色素上皮源性因子治疗糖尿病视网膜病变的机理研究

发布时间：2018-04-11 17:00

本文选题：1型糖尿病 + 糖尿病视网膜病变　；参考：《河北医科大学》2011年硕士论文

【摘要】：目的:糖尿病是一种常见病和多发病,已成为世界范围的威胁人类健康的疾病。每年约有数以百万糖尿病患者因其并发症而致残或死亡,导致严重的社会和经济负担。糖尿病视网膜病变(diabetic retinopathy,DR)是糖尿病最常见眼部微血管并发症,也是致盲的重要眼病之一。因此如何早期预防和诊治,减少DR的发生,降低致盲率,已成为世人尤以关注的社会问题。DR最主要的病理改变是新生血管形成。研究表明新生血管形成是多种血管生成因子和抑制因子相互拮抗协同作用的结果,其中色素上皮源性因子(pigment epithelium-derived factor, PEDF)被认为是“天然的新生血管抑制物”。PEDF是目前发现的最强大的新生血管抑制因子,其在糖尿病视网膜病变发病机制中具有重要的作用。本研究通过建立1型糖尿病SD大鼠模型,观察玻璃体内注射PEDF对早期糖尿病视网膜病变的防治作用,探讨PEDF在糖尿病视网膜病变的发生、发展中的作用及其机制。方法:选取8周龄的健康雄性SD大鼠40只,体重200-220g,随机分为4组:正常对照组(CON组)、糖尿病组(DM组)、糖尿病PEDF注射组(DM+PEDF组)和糖尿病生理盐水注射组(DM+NS组),每组10只。组间大鼠体重无统计学差异。实验期间各组大鼠常规饲料喂养。适应性喂养5天后,糖尿病组大鼠禁食12小时后左下腹腔给予链脲佐菌素( STZ,0.1mmol/L,柠檬酸钠配制,pH4.5)65mg/kg注射。注射后48小时选取尾静脉测血糖≥16.8mmol/L作为1型糖尿病动物模型。糖尿病模型建立后,正常对照组和DM组不做任何处理,DM+PEDF组在糖尿病模型建立后1周和2周时分别向玻璃体腔内注射PEDF 2ug/8ul,而DM+NS组注射同样体积的生理盐水。于糖尿病模型建立后3周时处死动物,摘除眼球制作标本,进行组织病理学及电镜观察。在光学显微镜下观察并计数突破视网膜内界膜的血管内皮细胞细胞核数目,电镜下观察视网膜组织超微结构的改变。并采用免疫组化方法检测视网膜PEDF、血管内皮生长因子(vascular endothelial growth factor , VEGF)、炎性相关因子-细胞间粘附分子1(intercellular adhesion molecule 1,ICAM-1)和单核细胞趋化蛋白1(monocyte chemotactic protein 1,MCP-1)以及细胞通透性相关因子-紧密连接蛋白1(zonula occludens 1,ZO-1)以及蛋白激酶B(protein kinase B,PKB,又称Akt)等在蛋白水平的表达。实验数据用x±SD(均数士标准差)表示,应用方差分析计算组间的差异。p0.05认为有统计学差异, P0.001被认为有显著统计学差异。结果: 1糖尿病大鼠的成模率和死亡率 30只大鼠接受STZ注射,28只成模,均为一次注射成模,成模率93.3%。DM+PEDF以及DM组分别有1只大鼠在试验中死亡。故糖尿病组大鼠死亡率为7.1%。 2糖尿病大鼠体重以及血糖的变化。同CON组大鼠相比,糖尿病组大鼠,即DM、DM+NS,DM+PEDF组大鼠的血糖在整个实验过程均中明显升高,而三组接受不同处理的糖尿病组大鼠间血糖水平无统计学差异。同正常同龄对照组大鼠相比,糖尿病大鼠体重明显降低,且这种体重的差异有随着病程的延长而增加的趋势。 3眼底表现同CON组大鼠相比,1型糖尿病程为3周的大鼠晶状体透明,眼底未见明显改变。 4视网膜超微结构的变化: 在透射电镜下,正常对照组大鼠视网膜神经节细胞结构清晰,细胞器结构完整,DM组大鼠视网膜可见神经节细胞水肿,线粒体肿胀变大,基质肿胀明显,可见大部分或全部的嵴消失,部分双侧膜融合,粗面内质网扩张。玻璃体内PEDF注射可以明显的改善这一病理改变,而玻璃体内注射生理盐水则无此作用。 5视网膜PEDF与VEGF的表达变化在正常对照组与3周1型糖尿病病程的大鼠中,PEDF与VEGF的表达都很少,阳性细胞主要位于神经节细胞层、内核层等细胞中。各组间阳性细胞数无明显统计学差异。表明3周糖尿病的病程尚不能引起视网膜表达PEDF以及VEGF的变化,玻璃体内PEDF注射似乎可以增加PEDF的表达,减少VEGF的表达,但相对于DM以及DM+NS无明显统计学差异。 6 PEDF干预可以减少炎性相关因子ICAM-1以及MCP-1的表达正常对照组大鼠视网膜中ICAM-1以及MCP-1表达极少,同正常对照组相比,3周糖尿病病程可以导致视网膜ICAM-1以及MCP-1的表达明显增加,玻璃体内PEDF注射可以明显减少ICAM-1以及MCP-1的表达,而注射NS则无此种作用。表明PEDF通过减少一些炎性因子的表达改善糖尿病视网膜病变的进展。 7 PEDF干预可以增加视网膜细胞连接蛋白ZO-1以及相关因子Akt的表达在正常组大鼠视网膜中,ZO-1蛋白主要表达于内核层的细胞以及神经节细胞,糖尿病3周时ZO-1蛋白的表达明显减少,而PEDF注射能增加内核层以及神经节细胞的ZO-1蛋白的表达。在正常大鼠的视网膜中,Akt主要表达于内丛状层以及脉络膜的细胞浆中。糖尿病3周时内丛状层中的Akt较正常对照组明显减少,PEDF治疗可使糖尿病大鼠视网膜内丛状层中Akt表达增加。结论: 1同正常对照组相比,糖尿病视网膜病变早期(3周病程)时视网膜VEGF以及PEDF均无表达变化; 2糖尿病视网膜病变早期时已出现视网膜神经节细胞超微结构的改变,而PEDF治疗可以改善视网膜神经节细胞的损伤并有一定保护作用; 3糖尿病视网膜病变早期时已出现视网膜炎性相关因子ICAM-1及MCP-1的表达增加,PEDF玻璃体内注射可以减少视网膜炎性相关因子的表达; 4糖尿病视网膜病变早期时视网膜ZO-1以及Akt的表达明显减少,而PEDF玻璃体内注射可以增加细胞连接蛋白以及相关因子的表达。 5以上结果表明PEDF可以明显的改善糖尿病视网膜病变早期时神经节细胞的损害,通过减少炎性因子和增加细胞连接蛋白而减轻血-视网膜屏障的破坏,从而对早期糖尿病视网膜病变起着一定的防治作用。
