糖尿病性视网膜病变发病相关机制研究

发布时间：2018-06-01 13:21

本文选题：糖尿病性视网膜病变 + 促红细胞生成素　；参考：《武汉大学》2011年博士论文

【摘要】：糖尿病性视网膜病变是以视网膜新生血管性增殖为特征的一种致盲性眼病,随着人们生活水平的提高,糖尿病日益成为我国威胁人类健康的常见多发病,与此相应,糖尿病性视网膜病变的发病也日益增多。迄今为止,糖尿病性视网膜病变的确切发病机制还不完全清楚,大量的研究认为视网膜新生血管形成的过程与酶、激酶的调节以及各种生长因子的作用有关,同时炎症及免疫反应也在此过程中扮演着重要的作用。第一部分增殖性糖尿病性视网膜病变患者血清及玻璃体样本中GRO-a及EPO的表达 [目的]通过检测糖尿病性视网膜病变(DR)患者的血浆、玻璃体样本内促红细胞生成素(EPO)、生长相关基因α(Gro-a)的含量,探讨其在DR发展变化中的可能作用。 [方法]该研究为病例对照研究。包括非增生型糖尿病性视网膜病变(NPDR)组、增生型糖尿病性视网膜病变(PDR)组与对照组。采用酶联免疫吸附试验(ELISA)检测各组血浆、玻璃体样本中EPO和Gro-a的含量。 [结果]血浆EPO含量：NPDR组(22.16±4.85) mIU/ml, PDR组(25.46±8.83)mIU/ml,对照组(23.52±7.27)mIU/ml。玻璃体EPO含量：PDR组(461.36±101.20)mIU/ml,对照组(36.78±10.19)mIU/ml。血浆Gro-a含量：NPDR组(81.95±38.08)pg/ml, PDR组(82.61±25.26)pg/ml,对照组(55.68±22.53) pg/ml。玻璃体Gro-a含量：PDR组(327.74±216.29) pg/ml,对照组(81.95±47.59)pg/ml。3组血浆样本间EPO含量差异无统计学意义(P0.05), Gro-a含量差异有统计学意义(P0.05)。3组玻璃体样本内PDR组与对照组2组间EPO、Gro-α差异均有显著统计学意义(P0.05)。各组血浆、玻璃体内EPO、Gro-α含量与相应糖化血红蛋白(HbA1c%)有不同程度正相关关系。 [结论]PDR患者血浆Gro-a含量,玻璃体EPO、Gro-a含量显著增高,EPO、Gro-a含量与患者HbA1c (%)值有关。第二部分GRO-a, EPO在糖尿病大鼠视网膜组织中的表达及塞来昔布对GRO-α、EPO表达的影响 [目的]观察GRO-a及EPO在糖尿病大鼠视网膜组织中的表达,以及选择性环氧合酶-2 (Cyclooxygenase-2, COX-2)抑制剂(塞来昔布)对糖尿病大鼠视网膜组织中GRO-α和EPO表达的影响。 [方法]将40只SD大鼠随机分为4组,即正常组、糖尿病组、糖尿病+蒸馏水灌胃组以及糖尿病+塞来昔布灌胃组,每组10只。通过腹腔注射链脲佐菌素(streptoz-otocin, STZ)诱导建立糖尿病大鼠动物模型,饲养12周后处死大鼠,取视网膜,SYBR荧光实时定量PCR检测视网膜组织中COX-2、血管内皮生长因子(vascular endothelial growth factor, VEGF)、EPO和GRO-a mRNA的表达变化,western-blotting检测大鼠视网膜中COX-2、VEGF、EPO和GRO-a蛋白的表达变化。通过Quantity One凝胶图像分析软件进行结果分析,行ANOVA方差分析,SNK法做组间两两比较,以P0.05作为差异有统计学意义。 [结果]12周后,COX-2, VEGF, EPO及GRO-a mRNA和蛋白质的表达在糖尿病组和糖尿病+蒸馏水组最高,糖尿病+塞来昔布灌胃组高于正常组而低于糖尿病组。糖尿病组和糖尿病+蒸馏水组间差异无统计学意义(P0.05),其余各组间比较均有显著性差异(P0.05)。 [结论]EPO和GRO-a在糖尿病大鼠视网膜新生血管生成中起到了重要作用,塞来昔布可能可以通过抑制糖尿病大鼠视网膜中EPO和GRO-a的表达,从而抑制糖尿病大鼠视网膜组织中VEGF的表达,最终起到抗新生血管形成的作用。第三部分塞来昔布对糖尿病大鼠视网膜组织中d114、Notch-1表达的影响 [目的]观察选择性环氧合酶-2 (Cyclooxygenase-2,COX-2)抑制剂(塞来昔布)对糖尿病大鼠视网膜组织中Delta样配体4- Notch信号(delta-like 4 ligan-Notch, D114/Notch-1)通路的影响。 [方法]将40只SD大鼠随机分为4组,即正常组、糖尿病组、糖尿病+蒸馏水灌胃组以及糖尿病+塞来昔布灌胃组,每组10只。通过腹腔注射链脲佐菌素(streptoz-otocin,STZ)诱导建立糖尿病动物模型,饲养12周后处死大鼠,取视网膜组织,SYBR荧光实时定量PCR检测视网膜组织中COX-2、血管内皮生长因子(vascular endothelial growth factor,VEGF)以及D114/Notch-1 mRNA的表达变化,western-blotting检测大鼠视网膜组织中COX-2、VEGF以及D114/Notch-1蛋白的表达。通过Quantity One凝胶图像分析软件进行结果分析,行ANOVA方差分析,SNK法做组间两两比较,以P0.05作为差异有统计学意义。 [结果]12周后,COX-2、VEGF、D114及Notch-1 mRNA和蛋白质的表达在糖尿病组和糖尿病+蒸馏水组间差异无统计学意义(P=0.121),其余各组间比较均有显著性差异(P0.05)。正常组表达最少,糖尿病组和糖尿病+蒸馏水组表达最高,COX-2、VEGF mRNA和蛋白质表达在塞来昔布用药组高于正常组,但低于糖尿病组以及糖尿病+蒸馏水组；而D114/Notch-1 mRNA和蛋白质在塞来昔布用药组表达最高。 [结论]D114和Notch-1在糖尿病大鼠视网膜的新生血管生成过程中起到了重要作用,选择性COX-2抑制剂塞来昔布可以通过激活糖尿病大鼠视网膜中的D114/ Notch-1表达,从而抑制糖尿病大鼠视网膜组织中的VEGF表达。
