去整合素Echistatin对糖尿病兔晶状体后囊膜混浊的作用研究

发布时间：2018-06-27 02:16

本文选题：糖尿病 + 后囊膜混浊　；参考：《广西医科大学》2014年博士论文

【摘要】：糖尿病性白内障是糖尿病第二位常见眼部并发症，，手术是其唯一有效治疗手段，后发性白内障（即后囊膜混浊(posterior capsular opacification,PCO)）是白内障摘除术后最常见并发症，不仅导致术后视力下降，还对糖尿病眼后段病变的诊断和治疗效果造成较大影响。晶状体上皮细胞增殖和上皮-间质转分化（epithelial-mesenchymal transition, EMT）在PCO的发生和发展中起重要作用，而细胞间及细胞与细胞外基质间粘附是其生存和增殖的基础。细胞粘附分子-整合素参与调控细胞粘附、迁移、增殖及分化等众多生理过程，对阻断整合素的调控作用是否可以有效抑制糖尿病性PCO发生和发展的研究目前甚少。前期实验已证实去整合素Echistatin (Ecs)可明显抑制体外培养的人晶状体上皮细胞(LensePitheliumcells,LECs)的增殖、粘附及移行；并发现糖尿病兔晶状体囊外摘除术后（extrocapsular lensextration，ECLE）出现PCO的最早时间（10天）和最显著时间（6周）均较正常血糖兔（14天，12周）提前，同期后囊膜混浊程度较正常血糖兔提高。因此为探索去整合素能否有效抑制糖尿病性PCO的发生发展，本实验拟利用糖尿病兔眼晶状体后囊膜混浊模型,观察去整合素Ecs对糖尿病性PCO分级的影响，及体内LECs增殖、细胞粘附和上皮-间质转分化（EMT）的变化；同时检测血清和房水中糖尿病并发症相关因子，即AGEs及IGF-1的含量变化，探讨Ecs是否通过对糖尿病并发症致病因子的调控从而影响PCO的发生发展；并进一步探讨Ecs对细胞内信号传导通路中的关键信号因子PI3K及其下游信号因子ILK基因表达的干预作用，初步明确Ecs在LECs中作用的分子途径，为去整合素Ecs防治糖尿病性后发性白内障提供有力的理论依据。第一部分Ecs对糖尿病兔PCO分级，LECs增殖、细胞间连接及EMT的影响目的： LECs在晶状体后囊膜上的增殖和上皮细胞-间质转化（EMT）是形成PCO的重要环节。细胞粘附则是LECs生存及出现其他生物学行为的基础，细胞间连接蛋白的表达可以在一定程度上反映细胞粘附的情况。亦是PCO形成的重要环节。整合素不仅介导了细胞的粘附，还在细胞增殖、EMT中发挥了重要作用，本实验拟通过观察整合素的阻滞剂-去整合素echistatin对糖尿病兔PCO形成，LECs增殖、粘附及间质转化的影响，了解去整合素是否能有效抑制糖尿病兔PCO的发生发展，并尝试阐述其分子机制。方法：应用四氧嘧啶建立糖尿病兔模型，每只兔右眼均行晶状体囊外摘除术（extrocapsular lens extration，ECLE），术毕随机分组，对照组在术眼晶状体囊袋内注入0.2ml高压蒸馏水，Ecs组则注入0.2ml10μg/ml Ecs。分别在术后10天（10d）及6周（6w）观察以下指标：⑴裂隙灯下观察术眼晶状体后囊膜混浊情况并分级；⑵应用免疫组化法检测Ecs对LECs中PCNA表达的影响。⑶应用免疫荧光法检测Ecs对晶状体后囊膜上LECs中连接蛋白43表达的影响；⑷应用免疫组化法和荧光定量PCR(Real-TimeQuantitative PCR, RT-PCR)法检测Ecs对晶状体后囊膜上α-SMA和Ⅳ型胶原表达的影响。结果： ⑴术后10天对照组和Ecs组间PCO分级无明显差异(P=0.495)，术后6周Ecs可明显降低糖尿病兔PCO分级(P=0.025)。⑵术后10天及6周Ecs 组LECs中PCNA的表达明显低于对照组（p=0.031,0.021）。⑶术后10天及6周对照组晶状体后囊膜上Cx43表达明显增强，而Ecs组仅见少量表达，两组间存在显著差异（p=0.007,0.001），提示Ecs可有效抑制糖尿病兔模型中LECs间连接蛋白43的表达。⑷术后10天，免疫组化及RT-PCR结果均显示α-SMA在晶状体后囊膜上的表达两组间无明显差异(p0.05)，术后6周对照组晶状体后囊膜上α-SMA表达显著增多，而Ecs组仅少量增加，表达较对照组明显降低(p0.05)，提示Ecs可有效抑制术后晚期α-SMA的表达。⑸RT-PCR结果显示晶状体后囊膜上Ⅳ型胶原表达，术后10天两组间无明显差异(p=0.157)，术后6周Ecs组较对照组表达明显增降低(p=0.049)；而免疫组化结果显示无论是10d或6w，Ecs组Ⅳ型胶原表达均较对照组显著减少（p=0.037,0.001）。提示Ecs可有效抑制Ⅳ型胶原的表达，可能部分Ⅳ型胶原mRNA未翻译为蛋白。结论：糖尿病兔PCO模型中，Ecs可有效抑制PCNA、Cx43、α-SMA、Ⅳ型胶原表达，降低PCO的分级，抑制PCO的发生和发展。第二部分Ecs对糖尿病相关因子AGEs、IGF-1及PI3K/ILK信号通路的影响研究目的：晚期糖基化终末产物（advanced glycation end products，AGEs）和胰岛素样生长因子-1（insulin-like growth actor1，IGF-1）与多种糖尿病并发症的发生均有密切关系，其是否也参与了糖尿病兔PCO的形成？ Ecs发挥抑制PCO的作用是否与改变以上两种糖尿病并发症相关因子的表达相关？整合素信号通路中哪些信号因子参与了上述作用的调控？本课题中我们通过一系列实验来探讨以上三个问题的答案，以期进一步了解Ecs抑制PCO发生发展的机制。方法：将兔子分为正常血糖对照组，糖尿病组及Ecs组，所有兔子行ECLE术，分别采集术后10天和6周血清及术眼房水，通过ELISA法检测各标本中AGEs和IGF-1含量的变化。取糖尿病组及Ecs组中兔术眼晶状体后囊膜，通过RT-PCR法检测ILK、PI3K的基因表达变化。结果： ELISA检测结果提示术后10d及6w，糖尿病组及Ecs组血清和房水中AGEs和IGF-1的含量均明显高于对照组（P0.05）；在糖尿病兔模型中，Ecs组房水中IGF-1含量明显低于糖尿病组（P0.05），但血清及房水中AGEs含量及血清中IGF-1含量Ecs组与糖尿病组间差异无统计学意义（P0.05）。RT-PCR检测结果显示术后10天及6周，与糖尿病组相比，Ecs组晶状体后囊膜上PI3K及ILK mRNA表达下调（P0.05）。结论： AGEs和IGF-1可能参与了糖尿病兔PCO的发生发展过程，Ecs对糖尿病兔房水中IGF-1的表达有明显抑制作用，可能是其抑制PCO发展的机制之一。Ecs通过下调糖尿病兔晶状体后囊膜上ILK和PI3K的表达，阻碍ILK和PI3K之间信号传导，从而抑制PCO发展。
