【作者】 王玲；

【导师】 王文吉； 莫晓芬； 徐格致； 江睿；

【作者基本信息】 复旦大学 ， 眼科学， 2005， 博士

【摘要】 目的：观察胰岛素原C肽对体外培养的视网膜Müller细胞和微血管内皮细胞的影响，及其与胰岛素的相互作用。并通过在体研究，观察早期单独应用生理剂量的重组人胰岛素原C肽对实验性糖尿病大鼠视网膜表达血管内皮生长因子（VEGF）及一氧化氮合酶（NOS）活力和一氧化氮合成（NO）的影响，及其对血-视网膜屏障功能的影响，探讨其作为预防糖尿病视网膜病变药物的前景。方法：（1）酶消化法分离培养大鼠视网膜Müller细胞及牛视网膜微血管内皮细胞。免疫细胞化学法及透射电镜鉴定细胞性质。培养细胞各组分别添加不同浓度的胰岛素原C肽或／和胰岛素。硝酸还原酶法测定上清NO含量。ELISA法测定上清VEGF含量。（2）链脲佐菌素诱导糖尿病大鼠模型。实验动物分三组，糖尿病组、糖尿病应用C肽组（C肽剂量：130nmol／KgBW皮下注射2次／日）、正常对照组，SPF环境饲养八周，每周测定体重及血糖。免疫组织化学法对眼球切片作视网膜VEGF表达的定性检测，ELISA法对视网膜匀浆作VEGF含量的定量检测。分型测定视网膜NOS活力。硝酸还原酶法测定视网膜匀浆NO含量。伊凡斯蓝示踪法测定血-视网膜屏障功能。结果：（一）离体试验：（1）C肽抑制Müller细胞NO合成：胰岛素组平均NO浓度明显高于正常对照组（t=4.111，P＜0.01，n=6）。C肽组低于正常组（t=2.932，P＜0.05）。联合应用组与对照组无差异（t=1.891，P＞0.1）。C肽作用与浓度有关。（2）单独应用胰岛素原C肽对Müller细胞表达VEGF无影响（F=0.58，P＞0.05），但等摩尔浓度(10^-5mol／L)的C肽联合胰岛素应用时，可以抑制胰岛素引起的VEGF表达增高，两者之间的交互作用有显著意义（F=7.50，P＜0.05）。（3）10^-5胰岛素使BREC合成NO增加（t=4.922，P＜0.001）；10^-5mol／L的胰岛素原C肽与胰岛素联合应用使BREC合成NO水平降低至胰岛素组与正常组之间(vs 10^-5M胰岛素组：t=2.318，P＜0.05；vs对照组：t=2.806，P＜0.02)；浓度10^-9-10^-5M的胰岛素原C肽可以抑制BREC合成NO，各浓度间无差异（P＞0.05）。（二）在体试验：（1）糖尿病组与糖尿病应用C肽组大鼠血糖维持在24.14-33.3mol／L的高水平，体重不随周龄增长，正常对照组大鼠血糖波动于正常范围，体重稳步增长。（2）三组视网膜均有VEGF阳性表达，阳性颗粒主要位于神经节细胞层与近内核层，其中糖尿病组视网膜内核层阳性颗粒密集，其他两组较少。三组视网膜VEGF含量分别为，正常对照组4.30±1.012，糖尿病组10.63±2.743，糖尿病C肽组7.49±2.262。三组间差别有统计意义（F=13.604，P＜0.01）。（3）视网膜匀浆的平均NO含量分别为：糖尿病组0.115±0.023，糖尿病用C肽组0.079±0.013，正常对照组0.042±0.017（μmol／gprot）。三组间差别有极显著意义（F=16.63，P＜0.01）。糖尿病组平均iNOS 2.662±0.355，cNOS 2.556±0.260，糖尿病用C肽组平均iNOS2.238±0.367，cNOS3.286±0.383，正常对照组平均iNOS 1.997±0.292，cNOS4.063±0.636（U／mgprot），三组间差别均有统计意义（iNOS：F=6.877，P＜0.01；cNOS：F=9.892，P＜0.01）。（4）三组视网膜EB渗漏分别为，糖尿病组186.74±16.00，糖尿病用C肽组141.47±33.48，正常对照组32.02±11.45，三组间差异有极显著的统计意义（F=47.17，P＜0.01）。结论：（1）联合应用等摩尔胰岛素原C肽与胰岛素可以纠正胰岛素引起的视网膜Müller细胞NO合成过度。（2）联合应用等摩尔胰岛素原C肽与胰岛素可以纠正胰岛素引起的Müller细胞VEGF表达过度。（3）胰岛素原C肽可以纠正胰岛素引起的牛视网膜微血管内皮细胞合成NO过度。（4）早期单独应用胰岛素原C肽可以部分改善糖尿病引起的视网膜VEGF高表达，调节视网膜NOS活力，部分抑制视网膜NO过度合成，并部分恢复血-视网膜屏障功能、减少白蛋白渗出。更多还原

