本文選題：糖尿病 + 后囊膜混濁　； 參考：《廣西醫(yī)科大學(xué)》2014年博士論文

【摘要】：糖尿病性白內(nèi)障是糖尿病第二位常見眼部并發(fā)癥，，手術(shù)是其唯一有效治療手段，后發(fā)性白內(nèi)障（即后囊膜混濁(posterior capsular opacification,PCO)）是白內(nèi)障摘除術(shù)后最常見并發(fā)癥，不僅導(dǎo)致術(shù)后視力下降，還對糖尿病眼后段病變的診斷和治療效果造成較大影響。晶狀體上皮細(xì)胞增殖和上皮-間質(zhì)轉(zhuǎn)分化（epithelial-mesenchymal transition, EMT）在PCO的發(fā)生和發(fā)展中起重要作用，而細(xì)胞間及細(xì)胞與細(xì)胞外基質(zhì)間粘附是其生存和增殖的基礎(chǔ)。細(xì)胞粘附分子-整合素參與調(diào)控細(xì)胞粘附、遷移、增殖及分化等眾多生理過程，對阻斷整合素的調(diào)控作用是否可以有效抑制糖尿病性PCO發(fā)生和發(fā)展的研究目前甚少。前期實(shí)驗(yàn)已證實(shí)去整合素Echistatin (Ecs)可明顯抑制體外培養(yǎng)的人晶狀體上皮細(xì)胞(LensePitheliumcells,LECs)的增殖、粘附及移行；并發(fā)現(xiàn)糖尿病兔晶狀體囊外摘除術(shù)后（extrocapsular lensextration，ECLE）出現(xiàn)PCO的最早時間（10天）和最顯著時間（6周）均較正常血糖兔（14天，12周）提前，同期后囊膜混濁程度較正常血糖兔提高。因此為探索去整合素能否有效抑制糖尿病性PCO的發(fā)生發(fā)展，本實(shí)驗(yàn)擬利用糖尿病兔眼晶狀體后囊膜混濁模型,觀察去整合素Ecs對糖尿病性PCO分級的影響，及體內(nèi)LECs增殖、細(xì)胞粘附和上皮-間質(zhì)轉(zhuǎn)分化（EMT）的變化；同時檢測血清和房水中糖尿病并發(fā)癥相關(guān)因子，即AGEs及IGF-1的含量變化，探討Ecs是否通過對糖尿病并發(fā)癥致病因子的調(diào)控從而影響PCO的發(fā)生發(fā)展；并進(jìn)一步探討Ecs對細(xì)胞內(nèi)信號傳導(dǎo)通路中的關(guān)鍵信號因子PI3K及其下游信號因子ILK基因表達(dá)的干預(yù)作用，初步明確Ecs在LECs中作用的分子途徑，為去整合素Ecs防治糖尿病性后發(fā)性白內(nèi)障提供有力的理論依據(jù)。 第一部分Ecs對糖尿病兔PCO分級，LECs增殖、細(xì)胞間連接及EMT的影響 目的： LECs在晶狀體后囊膜上的增殖和上皮細(xì)胞-間質(zhì)轉(zhuǎn)化（EMT）是形成PCO的重要環(huán)節(jié)。細(xì)胞粘附則是LECs生存及出現(xiàn)其他生物學(xué)行為的基礎(chǔ)，細(xì)胞間連接蛋白的表達(dá)可以在一定程度上反映細(xì)胞粘附的情況。亦是PCO形成的重要環(huán)節(jié)。整合素不僅介導(dǎo)了細(xì)胞的粘附，還在細(xì)胞增殖、EMT中發(fā)揮了重要作用，本實(shí)驗(yàn)擬通過觀察整合素的阻滯劑-去整合素echistatin對糖尿病兔PCO形成，LECs增殖、粘附及間質(zhì)轉(zhuǎn)化的影響，了解去整合素是否能有效抑制糖尿病兔PCO的發(fā)生發(fā)展，并嘗試闡述其分子機(jī)制。 方法： 應(yīng)用四氧嘧啶建立糖尿病兔模型，每只兔右眼均行晶狀體囊外摘除術(shù)（extrocapsular lens extration，ECLE），術(shù)畢隨機(jī)分組，對照組在術(shù)眼晶狀體囊袋內(nèi)注入0.2ml高壓蒸餾水，Ecs組則注入0.2ml10μg/ml Ecs。分別在術(shù)后10天（10d）及6周（6w）觀察以下指標(biāo)：⑴裂隙燈下觀察術(shù)眼晶狀體后囊膜混濁情況并分級；⑵應(yīng)用免疫組化法檢測Ecs對LECs中PCNA表達(dá)的影響。⑶應(yīng)用免疫熒光法檢測Ecs對晶狀體后囊膜上LECs中連接蛋白43表達(dá)的影響；⑷應(yīng)用免疫組化法和熒光定量PCR(Real-TimeQuantitative PCR, RT-PCR)法檢測Ecs對晶狀體后囊膜上α-SMA和Ⅳ型膠原表達(dá)的影響。 結(jié)果： ⑴術(shù)后10天對照組和Ecs組間PCO分級無明顯差異(P=0.495)，術(shù)后6周Ecs可明顯降低糖尿病兔PCO分級(P=0.025)。⑵術(shù)后10天及6周Ecs 組LECs中PCNA的表達(dá)明顯低于對照組（p=0.031,0.021）。⑶術(shù)后10天及6周對照組晶狀體后囊膜上Cx43表達(dá)明顯增強(qiáng)，而Ecs組僅見少量表達(dá)，兩組間存在顯著差異（p=0.007,0.001），提示Ecs可有效抑制糖尿病兔模型中LECs間連接蛋白43的表達(dá)。