本文選題：SIRT1　切入點：mi　出處：《青島大學》2015年博士論文

【摘要】：目的:糖尿病角膜神經(jīng)病變屬于糖尿病周圍神經(jīng)病變,是引發(fā)糖尿病角膜病變的高危因素,充分闡明糖尿病角膜神經(jīng)損害的發(fā)生機制,為全面闡明糖尿病周圍神經(jīng)病變的發(fā)病機制提供了全新的內(nèi)容。本研究通過沉默信號調(diào)控因子(silence signal regulating factor 1,Sirt1)作用于微小RNA(mi RNA),并靶向NOX4基因,實現(xiàn)對糖尿病角膜神經(jīng)損害的保護作用及其分子機制進行研究。方法:本研究選用2型糖尿病模型BKS.Cg-m+/+Leprdb/J小鼠(簡稱BKS-db/db小鼠)作為動物模型,首先探討了SIRT1在三叉神經(jīng)節(jié)(trigeminal ganglion,TG)組織上的表達。采用腺病毒系統(tǒng),構建了過量表達SIRT1基因的腺病毒,通過結膜下注射的方法,使SIRT1在TG組織中過量表達,然后采用mi RNA Array基因芯片技術,檢測TG組織中受到SIRT1調(diào)控的一系列m RNAs。通過生物信息學的分析,探討mi R-182可能的靶基因,并在體外培養(yǎng)的TG神經(jīng)細胞上進行驗證。體外原代培養(yǎng)小鼠TG三叉神經(jīng)節(jié)細胞,檢測mi R-182 agomir對體外原代培養(yǎng)的TG細胞突觸生長及存活情況的影響。建立BKS-db/db小鼠角膜損傷的動物模型,結膜下注射mi R-182的模擬物或者抑制劑,72小時后收集角膜標本,檢測上皮細胞與神經(jīng)修復相關基因與蛋白的表達,并行角膜神經(jīng)染色,觀察角膜神經(jīng)的變化。通過Targetscan等軟件,預測mi R-182的靶基因。篩選驗證NOX4為mi R-182的目標基因。通過結膜下注射mi R-182模擬及和NOX4過表達腺病毒和NOX4干擾RNA的方法,觀察角膜上皮愈合的情況,通過角膜知覺、以及β-tublin角膜神經(jīng)染色客觀評價角膜末梢神經(jīng)修復情況。結果:通過western blot以及定量PCR技術,結合免疫熒光共定位技術,發(fā)現(xiàn)SIRT1在2型糖尿病小鼠TG神經(jīng)節(jié)的表達顯著下降。通過mi RNA Array基因芯片技術,共發(fā)現(xiàn)了受到SIRT1調(diào)控的差異表達明顯的mi RNA共29條,其中表達上調(diào)基因19條,表達下調(diào)基因11條。選取其中隨SIRT1表達升高,差異表達最顯著的mi R-182為研究對象進行進一步研究。體外培養(yǎng)TG細胞,應用SIRT1的兩種激活劑,分別為SRT1720與白藜蘆醇(RSV),處理原代培養(yǎng)的TG細胞后,發(fā)現(xiàn)隨SIRT1的表達顯著升高,mi R-182的表達也顯著升高。為進一步驗證這個結果,構建了過量表達SIRT1的腺病毒系統(tǒng),同時也構建了SIRT1干擾的腺病毒系統(tǒng),分別作用于體外原代培養(yǎng)的TG細胞,發(fā)現(xiàn)隨SIRT1的表達顯著升高,mi R-182的表達也顯著升高;隨SIRT1的表達顯著降低,mi R-182的表達也顯著降低。由此說明SIRT1調(diào)控mi R-182的表達。應用mi R-182 agomir處理原代培養(yǎng)的三叉神經(jīng)節(jié)細胞,結果顯示,與正常培養(yǎng)三叉神經(jīng)節(jié)細胞相比,mi R-182 agomir處理后三叉神經(jīng)節(jié)細胞突觸長度顯著增加。與對照組BKS-db/+小鼠相比,24周齡的Ⅱ型糖尿病BKS-db/db小鼠一致維持高血糖、高糖化血紅蛋白、多飲、多食、多尿等糖尿病基本體征;通過免疫熒光、免疫組化、q RT-PCR檢測顯示,與db/+對照小鼠比較,SIRT1在BKS-db/db小鼠的TG和角膜中表達顯著下調(diào)。結膜下注射mi R-182 agomir后可以顯著促進BKS-db/db小鼠角膜上皮損傷后的愈合,并顯著促進角膜神經(jīng)的修復,恢復角膜敏感度。通過免疫熒光、免疫組化、q RT-PCR檢測顯示,與db/+對照小鼠比較,NOX4在BKS-db/db小鼠的TG細胞和角膜中表達顯著上調(diào)。通過結膜下注射過表達NOX4腺病毒,可以顯著阻斷mi R-182的上皮和神經(jīng)保護作用,而通過結膜下注射NOX4的干擾RNA,可以顯著促進BKS-db/db小鼠角膜上皮損傷后的愈合,并顯著促進角膜神經(jīng)的修復。結論:本研究發(fā)現(xiàn)了對受Sirt1調(diào)控的mi R-182通過直接靶向NOX4具有促進三叉神經(jīng)節(jié)神經(jīng)元和糖尿病角膜神經(jīng)病變的神經(jīng)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.

【學位授予單位】：青島大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R587.2;R772.2

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