[Abstract]:Diabetic retinopathy is a kind of blinding eye disease characterized by retinal neovascularization. With the improvement of people's living standard, diabetes is becoming a common and frequently occurring disease which threatens human health in our country. Accordingly, the incidence of diabetic retinopathy is increasing. So far, diabetic retinopathy The exact pathogenesis of the change is not completely clear. A large number of studies suggest that the formation of retinal neovascularization is related to the regulation of enzymes and kinases and the effects of various growth factors, while inflammation and immune responses also play an important role in this process.
Part 1 expression of GRO-a and EPO in serum and vitreous samples from patients with proliferative diabetic retinopathy
[Objective] to investigate the possible role of erythropoietin (EPO) and growth related gene alpha (Gro-a) in the plasma of patients with diabetic retinopathy (DR) in the development and changes of DR.
[Methods] the study was a case-control study, including non proliferative diabetic retinopathy (NPDR) group, proliferative diabetic retinopathy (PDR) group and control group. The levels of plasma, EPO and Gro-a in the vitreous samples were detected by enzyme linked immunosorbent assay (ELISA).
[results] the plasma EPO content: NPDR group (22.16 + 4.85) mIU/ml, PDR group (25.46 + 8.83) mIU/ml, control group (23.52 + 7.27) mIU/ml., EPO content of vitreous body: PDR group (461.36 + 101.20) mIU/ml, and control group (36.78 + 10.19) mIU/ml. plasma Gro-a content: NPDR group (81.95 + 38.08) The content of body Gro-a: PDR group (327.74 + 216.29) pg/ml, and there was no significant difference between the plasma samples of the control group (81.95 + 47.59) pg/ml.3 (P0.05), and the difference of Gro-a content was statistically significant (P0.05) in the.3 group, the PDR group and the control group 2 groups were EPO, and the Gro- alpha difference was significant statistically significant. There was a positive correlation between EPO and Gro- alpha levels and corresponding glycosylated hemoglobin (HbA1c%) levels in vivo.
