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(p>0.05)，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(p<0.05)，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（P<0.05）；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（P<0.05），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（P>0.05）。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（P<0.05）。
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。
Diabetes is a kind of chronic disease with high incidence in the world.There are2.8%of people with it in global population, and it will increase to4.4%by2030. Including China, the United States and India occupied the topthree of the world. Diabetes usely incorporates many kinds of complications.Among them, diabetic cataract is the second common eye diabetes complication.Although the control of blood glucose can slow down its development, once thevision affected significantly, surgery is the only effective treatment. Aftercataract is the most common complication late surgery causing the restoredvision falling again. It affects the diabetic fundus examination, retinalphotocoagulation and vitreous surgery et al. Based on the status quo, muchattention has been paid to the solution that how to restore the best vision forthese patients and how to reduce the PCO formation.
Current research suggests the main reason causing posterior capsularopacity is the lens epithelial cells residualed in anterior lens capsule and theequator postoperative proliferates, migrates and adheres to the posterior capsular,and epithelial-mesenchymal transition (EMT). Adhesion between cells to cellsand cells to extracellular matrix (ECM) is the foundation of their survival and proliferation, migration. And this kind of adhesive is regulated by cell adhesionmolecules. In cell adhesion molecule super family, integrin is considered thattake the most important role in above adhesion function. Integrin has been foundthat it not only participates in adhesion between cells and cells with extracellularmatrix, also affect cell proliferation, differentiation, survival and apoptosisprocess, etc. It is not only the essential cell molecular in embryonic period forlens epithelial cells normal growth and maintain normal phenotype, but alsoparticipated in the adhesion, migration, proliferation and epithelial-mesenchymal transition of lens epithelial cells after cataract surgery andpromoted the PCO occurrence and development.
In addition to the LECs anomalies biological behavior stimulated bysurgical trauma and inflammation, high glucose and many diabetes relatedpathogenic factors all make the incidence of diabetic PCO significantly higherthan PCO with normal blood glucose. Whether integrin plays an important rolein it? Whether blocking integrin can effectively restrain the development andprogression of the diabetic PCO? Related research is currently very little.Disintegrin is a kind of protein molecule consists of49-84amino acids. It is richin the arginine-glycine-aspartate (Arg-Gly-Asp, RGD) sequence or glycine–aspartic-acid lysine (Lys-Gly-Asp, KGD is) sequence which can be identifiedby integrin. Through them, disintegrin bind with integrin receptors on cells andcompetitively inhibiting the connection between integrins and extracellularmatrix which owns RGD or KGD sequence, thereby inhibiting the physiologicalfunctions of integrins in cell adhesion, migration and other.
Our previous studies have confirmed that disintegrin Echistatin (Ecs) caninhibit the adhesion, migration and proliferation of human LECs in vitro. Nowwe took advantage of diabetic rabbits PCO model to study the role of Ecs on PCO, LECs proliferation, cells connection, EMT and the levels change ofdiabetes complications related factors of diabetic rabbits（AGEs, IGF–1）andthe key signal factor (PI3K, ILK) to provide a theoretical basis for applyingdisintegrin Ecs on prevention and treatment diabetic PCO.
PART Ⅰ the role of Ecs in PCO grading and proliferating cell nuclearantigen(PCNA), Cx43, nchymal-epithelial transition (EMT) on LECs ofdiabetic rabbit
Objective:
LECs proliferation and epithelial-mesenchymal transition (EMT) is closelyrelated to the formation of PCO. Migration of the residual LECs in capsular bagafter surgery to posterior capsular and the proliferation, EMT, abnormalsynthesis and secretion of a large number of extracellular cell matrix(ECM) andcollagen deposition cause opacity and shrinkage of the posterior capsular,followed by the formation of PCO. Currently, integrin, the cell adhesionmolecules, is considered not only mediated cell adhesion, also plays animportant role in PCO. In this study we proposed to understand whetherdisintegrin can effectively inhibit the diabetic PCO and try to expound itsmolecular mechanism by observing the role of disintegrin echistatin in PCOgrade, LECs PCNA, cell connection and EMT in diabetic rabbit PCO model.
Method:
Rabbits were induced diabetes with alloxan and implemented lensextraction surgery. Then the rabbits in control group or Ecs group were injectedsterilization distilled water or10μg/ml echistatin in len capsule respectively.Ten days after surgery (10d) and6weeks (6w), the following indicators were observed: the posterior capsular opacity grade were assessed by slit lamp. Theexpression of LECs PCNA was determined by immunohistochemistry. Theexpression of connection43on posterior capsular was evaluated byimmunofluorescence. The expressions of α-SMA and collagen type Ⅳweredetermined by immunohistochemistry and RT-PCR.
Results:
The PCO grading of Ecs group was significantly lower than that of thecontrol group at6weeks after surgery though no significant difference betweenthem at10days.. The expression of PCNA and Cx43was decreased in Ecsgroup at10d and6w postoperatively. Immunohistochemistry and RT-PCRresults indicated that, at6weeks after surgery, α-SMA was down-regulated inEcs group. RT-PCR results showed that, compared with the control group，theexpression of collagen type Ⅳon lens posterior capsular membrane in Ecsgroup has no significant difference at10days postoperatively, but at6weekswhich was significantly increased; while immunohistochemistry showed either10days or6weeks, the expression of collagen type Ⅳ in Ecs group wassignificantly reduced. It is suggesting that maybe part collagen type Ⅳ mRNAwas not translated into proteins ultimately.
Conclusion:
In diabetic rabbit model, PCNA, Cx43, α-SMA, Ⅳcollagen expression onlens posterior capsule membrane can be effectively inhibited by Ecs. This maythe reason that Ecs restrain the development of PCO and decrease it grading.
PART Ⅱ Impact of study Ecs on diabetes-related factors AGES, IGF-1and PI3K/ILK signaling pathways
Purpose:
Studies have shown that advanced glycation end products (AGEs) andinsulin-like growth factor-1(IGF-1) are closely linked with the occurrence ofvarious complications of diabetes. whether they are involved in the formation ofPCO diabetic rabbit? Whether Ecs played a role in suppressing PCO are relatedwith the content change of of these two factors and what signal factors involvedin the above process?In this study, we designed a series of experiments toexplore the answers to these questions in order to further understanding themolecular mechanism of Ecs inhibition of PCO development.
Methods:
rabbits were divided into nondiabetes group(NDM), diabetes group(DM)and Ecs group. Ten days or6weeks after extrocapsular lens extration (ECLE),all rabbits were executed and collected serum and aqueous humor, which weredetected the content change of AGEs and IGF-1by ELISA method. Theexpression of ILK, PI3K on diabetic rabbits lens posterior capsule membrane indiabetes group and Ecs group were determined by RT-PCR.
Results:
ELISA results indicated, at either10days or6weeks postoperative, theAGEs and IGF-1levels in serum and aqueous humor of diabetes rabbits weresignificantly higher than that of nondiabetes rabbit. Compared with diabetesgroup, the IGF-1in aqueous humor of Ecs group was significantly decreasedand the expressions of ILK and PI3K on posterior capsular were down-regulation.
Conclusion:
AGEs and IGF-1may be involved in the development of diabetic rabbitPCO, Ecs decreased the content of IGF1in aqueous humor may be one of thereasons for prevention PCO. ILK and PI3K are downstream signaling moleculesof integrin. Ecs inhibited the development of PCO by down-regulating ILK andPI3K expression.
引文
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