⑷術(shù)后10天，免疫組化及RT-PCR結(jié)果均顯示α-SMA在晶狀體后囊膜上的表達(dá)兩組間無明顯差異(p0.05)，術(shù)后6周對照組晶狀體后囊膜上α-SMA表達(dá)顯著增多，而Ecs組僅少量增加，表達(dá)較對照組明顯降低(p0.05)，提示Ecs可有效抑制術(shù)后晚期α-SMA的表達(dá)。⑸RT-PCR結(jié)果顯示晶狀體后囊膜上Ⅳ型膠原表達(dá)，術(shù)后10天兩組間無明顯差異(p=0.157)，術(shù)后6周Ecs組較對照組表達(dá)明顯增降低(p=0.049)；而免疫組化結(jié)果顯示無論是10d或6w，Ecs組Ⅳ型膠原表達(dá)均較對照組顯著減少（p=0.037,0.001）。提示Ecs可有效抑制Ⅳ型膠原的表達(dá)，可能部分Ⅳ型膠原mRNA未翻譯為蛋白。 結(jié)論： 糖尿病兔PCO模型中，Ecs可有效抑制PCNA、Cx43、α-SMA、Ⅳ型膠原表達(dá)，降低PCO的分級，抑制PCO的發(fā)生和發(fā)展。 第二部分Ecs對糖尿病相關(guān)因子AGEs、IGF-1及PI3K/ILK信號通路的影響研究 目的： 晚期糖基化終末產(chǎn)物（advanced glycation end products，AGEs）和胰島素樣生長因子-1（insulin-like growth actor1，IGF-1）與多種糖尿病并發(fā)癥的發(fā)生均有密切關(guān)系，其是否也參與了糖尿病兔PCO的形成？ Ecs發(fā)揮抑制PCO的作用是否與改變以上兩種糖尿病并發(fā)癥相關(guān)因子的表達(dá)相關(guān)？整合素信號通路中哪些信號因子參與了上述作用的調(diào)控？本課題中我們通過一系列實(shí)驗(yàn)來探討以上三個問題的答案，以期進(jìn)一步了解Ecs抑制PCO發(fā)生發(fā)展的機(jī)制。 方法： 將兔子分為正常血糖對照組，糖尿病組及Ecs組，所有兔子行ECLE術(shù)，分別采集術(shù)后10天和6周血清及術(shù)眼房水，通過ELISA法檢測各標(biāo)本中AGEs和IGF-1含量的變化。取糖尿病組及Ecs組中兔術(shù)眼晶狀體后囊膜，通過RT-PCR法檢測ILK、PI3K的基因表達(dá)變化。 結(jié)果： ELISA檢測結(jié)果提示術(shù)后10d及6w，糖尿病組及Ecs組血清和房水中AGEs和IGF-1的含量均明顯高于對照組（P0.05）；在糖尿病兔模型中，Ecs組房水中IGF-1含量明顯低于糖尿病組（P0.05），但血清及房水中AGEs含量及血清中IGF-1含量Ecs組與糖尿病組間差異無統(tǒng)計學(xué)意義（P0.05）。RT-PCR檢測結(jié)果顯示術(shù)后10天及6周，與糖尿病組相比，Ecs組晶狀體后囊膜上PI3K及ILK mRNA表達(dá)下調(diào)（P0.05）。 結(jié)論： AGEs和IGF-1可能參與了糖尿病兔PCO的發(fā)生發(fā)展過程，Ecs對糖尿病兔房水中IGF-1的表達(dá)有明顯抑制作用，可能是其抑制PCO發(fā)展的機(jī)制之一。Ecs通過下調(diào)糖尿病兔晶狀體后囊膜上ILK和PI3K的表達(dá)，阻礙ILK和PI3K之間信號傳導(dǎo)，從而抑制PCO發(fā)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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R587.2;R776.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 李祖國,張進(jìn)華,鄧永健,丁彥青;整合素β_1對大腸癌細(xì)胞與血管內(nèi)皮細(xì)胞間細(xì)胞通訊的影響[J];第一軍醫(yī)大學(xué)學(xué)報;2001年03期

2 陳衛(wèi);蘇踴躍;梁光萍;陳建;張曼輝;羅向東;;人β1整合素近端和遠(yuǎn)端啟動子報告基因載體的構(gòu)建和鑒定及啟動活性分析[J];醫(yī)學(xué)研究生學(xué)報;2007年02期

3 談艷,杜勤,盛凈;高糖對大鼠GMC表面β_1整合素表達(dá)和ECM分泌的影響[J];上海第二醫(yī)科大學(xué)學(xué)報;2004年08期

4 朱玉廣;朱艷;王杰;鐘瑩瑩;杜孝楠;張榮;;整合素-β1在轉(zhuǎn)化生長因子-β2誘導(dǎo)晶狀體上皮細(xì)胞轉(zhuǎn)化中的作用[J];山東大學(xué)學(xué)報(醫(yī)學(xué)版);2012年11期

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