[Conclusion plasma levels of Gro-a and vitreous EPO and Gro-a in patients with]PDR were significantly increased, and the contents of EPO and Gro-a were related to HbA1c (%) value of patients.
The second part is the expression of GRO-a and EPO in retina of diabetic rats and celecoxib.
Influence on the expression of GRO- alpha and EPO
[Objective] to observe the expression of GRO-a and EPO in the retinal tissue of diabetic rats and the effect of selective cyclooxygenase -2 (Cyclooxygenase-2, COX-2) inhibitor (celecoxib) on the expression of GRO- alpha and EPO in the retina of diabetic rats.
[Methods] 40 SD rats were randomly divided into 4 groups: normal group, diabetic group, diabetes + distilled water gavage group and diabetic + celecoxib gavage group, 10 rats in each group were induced by intraperitoneal injection of streptozotocin (streptoz-otocin, STZ) to establish diabetic rat model. After 12 weeks of feeding, rats were killed, retina was taken and SYBR fluorescence was determined in real time. The expression of COX-2, vascular endothelial growth factor (vascular endothelial growth factor, VEGF), EPO and GRO-a mRNA in the retina of the retina was measured by PCR. Analysis, SNK method to do 22 comparisons between groups, P0.05 as the difference was statistically significant.
[results after]12 weeks, the expression of COX-2, VEGF, EPO and GRO-a mRNA and protein were highest in the diabetic group and the diabetic + distilled water group. The diabetes + celecoxib group was higher than the normal group but lower than the diabetic group. There was no significant difference between the diabetic group and the diabetes + distilled water group (P0.05), and the other groups had significant differences (P0). .05).
[conclusion]EPO and GRO-a play an important role in the formation of retinal neovascularization in diabetic rats. Celecoxib may inhibit the expression of EPO and GRO-a in the retina of diabetic rats and inhibit the expression of VEGF in the retina tissue of diabetic rats and eventually play a role in anti angiogenesis.
The third part is the effect of celecoxib on the expression of D114 and Notch-1 in retina of diabetic rats.
[Objective] to observe the effect of selective cyclooxygenase -2 (Cyclooxygenase-2, COX-2) inhibitor (celecoxib) on the Delta like ligand 4- Notch signal (Delta-like 4 ligan-Notch, D114/Notch-1) pathway in the retinal tissue of diabetic rats.
[Methods] 40 SD rats were randomly divided into 4 groups: normal group, diabetic group, diabetes + distilled water gavage group and diabetic + celecoxib gavage group, 10 rats in each group were induced by intraperitoneal injection of streptozotocin (streptoz-otocin, STZ) to establish diabetic animal model. After 12 weeks of feeding, rats were killed, retina tissue was taken and SYBR fluorescence was determined in real time. COX-2, vascular endothelial growth factor (VEGF) and the expression of D114/Notch-1 mRNA in retinal tissue were measured by PCR. Western-blotting was used to detect the COX-2, VEGF, and protein expression in the retina tissue of rats. Analysis of variance, SNK method made 22 comparisons between groups, P0.05 as the difference was statistically significant.
[results after]12 weeks, there was no significant difference in the expression of COX-2, VEGF, D114 and Notch-1 mRNA and protein between the diabetic group and the diabetic + distilled water group (P=0.121). There was a significant difference between the other groups (P0.05). The expression of the normal group was least, and the highest expression in the diabetic group and the diabetic + distilled water group, COX-2, VEGF mRNA and protein table. The celecoxib group was higher than the normal group, but lower than the diabetic group and the diabetes + distilled water group, and the highest expression of D114/Notch-1 mRNA and protein in the celecoxib group.
[conclusion]D114 and Notch-1 have played an important role in the formation of retinal neovascularization in diabetic rats. Selective COX-2 Inhibitor Celecoxib can inhibit the expression of VEGF in retina of diabetic rats by activating the D114/ Notch-1 expression.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R774.1

【共引文献】