本文選題：1型糖尿病 + 糖尿病視網(wǎng)膜病變�。� 參考：《河北醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的:糖尿病是一種常見病和多發(fā)病,已成為世界范圍的威脅人類健康的疾病。每年約有數(shù)以百萬糖尿病患者因其并發(fā)癥而致殘或死亡,導(dǎo)致嚴(yán)重的社會(huì)和經(jīng)濟(jì)負(fù)擔(dān)。糖尿病視網(wǎng)膜病變(diabetic retinopathy,DR)是糖尿病最常見眼部微血管并發(fā)癥,也是致盲的重要眼病之一。因此如何早期預(yù)防和診治,減少DR的發(fā)生,降低致盲率,已成為世人尤以關(guān)注的社會(huì)問題。DR最主要的病理改變是新生血管形成。研究表明新生血管形成是多種血管生成因子和抑制因子相互拮抗協(xié)同作用的結(jié)果,其中色素上皮源性因子(pigment epithelium-derived factor, PEDF)被認(rèn)為是“天然的新生血管抑制物”。PEDF是目前發(fā)現(xiàn)的最強(qiáng)大的新生血管抑制因子,其在糖尿病視網(wǎng)膜病變發(fā)病機(jī)制中具有重要的作用。本研究通過建立1型糖尿病SD大鼠模型,觀察玻璃體內(nèi)注射PEDF對(duì)早期糖尿病視網(wǎng)膜病變的防治作用,探討PEDF在糖尿病視網(wǎng)膜病變的發(fā)生、發(fā)展中的作用及其機(jī)制。 方法:選取8周齡的健康雄性SD大鼠40只,體重200-220g,隨機(jī)分為4組:正常對(duì)照組(CON組)、糖尿病組(DM組)、糖尿病PEDF注射組(DM+PEDF組)和糖尿病生理鹽水注射組(DM+NS組),每組10只。組間大鼠體重?zé)o統(tǒng)計(jì)學(xué)差異。實(shí)驗(yàn)期間各組大鼠常規(guī)飼料喂養(yǎng)。適應(yīng)性喂養(yǎng)5天后,糖尿病組大鼠禁食12小時(shí)后左下腹腔給予鏈脲佐菌素( STZ,0.1mmol/L,檸檬酸鈉配制,pH4.5)65mg/kg注射。注射后48小時(shí)選取尾靜脈測(cè)血糖≥16.8mmol/L作為1型糖尿病動(dòng)物模型。糖尿病模型建立后,正常對(duì)照組和DM組不做任何處理,DM+PEDF組在糖尿病模型建立后1周和2周時(shí)分別向玻璃體腔內(nèi)注射PEDF 2ug/8ul,而DM+NS組注射同樣體積的生理鹽水。于糖尿病模型建立后3周時(shí)處死動(dòng)物,摘除眼球制作標(biāo)本,進(jìn)行組織病理學(xué)及電鏡觀察。在光學(xué)顯微鏡下觀察并計(jì)數(shù)突破視網(wǎng)膜內(nèi)界膜的血管內(nèi)皮細(xì)胞細(xì)胞核數(shù)目,電鏡下觀察視網(wǎng)膜組織超微結(jié)構(gòu)的改變。并采用免疫組化方法檢測(cè)視網(wǎng)膜PEDF、血管內(nèi)皮生長(zhǎng)因子(vascular endothelial growth factor , VEGF)、炎性相關(guān)因子-細(xì)胞間粘附分子1(intercellular adhesion molecule 1,ICAM-1)和單核細(xì)胞趨化蛋白1(monocyte chemotactic protein 1,MCP-1)以及細(xì)胞通透性相關(guān)因子-緊密連接蛋白1(zonula occludens 1,ZO-1)以及蛋白激酶B(protein kinase B,PKB,又稱Akt)等在蛋白水平的表達(dá)。實(shí)驗(yàn)數(shù)據(jù)用x±SD(均數(shù)士標(biāo)準(zhǔn)差)表示,應(yīng)用方差分析計(jì)算組間的差異。p0.05認(rèn)為有統(tǒng)計(jì)學(xué)差異, P0.001被認(rèn)為有顯著統(tǒng)計(jì)學(xué)差異。 結(jié)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,均為一次注射成模,成模率93.3%。DM+PEDF以及DM組分別有1只大鼠在試驗(yàn)中死亡。故糖尿病組大鼠死亡率為7.1%。 2糖尿病大鼠體重以及血糖的變化。 同CON組大鼠相比,糖尿病組大鼠,即DM、DM+NS,DM+PEDF組大鼠的血糖在整個(gè)實(shí)驗(yàn)過程均中明顯升高,而三組接受不同處理的糖尿病組大鼠間血糖水平無統(tǒng)計(jì)學(xué)差異。同正常同齡對(duì)照組大鼠相比,糖尿病大鼠體重明顯降低,且這種體重的差異有隨著病程的延長(zhǎng)而增加的趨勢(shì)。 