| Objective: With changing lifestyles and the extension of human life, the incidence of diabetes increased, The data from the Chinese Guideline of Type 2 Diabetes Prevention and Treatment(forum of 2010)shows: at present, the overall incidence of diabetes has reached 9.7% ,and the incidence of pre-diabetes is 15% , the number of the latter has more than 1.48*108, which is higher than the level of Europe and America, China now has more than 9.24*107 diabetes and has become a global superpower of diabetes. In the coming decades diabetes is still a serious public health diseases. As a chronic disease, diabetic complications can be divided into two kinds of acute complications and chronic complications, diabetic retinopathy (DR) as one of the chronic complications; the harm which cannot be ignored can cause decreased vision and irreversible loss of vision. Now that the incidence of diabetic retinopathy is the result of retinal tissue hypoxia and impaired microcirculation, its development is closely related to the degree of glycemic control and diabetes duration, the longer the duration, the higher the incidence rate of the DR. Current drug treatment of diabetic retinopathy is not yet mature, and laser photocoagulation therapy is generally acceptable to delay the development, but its role in improving eyesight is weak. In order to find effective approach to delay the development of diabetic retinopathy, it is imperative to understand its pathogenesis. The current research suggest that the incidence of DR is related to these factors, as follows :glucose metabolism disorders, polyol - inositol pathway abnormalities, non-enzymatic glycosylation, protein kinase C activation, vasodilation prostate product, hemodynamic changes, decreased activity of superoxide dismutase and the changes of the growth factors'level in the retina ,especially vascular endothelial growth factor (VEGF) is growing concern. As a growth factor,VEGF has the role of increasing the vascular permeability, and is considered as the known strongest selective endothelial cell mitogenic factor and vascular endothelial growth factor. Variety of cells in vivo, including retinal pigment epithelial cells can secrete VEGF.In normal circumstances, a small amount of VEGF can be secreted by retinal pigment epithelial cells, high glucose can promote the secretion of VEGF within the retina, studies have confirmed that VEGF is closely related with the DR. Li Cai Rui, etc. [1] discussed the VEGF expression in the serum and retinal of non-obese diabetic mice, and discussed the relationship between the two, it was found that with the development of diabetes, the diabetic retinopathy aggravated, and the serum VEGF levels increased, and the longer the duration, the higher the VEGF levels in serum ,this result revealed that it had significantly correlated between the VEGF content and duration of diabetes. This result was consistent with the research result that the incidence of diabetic retinopathy in these patients whose duration of diabetes more than 10 years was 50%[2]. Blood glucose of the diabetic patients is not static, it will be changed with a number of factors, including poor control of postprandial hyperglycemia and the unreasonable application of insulin or insulin secretagogues[3]. With the increasing study of the high glucose's toxicity, the fluctuation of the high glucose is increasingly become a hot spot. This stutdy simulated the status of the acute blood glucose fluctuation in diabetic patients , compared the effects of intermittent between constant high glucose media on VEGF level in human retinal pigment epithelial (hRPE) cells ,so as to discuss whether the intermittent high glucose had the greater contribution to the development of diabetic retinopathy.Methods: Cultured human retinal pigment epithelial (hRPE) cells were divided into four groups, each with 6 holes. Experimental groups as follows: A normal control group: glucose concentration was 5.55mmol/L of DMEM culture medium; B constant high glucose group: glucose concentration was 25mmol/L of the DMEM culture medium; C intermittent high glucose group: two conditions alternating DMEM culture medium in which the glucose concentration were 5.55mmol/L and 25mmol/L respectively, alternate culture 2 hours in high glucose and 3 hours in normal glucose ,overnight in the high glucose; D hypertonic control group: the concentration of mannitol was 19.45mmol/L plus the glucose concentration was 5.55mmol/L of the DMEM culture medium. This four groups were cultured respectively.Cell morphology were observed at 24 hours, 48 hours and 72 hours .Cell supernatants were collected at 24 hours, 48 hours and 72 hours, and were saved at -20℃. The VEGF levels in the collected supernatant were detected by using the VEGF ELISA kit according to the instructions.Results:1 After 24h: the morphology of the cells in each group were observed over the previous form that did not change significantly .The VEGF levels in the supernatant were not significantly different among the four groups(P>0.05);2 After 48h: under inverted microscope the morphology of the cells did not change significantly compared with before and among each groups. The VEGF levels in the supernatant were not significantly different among the four groups (P>0.05);3 After 72h:Compared with the normal control group ,the hypertonic control group cells were still observed spindle or polygonal-based under the inverted microscope, and there was no significant difference on VEGF levels between the two groups (P>0.05);Compared with the normal control group ,the constant high glucose group cells were observed became thinner in the cell body, irregular cells increased, and the constant high glucose group had significantly higher VEGF levels, the difference of VEGF levels between the two groups was significant (P<0.001);Compared with the normal control group ,the intermittent high glucose group were also seen that the cell were thin and irregular, the intermittent high glucose group had obviously higher VEGF levels which was significant difference between this two groups (P<0.001);Compared with the constant high glucose group,the intermittent high glucose group had the more irregular cells and increased VEGF levels .The difference of VEGF levels between the two groups was statistically (P<0.001).Conclusion:1 The constant high glucose could cause human retinal pigment epithelial cells into thinner and irregular, while increasing the secretion of VEGF, this is consistent with previous studies;2 Compared with constant high glucose,the intermittent high glucose had greater effects on the cell morphology and the VEGF level which were secreted by human retinal pigment epithelial cells.This research suggested that glucose fluctuation may be further promote the development of diabetic retinopathy by increasing the secretion of VEGF within the retina.
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