[Abstract]:Diabetic cataract is the second common ocular complication of diabetes. Surgery is the only effective treatment. Posterior capsular opacification (posterior capsular opacification, PCO) is the most common complication after cataract extraction, which not only leads to postoperative visual loss, but also the diagnosis and treatment of diabetic posterior segment lesions. The proliferation of lens epithelial cells and epithelial mesenchymal transition (epithelial-mesenchymal transition, EMT) play an important role in the development and development of PCO, and the adhesion between cells and cells and extracellular matrix is the basis for its survival and proliferation. Cell adhesion molecules integrin is involved in regulating cell adhesion and migration. Many physiological processes, such as proliferation and differentiation, are very rare in the study of whether the regulation of integrin can effectively inhibit the occurrence and development of diabetic PCO. Earlier experiments have proved that de integrin Echistatin (Ecs) can obviously inhibit the proliferation, adhesion and migration of human lens epithelial cells (LensePitheliumcells, LECs) in vitro. It was found that the earliest time (10 days) and the most significant time (6 weeks) of PCO in extrocapsular lensextration (ECLE) after extracapsular extracapsular extirpation were higher than that of normal blood glucose rabbits (14 days, 12 weeks), and the degree of posterior capsule opacification was higher than that of normal blood glucose rabbits. The effect of integrin Ecs on diabetic PCO classification and the changes of LECs proliferation, cell adhesion and epithelial mesenchymal transition (EMT) were observed in the diabetic rabbit eye lens posterior capsule opacification model, and the changes in serum and atrial diabetic complications related factors, namely, AGEs and IGF-1 content, were detected. To investigate whether Ecs affects the occurrence and development of PCO by regulating the pathogenic factors of diabetic complications and further exploring the intervention of Ecs on the key signal factor PI3K and its downstream signal factor ILK gene expression in the intracellular signal transduction pathway, and preliminarily clarified the molecular pathway of the action of Ecs in LECs for the prevention of integrin Ecs. It provides a powerful theoretical basis for the treatment of diabetic cataract.
The first part is the effect of Ecs on PCO grading, LECs proliferation, intercellular connection and EMT in diabetic rabbits.
Objective:
The proliferation of LECs in the posterior capsule and the epithelial cell mesenchymal transition (EMT) are important links in the formation of PCO. Cell adhesion is the basis for the survival and other biological behavior of LECs. The expression of intercellular connexin can reflect the cell adhesion to a certain extent. It is also an important link in the formation of PCO. Integrin is not only a mediating agent. To guide cell adhesion and also play an important role in cell proliferation, EMT plays an important role. This experiment is to observe the effect of integrin blocker, de integrin Echistatin, on the formation of PCO, LECs proliferation, adhesion and interstitial transformation in diabetic rabbits, to understand whether integrin can effectively inhibit the development of PCO in diabetic rabbits and try to elaborate on its molecules. Mechanism.
