, 3d, 4d, 5d and 6d through immunohistochemistry determination to find the time achieve the highest concentration of VEGF.(5)The relationship between Zinc and Müller cells secretion of VEGF and dose effect: In high glucose with zinc groups, determine VEGF secretion in time effect point to make clear the relationship between zinc and secretion of VEGF, and charify the dose of the biggest influence and whether it has toxic effects.(6) Immunofluorescence cytochemistry(SP):The cells in the cytoplasm and cell membrane, nuclear parts appear yellowish-brown particles considered to be positive(7)Image collection, data sorting and statistically analysis.Results:VEGF immunohistochemical staining showed that: VEGF of negative controled Müller cells dose not express. Compared with the high concentration glucose group the expression of VEGF in Müller cells cultured in normal group, on 1, 2, 3, 4, and 5 day the Müller cells VEGF level is significantly higher, on 6 day come down to the basic as normal,in the fourth day gets to the peak,they have significant differences(P<0.01). The high glucose group is higher than the zinc supplementing group on 1, 2, 3, 4, and 5 day, and become equal even below zinc supplementing group on the sixth day, they have significant differences (P<0.01). The normal group , high glucose with zinc supplementing group and normal glucose with zinc supplementing group do not have statistical differences (P>0.05). Conclusions:(1) The secretion of VEGF in Müller cells in the condition of high glucose is obviously higherand has time dependence, getting the peak on the fourth day.(2) Adding zinc groups has inhibition effect on VEGF secretion in Müller cells, we speculate zinc supplement has prevention and cure function to DR.(3) The normal group , high glucose with zinc supplementing groups and normal glucose with zinc supplementing group do not have statistical differences, showed that the concentration of zinc has to Müller cells cultured in vitro. |
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