3眼底表現(xiàn) 同CON組大鼠相比,1型糖尿病程為3周的大鼠晶狀體透明,眼底未見明顯改變。 4視網(wǎng)膜超微結(jié)構(gòu)的變化: 在透射電鏡下,正常對(duì)照組大鼠視網(wǎng)膜神經(jīng)節(jié)細(xì)胞結(jié)構(gòu)清晰,細(xì)胞器結(jié)構(gòu)完整,DM組大鼠視網(wǎng)膜可見神經(jīng)節(jié)細(xì)胞水腫,線粒體腫脹變大,基質(zhì)腫脹明顯,可見大部分或全部的嵴消失,部分雙側(cè)膜融合,粗面內(nèi)質(zhì)網(wǎng)擴(kuò)張。玻璃體內(nèi)PEDF注射可以明顯的改善這一病理改變,而玻璃體內(nèi)注射生理鹽水則無此作用。 5視網(wǎng)膜PEDF與VEGF的表達(dá)變化 在正常對(duì)照組與3周1型糖尿病病程的大鼠中,PEDF與VEGF的表達(dá)都很少,陽(yáng)性細(xì)胞主要位于神經(jīng)節(jié)細(xì)胞層、內(nèi)核層等細(xì)胞中。各組間陽(yáng)性細(xì)胞數(shù)無明顯統(tǒng)計(jì)學(xué)差異。表明3周糖尿病的病程尚不能引起視網(wǎng)膜表達(dá)PEDF以及VEGF的變化,玻璃體內(nèi)PEDF注射似乎可以增加PEDF的表達(dá),減少VEGF的表達(dá),但相對(duì)于DM以及DM+NS無明顯統(tǒng)計(jì)學(xué)差異。 6 PEDF干預(yù)可以減少炎性相關(guān)因子ICAM-1以及MCP-1的表達(dá) 正常對(duì)照組大鼠視網(wǎng)膜中ICAM-1以及MCP-1表達(dá)極少,同正常對(duì)照組相比,3周糖尿病病程可以導(dǎo)致視網(wǎng)膜ICAM-1以及MCP-1的表達(dá)明顯增加,玻璃體內(nèi)PEDF注射可以明顯減少ICAM-1以及MCP-1的表達(dá),而注射NS則無此種作用。表明PEDF通過減少一些炎性因子的表達(dá)改善糖尿病視網(wǎng)膜病變的進(jìn)展。 7 PEDF干預(yù)可以增加視網(wǎng)膜細(xì)胞連接蛋白ZO-1以及相關(guān)因子Akt的表達(dá) 在正常組大鼠視網(wǎng)膜中,ZO-1蛋白主要表達(dá)于內(nèi)核層的細(xì)胞以及神經(jīng)節(jié)細(xì)胞,糖尿病3周時(shí)ZO-1蛋白的表達(dá)明顯減少,而PEDF注射能增加內(nèi)核層以及神經(jīng)節(jié)細(xì)胞的ZO-1蛋白的表達(dá)。在正常大鼠的視網(wǎng)膜中,Akt主要表達(dá)于內(nèi)叢狀層以及脈絡(luò)膜的細(xì)胞漿中。糖尿病3周時(shí)內(nèi)叢狀層中的Akt較正常對(duì)照組明顯減少,PEDF治療可使糖尿病大鼠視網(wǎng)膜內(nèi)叢狀層中Akt表達(dá)增加。 結(jié)論: 1同正常對(duì)照組相比,糖尿病視網(wǎng)膜病變?cè)缙?3周病程)時(shí)視網(wǎng)膜VEGF以及PEDF均無表達(dá)變化; 2糖尿病視網(wǎng)膜病變?cè)缙跁r(shí)已出現(xiàn)視網(wǎng)膜神經(jīng)節(jié)細(xì)胞超微結(jié)構(gòu)的改變,而PEDF治療可以改善視網(wǎng)膜神經(jīng)節(jié)細(xì)胞的損傷并有一定保護(hù)作用; 3糖尿病視網(wǎng)膜病變?cè)缙跁r(shí)已出現(xiàn)視網(wǎng)膜炎性相關(guān)因子ICAM-1及MCP-1的表達(dá)增加,PEDF玻璃體內(nèi)注射可以減少視網(wǎng)膜炎性相關(guān)因子的表達(dá); 4糖尿病視網(wǎng)膜病變?cè)缙跁r(shí)視網(wǎng)膜ZO-1以及Akt的表達(dá)明顯減少,而PEDF玻璃體內(nèi)注射可以增加細(xì)胞連接蛋白以及相關(guān)因子的表達(dá)。 5以上結(jié)果表明PEDF可以明顯的改善糖尿病視網(wǎng)膜病變?cè)缙跁r(shí)神經(jīng)節(jié)細(xì)胞的損害,通過減少炎性因子和增加細(xì)胞連接蛋白而減輕血-視網(wǎng)膜屏障的破壞,從而對(duì)早期糖尿病視網(wǎng)膜病變起著一定的防治作用。
[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.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R774.1

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