Sirt1介导miR-182对糖尿病角膜神经的保护作用及其分子机制的研究

发布时间：2018-04-01 03:35

本文选题：SIRT1　切入点：mi　出处：《青岛大学》2015年博士论文

【摘要】：目的:糖尿病角膜神经病变属于糖尿病周围神经病变,是引发糖尿病角膜病变的高危因素,充分阐明糖尿病角膜神经损害的发生机制,为全面阐明糖尿病周围神经病变的发病机制提供了全新的内容。本研究通过沉默信号调控因子(silence signal regulating factor 1,Sirt1)作用于微小RNA(mi RNA),并靶向NOX4基因,实现对糖尿病角膜神经损害的保护作用及其分子机制进行研究。方法:本研究选用2型糖尿病模型BKS.Cg-m+/+Leprdb/J小鼠(简称BKS-db/db小鼠)作为动物模型,首先探讨了SIRT1在三叉神经节(trigeminal ganglion,TG)组织上的表达。采用腺病毒系统,构建了过量表达SIRT1基因的腺病毒,通过结膜下注射的方法,使SIRT1在TG组织中过量表达,然后采用mi RNA Array基因芯片技术,检测TG组织中受到SIRT1调控的一系列m RNAs。通过生物信息学的分析,探讨mi R-182可能的靶基因,并在体外培养的TG神经细胞上进行验证。体外原代培养小鼠TG三叉神经节细胞,检测mi R-182 agomir对体外原代培养的TG细胞突触生长及存活情况的影响。建立BKS-db/db小鼠角膜损伤的动物模型,结膜下注射mi R-182的模拟物或者抑制剂,72小时后收集角膜标本,检测上皮细胞与神经修复相关基因与蛋白的表达,并行角膜神经染色,观察角膜神经的变化。通过Targetscan等软件,预测mi R-182的靶基因。筛选验证NOX4为mi R-182的目标基因。通过结膜下注射mi R-182模拟及和NOX4过表达腺病毒和NOX4干扰RNA的方法,观察角膜上皮愈合的情况,通过角膜知觉、以及β-tublin角膜神经染色客观评价角膜末梢神经修复情况。结果:通过western blot以及定量PCR技术,结合免疫荧光共定位技术,发现SIRT1在2型糖尿病小鼠TG神经节的表达显著下降。通过mi RNA Array基因芯片技术,共发现了受到SIRT1调控的差异表达明显的mi RNA共29条,其中表达上调基因19条,表达下调基因11条。选取其中随SIRT1表达升高,差异表达最显著的mi R-182为研究对象进行进一步研究。体外培养TG细胞,应用SIRT1的两种激活剂,分别为SRT1720与白藜芦醇(RSV),处理原代培养的TG细胞后,发现随SIRT1的表达显著升高,mi R-182的表达也显著升高。为进一步验证这个结果,构建了过量表达SIRT1的腺病毒系统,同时也构建了SIRT1干扰的腺病毒系统,分别作用于体外原代培养的TG细胞,发现随SIRT1的表达显著升高,mi R-182的表达也显著升高;随SIRT1的表达显著降低,mi R-182的表达也显著降低。由此说明SIRT1调控mi R-182的表达。应用mi R-182 agomir处理原代培养的三叉神经节细胞,结果显示,与正常培养三叉神经节细胞相比,mi R-182 agomir处理后三叉神经节细胞突触长度显著增加。与对照组BKS-db/+小鼠相比,24周龄的Ⅱ型糖尿病BKS-db/db小鼠一致维持高血糖、高糖化血红蛋白、多饮、多食、多尿等糖尿病基本体征;通过免疫荧光、免疫组化、q RT-PCR检测显示,与db/+对照小鼠比较,SIRT1在BKS-db/db小鼠的TG和角膜中表达显著下调。结膜下注射mi R-182 agomir后可以显著促进BKS-db/db小鼠角膜上皮损伤后的愈合,并显著促进角膜神经的修复,恢复角膜敏感度。通过免疫荧光、免疫组化、q RT-PCR检测显示,与db/+对照小鼠比较,NOX4在BKS-db/db小鼠的TG细胞和角膜中表达显著上调。通过结膜下注射过表达NOX4腺病毒,可以显著阻断mi R-182的上皮和神经保护作用,而通过结膜下注射NOX4的干扰RNA,可以显著促进BKS-db/db小鼠角膜上皮损伤后的愈合,并显著促进角膜神经的修复。结论:本研究发现了对受Sirt1调控的mi R-182通过直接靶向NOX4具有促进三叉神经节神经元和糖尿病角膜神经病变的神经再生的作用。
[Abstract]:Objective: diabetic corneal neuropathy of diabetic peripheral neuropathy is the risk factors of diabetic keratopathy, the mechanism to fully elucidate the corneal nerve damage in diabetes, and provide a new content for the elucidation of the pathogenesis of diabetic peripheral neuropathy. The silencing signal regulation factor (silence signal regulating factor 1, Sirt1) the role of RNA (Mi RNA) in tiny, and targeting NOX4 gene, the protective effect and molecular mechanism of corneal nerve damage in diabetes research. Methods: This study selected 2 BKS.Cg-m+/+Leprdb/J diabetic mice (BKS-db/db mice) as the animal model, the research of the SIRT1 in the trigeminal ganglion (trigeminal ganglion TG) expression tissue. By adenovirus system, overexpression of SIRT1 adenovirus, through the method of subconjunctival injection, The overexpression of SIRT1 in TG tissues, and then the MI RNA Array gene chip technology, bioinformatics analysis through a series of M RNAs. SIRT1 regulated by detection TG organization, to explore the target genes of MI R-182, and verified in the TG neural cells cultured in vitro. The cultured mouse trigeminal TG ganglion cells, growth and survival of MI R-182 agomir on the detection of in vitro cultured TG cell synapse. To establish an animal model of BKS-db/db mice corneal injury, subconjunctival injection of MI R-182 mimics or inhibitors, 72 hours after collection of corneal specimens to detect the expression of epithelial cells and neural repair related genes and proteins, parallel