[Abstract]:Objective: diabetes is a common disease, has become a worldwide threat to human health. There are about hundreds of millions of patients with diabetes and disability or death due to the complications each year, leading to serious social and economic burden of diabetic retinopathy (diabetic retinopathy DR) is the most common ocular microvascular complication of diabetes. One of the most important eye is blind. So how to early prevention and treatment, reduce the incidence of DR, reduce the blindness rate, especially in the world has become a social problem on.DR the main pathological change is the formation of new blood vessels. The results show that the formation of new blood vessels is a variety of angiogenic factors and factor antagonistic inhibition result of synergy, which pigment epithelium derived factor (pigment epithelium-derived, factor, PEDF) is considered to be the "angiogenesis inhibitor natural.PEDF has been found to be the most The powerful angiogenesis inhibiting factor, which plays an important role in the pathogenesis of diabetic retinopathy. In this study, through the establishment of type 1 diabetic SD rat model, the effect of intravitreal injection of PEDF in early diabetic retinopathy, explore the role of PEDF in the occurrence of diabetic retinopathy, the effect and mechanism of development.
Methods: 40 healthy male SD rats, weighing 200-220g were selected from 8 weeks of age, were randomly divided into 4 groups: normal control group (CON group), diabetic group (DM group), diabetic PEDF injection group (DM+PEDF group) and diabetic saline group (DM+NS group), 10 rats in each group. There was no significant difference in body weight of rats. During the experiment, the rats fed with normal diet. After 5 days adaptive feeding fasting diabetic rats after 12 hours left under intraperitoneal injection of streptozotocin (STZ, 0.1mmol/L, sodium citrate compound, pH4.5) injection of 65mg/kg. 48 hours after injection from tail vein blood glucose was 16.8mmol/L as the animal model of type 1 diabetes. Diabetes model, normal control group and DM group without any treatment, DM+PEDF group in the diabetic model after 1 weeks and 2 weeks respectively to the intravitreal injection of PEDF 2ug/8ul, and DM+NS normal saline group were injected with same volume. In the diabetic model At the 3 week after the death of animal specimen were enucleated, and electron microscope observation and histological examination. The number of nuclei of endothelial cell count of retinal in the optical microscope to observe the ultrastructural changes of retinal tissue under electron microscope. And the method of detection of retinal PEDF by immunohistochemistry, vascular endothelial growth factor (vascular endothelial growth factor, VEGF), inflammatory factors and intercellular adhesion molecule 1 (intercellular adhesion 1 molecule, ICAM-1) and monocyte chemotactic protein 1 (monocyte chemotactic 1 protein, MCP-1) and cell permeability factor - tight junction protein 1 (zonula occludens 1, ZO-1) and protein kinase B (protein kinase B, PKB, and Akt) expression in protein level by X + SD. The experimental data (mean SD) said that the application of variance analysis and calculation of the difference between the groups Different.P0.05 thought there were statistical differences, and P0.001 was considered to have significant statistical differences.