Method:
The diabetic rabbit model was established with four pyrimidine. The right eye of each rabbit was treated with extrocapsular lens extration (ECLE), and the control group was randomly divided into two groups. The control group injected 0.2ml high pressure distilled water into the lens capsule of the operation eye, and the Ecs group injected 0.2ml10 mu g/ml Ecs. respectively to observe the following indexes at 10 days (10d) and 6 weeks (6W) after the operation, respectively: (1) The effect of Ecs on the expression of PCNA in LECs was detected by immunohistochemical method. (3) the effect of Ecs on the expression of connexin 43 in LECs on the posterior capsule of the lens was detected by immunofluorescence. (4) immunohistochemistry and fluorescein quantitative PCR (Real-TimeQuantitative PCR, RT-P) were used. CR) to detect the effect of Ecs on the expression of alpha -SMA and type IV collagen in posterior capsular lens.
Result:
(1) there was no significant difference in PCO classification between the control group and the Ecs group at 10 days after the operation (P=0.495). The PCO grade (P=0.025) of the diabetic rabbits was significantly reduced by Ecs after 6 weeks of operation. (2) 10 and 6 weeks after the operation, Ecs
The expression of PCNA in the group LECs was significantly lower than that of the control group (p=0.031,0.021). (3) the expression of Cx43 in the posterior capsule of the lens was markedly enhanced at 10 and 6 weeks after the operation, while the Ecs group only found a small amount of expression, and there was a significant difference between the two groups (p=0.007,0.001), suggesting that Ecs could effectively inhibit the expression of the connexin 43 between LECs in the diabetic rabbit model. (4) 10 days after the operation, it was exempt. The immunohistochemical and RT-PCR results showed that there was no significant difference between the two groups of the expression of alpha -SMA on the posterior capsule of the lens (P0.05). The expression of alpha -SMA in the posterior capsule of the lens was significantly increased at 6 weeks after the operation, while the Ecs group increased only a little, and the expression was significantly lower than that of the control group (P0.05), suggesting that Ecs could effectively inhibit the expression of late alpha -SMA after the operation. The expression of type IV collagen on the posterior capsule of the lens showed no significant difference between the two groups after 10 days (p=0.157). The expression of Ecs in the group was significantly lower than that of the control group at 6 weeks after the operation (p=0.049), and the immunohistochemical results showed that the expression of type IV collagen in group Ecs was significantly lower than that in the control group (p=0.037,0.001). It suggested that Ecs could effectively inhibit the type IV glue. The expression of mRNA may not be translated into protein.
Conclusion:
In the PCO model of diabetic rabbits, Ecs can effectively inhibit the expression of PCNA, Cx43, -SMA and type IV collagen, reduce the classification of PCO, and inhibit the occurrence and development of PCO.
The second part is about the effect of Ecs on the AGEs, IGF-1 and PI3K/ILK signaling pathways of diabetes mellitus.
Objective:
The late glycosylation end products (advanced glycation end products, AGEs) and insulin like growth factor -1 (insulin-like growth actor1, IGF-1) are closely related to the occurrence of a variety of diabetic complications. Is it also involved in the formation of diabetic rabbit PCO? What are the signal factors in the integrin signaling pathway involved in the regulation of the role of the integrin signaling pathway? In this subject, we explore the answers to these three questions by a series of experiments to further understand the mechanism of Ecs inhibition of the development of PCO.
Method:
Rabbits were divided into normal blood glucose control group, diabetic group and Ecs group. All rabbits were treated with ECLE. The serum and ocular aqueous humor were collected 10 and 6 weeks after the operation. The changes of AGEs and IGF-1 were detected by ELISA method. The posterior capsule of the lens of the rabbit eyes in the diabetic and Ecs group was taken and the gene expression of ILK and PI3K were detected by RT-PCR method.
Result:
The results of ELISA test showed that the content of serum and aqueous AGEs and IGF-1 in serum and aqueous humor of diabetic group and Ecs group were significantly higher than that of control group (P0.05) after operation (P0.05). In diabetic rabbit model, IGF-1 content in aqueous humor of Ecs group was significantly lower than that of diabetes group (P0.05), but the difference between serum and atrial water AGEs content and serum IGF-1 content in serum and serum was different from that of diabetes group. The results of.RT-PCR detection without statistical significance (P0.05) showed that the expression of PI3K and ILK mRNA in the posterior capsule of the Ecs group was down (P0.05) compared with the diabetic group at 10 and 6 weeks after the operation.
Conclusion:
AGEs and IGF-1 may be involved in the development of PCO in diabetic rabbits. Ecs can inhibit the expression of IGF-1 in aqueous humor of diabetic rabbits. It may be one of the mechanisms to inhibit the development of PCO..Ecs can inhibit the signal transduction between ILK and PI3K by reducing the expression of ILK and PI3K on the posterior capsule of the diabetic rabbit lens, thus inhibiting the development of PCO.
【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R587.2;R776.1

【参考文献】