corneal nerve staining, observe the changes of corneal nerve. By Targetscan software, the predicted target gene of MI R-182. Screening and verification of NOX4 as the target gene mi R-182. By subconjunctival injection of M I R-182 and NOX4 simulation and method of expression of adenovirus and NOX4 interference RNA, observe the corneal epithelial healing, corneal through perception, and beta -tublin staining of corneal nerve objective evaluation of corneal nerve repair. Results: by Western blot and quantitative PCR technology, CO localization technology combined with immunofluorescence, found the expression of SIRT1 in 2 diabetic mice TG ganglion significantly decreased. Through the MI RNA Array gene chip technology, the differences were found by the regulation of SIRT1 expression of MI RNA was 29, of which 19 genes were upregulated and 11 downregulated genes were selected. With the increased expression of SIRT1, expression of the most significant mi R-182 as the research object for the further study. TG cells were cultured in vitro, using SIRT1 two activator, respectively SRT1720 and resveratrol (RSV), the primary cultured TG cells after expression with SIRT1 Increased expression of MI R-182 was also increased. In order to further verify the results, we constructed the overexpression adenovirus SIRT1 system, but also to construct adenovirus system SIRT1 interference, respectively. The effect of in vitro cultured TG cell, with the expression of SIRT1 was significantly increased, the expression of MI R-182 significantly increased with the expression of SIRT1 decreased significantly; the expression of MI, R-182 were significantly decreased. The expression of SIRT1 regulatory mi R-182. The application results of MI R-182 agomir in primary cultured trigeminal ganglion cells showed that compared with normal cultured trigeminal ganglion cells, trigeminal ganglion cell synapse length increased significantly mi R-182 after agomir treatment. Compared with the control group of BKS-db/+ mice, 24 weeks of age of type II diabetic BKS-db/db mice maintained high blood glucose, HbA1c, polydipsia, polyphagia, polyuria, diabetes group by body syndrome; Immunofluorescence, immunohistochemistry, Q RT-PCR detection, and db/+ control mice, the expression of SIRT1 in BKS-db/db mice and TG in corneas were significantly reduced. Subconjunctival injection of MI R-182 after agomir can significantly promote BKS-db/db murine corneal epithelial injury healing, and promote repair of corneal nerve. The recovery of corneal sensitivity through. Immunofluorescence, immunohistochemistry, q RT-PCR detection, and db/+ control mice, NOX4 expression was significantly up-regulated in TG cells and corneal BKS-db/db mice. By subconjunctival injection of overexpression of NOX4 adenovirus can significantly block epithelial mi R-182 and neuroprotective effects, and by interfering with RNA subconjunctival injection of NOX4. BKS-db/db can significantly promote murine corneal epithelial healing after injury, and promote repair of corneal nerve. Conclusion: This study found that the Sirt1 regulated mi R-182 by directly targeting NOX4 It has the effect of promoting nerve regeneration of trigeminal ganglion neurons and diabetic corneal neuropathy.

【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R587.2;R772.2

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