Result:
Model rate and mortality rate of 1 diabetic rats
30 rats received STZ injection, and 28 rats were moulded, all of them were injected once a single time, the rate of mold formation was 93.3%.DM+PEDF, and 1 rats in group DM died in the experiment. So the mortality of diabetic group was 7.1%..
2 the changes of body weight and blood sugar in diabetic rats.
Compared with CON rats, diabetic rats, DM, DM+NS, DM+PEDF rats blood glucose during the whole experiment were significantly increased, while the three group received diabetic rats in different treatment between the blood glucose level was not statistically significant. Compared with normal control rats, body weight of diabetic rats decreased obviously, increases with the extension of the course of the trend and the difference in weight.
3 fundus performance
Compared with the CON group, the lens of the rats with type 1 diabetes 3 weeks was transparent and the fundus had no obvious change.
4 changes in the ultrastructure of the retina:
Under transmission electron microscope, the normal control group rat retinal ganglion cell structure clear, organelle structure integrity, DM group of rat retinal ganglion cells showed edema, mitochondrial swelling increases, matrix swelling significantly, most or all of the visible part of bilateral cristae and membrane fusion, rough endoplasmic reticulum. Intravitreal injection of PEDF significantly the improvement of the pathological change, and intravitreal injection of saline had no such effect.
5 changes in the expression of PEDF and VEGF in the retina
In the normal control group and 3 weeks duration of type 1 diabetes in rats, the expression of PEDF and VEGF are few, the positive cells were mainly located in the ganglion cell layer, inner nuclear layer cells. The number of positive cells between groups had no significant difference. 3 weeks showed that the duration of diabetes is not caused by retinal expression of PEDF and VEGF change, intravitreal injection of PEDF may increase the expression of PEDF, reduce the expression of VEGF, DM and DM+NS compared with no significant difference.
6 PEDF intervention can reduce the expression of inflammatory related factors ICAM-1 and MCP-1
ICAM-1 and MCP-1 in normal control group rarely expressed in retina of rats, compared with normal control group, 3 week duration of diabetes can lead to retinal ICAM-1 and MCP-1 expression increased significantly, intravitreal injection of PEDF can significantly reduce the expression of MCP-1 and ICAM-1, and the injection of NS without this kind of role. That PEDF progresses through reduced expression of some inflammatory the improvement factor of diabetic retinopathy.
7 PEDF intervention can increase the expression of retina cell connexin ZO-1 and related factor Akt
In the retina of normal rats, ZO-1 protein was mainly expressed in inner nuclear layer cells and ganglion cells, the expression of ZO-1 protein after 3 weeks of diabetes decreased significantly, while PEDF injection can increase the expression of the kernel layer and ganglion cells. ZO-1 protein in the normal rat retina, Akt is mainly expressed in cytoplasm in the plexus the shape layer and choroidal plexiform layer. After 3 weeks of diabetes in Akt compared with normal control group decreased significantly, PEDF treatment can increase the expression of Akt in retina of diabetic rats in the inner plexiform layer.
Conclusion:
1 compared with the normal control group, there were no changes in the VEGF and PEDF in the retina and VEGF at the early stage of diabetic retinopathy (3).
2, the ultrastructural changes of retinal ganglion cells have been observed in the early stage of diabetic retinopathy. PEDF treatment can improve retinal ganglion cell injury and protect it.
3, the expression of ICAM-1 and MCP-1 has been increased in the early stage of diabetic retinopathy. PEDF intravitreal injection can reduce the expression of retinal inflammatory factors.
4, the expression of ZO-1 and Akt in retina decreased significantly at early stage of diabetic retinopathy, while PEDF intravitreal injection increased the expression of connexin and related factors.
5 the above results show that PEDF can improve the early ganglion cells of diabetic retinopathy obvious damage, and reduce the blood retinal barrier by reducing inflammatory factors and increase cell junction protein damage to early diabetic retinopathy plays a certain role in the prevention and treatment.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R774.1

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