【Abstract】 Objective: To investigate the effects of equal mole of insulin and proinsulin C-peptide on the functions of cultured rat retinal Muller cells and bovine retinal microvascular endothelial cells. And to evaluate the effects of proinsulin C-peptide on the vascular endothelial growth factor （VEGF） expression, the nitric oxide （NO） synthesis of diabetic rat retina and its protective effect on the blood-retinal barrier （BRB） function. Methods: （1） Rat retinal Muller cells and bovine retinal microvascular endothelial cells were cultured by enzyme digest method. Immunocytochemistry and electronic microscopy were used for cell identification. Different concentrations of insulin, proinsuling C-peptide were added to the cells separately or combined 24 hours before measuring. The nitric oxide level of the supernatant was measured by nitrate reductase method and VEGF concentration by ELISA. （2） Diabetic rats were induced by streptozotocin intravenously injected of 50mg/Kg body weight. All the animals （diabetic group, C-peptide interfering diabetic group, and control group） were fed in a specific pathogen free environment for eight weeks. The dose of C-peptide was 130nmol/Kg body weight by subcutaneous injection twice a day. The blood glucose level and the body weight were measured every week. Immunohistochemistry method was used to qualitatively detect the VEGF reaction on the retinal section. The VEGF concentration was measured by enzyme-linked immunoabsorption assay （ELISA） method. The vitality of different type of nitric oxide synthase was measured separately and NO level was measured by nitrate reductase method. And the BRB function was measured by Evans blue quantitatively.Results: In vitro: （1） The retinal Muller cells NO content was increased by insulin （p<0.01） and decreased by C-peptide （P<0.05） in the concentration of 10^-5M. And the efficacy of C-peptide correlated with the concentration. （2） The VEGF expression was increased by insulin, and decreased to normal level by combined using equal mole of insulin and C-peptide in cultured Muller cells. While it was not changed by C-peptidesolely. （3） The NO over synthesis caused by insulin （t=4.922, P<0.001） in BREC could be corrected by combined with equal mole of proinsulin C-peptide （vs Insulin: t=2.318, P<0.05; vs control: t=2.806, P<0.02）. There is no statistically significant difference among different concentration of C-peptide （P>0.05）. In vivo: （1） The blood glucose was maintained in a very high level ranged from 24.14 to 33.3mol/L in the two diabetic groups （with/without C-peptide interfering） and the body weight was not increased with the age. While the blood glucose level was normal and the body weight was growing steadily in control group. （2） The VEGF immuno-reaction was detectable on the retinal ganglion cell layer and near the inner nuclei layer in all three groups. And the positive reaction was more distinctive in the diabetic without C-peptide interfering group than the other twos. The difference of VEGF concentrations among the three groups was statistically significant （F-13.604, P＜0.01）. The C-peptide interfering diabetic group had the concentration that is lower than diabetic group （P<0.05） but higher than control group （P<0.05）. （3） The C-peptide group had the vitality of eNOS, iNOS and NO content between the other two groups. （4） The Evans blue exudation were 186.74±16.00 in diabetic group, 141.47±33.48 in C-peptide group, and 32.02±11.45 in control group. The EB level in C-peptide group was lower than diabetic group （P<0.05） but still higher than control group （P<0.01）.Conclusion: （1） The nitric oxide over synthesis in Muller cells caused by insulin can return to normal level by combined using equal mole of insulin and proinsulin C-peptide. （2） The VEGF over expression in Muller cells caused by insulin can also return to normal by combined using. （3） The NO over synthesis caused by insulin in BREC could be corrected by combined with equal mole of proinsulin C-peptide. （4） Early treatment of proinsulin C-peptide solely can partially inhibit the VEGF over expression, regulate NOS vitality, inhibit NO over synthesis, ameliorate the albumin exudation in diabetic rat retina, and restore the blood-retinal barrier function.更多还原