本文選題：MicroRNA-204-5p + SIRT1�。� 參考：《華中科技大學(xué)》2015年博士論文

【摘要】：糖尿病(Diabetes Mellitus, DM)是一組以慢性血糖水平增高為特征的代謝性疾病,是由于胰島素分泌和(或)作用缺陷所引起。近年來糖尿病發(fā)病率持續(xù)增高,人們?nèi)找鎸?duì)糖尿病及其并發(fā)癥防治予以重視。在眼科領(lǐng)域,糖尿病角膜病變?cè)谔悄虿』颊弑容^常見,糖尿病患者眼部手術(shù)術(shù)后角膜上皮的遷延不愈是臨床棘手的難題。糖尿病角膜病變主要臨床表現(xiàn)為淺層點(diǎn)狀角膜炎或持續(xù)性上皮缺損以及角膜知覺的減退。很多研究表明角膜上皮基底膜的異常增厚和角膜上皮細(xì)胞功能改變共同導(dǎo)致了糖尿病角膜病變。目前糖尿病角膜病變的發(fā)病機(jī)制尚不明確,其發(fā)生的機(jī)制以及治療對(duì)策仍是臨床中亟待解決的科學(xué)問題。 沉默信號(hào)調(diào)控因子1(silence signal regulating factor1, SlRT1)是一種組蛋白脫乙�；�,參與糖代謝和胰島素分泌等多條代謝通路,被認(rèn)為是治療糖尿病的新靶點(diǎn)。已有研究證實(shí)SIRT1在糖尿病角膜中表達(dá)顯著下調(diào),過量表達(dá)SIRT1后可以顯著促進(jìn)糖尿病小鼠角膜上皮的損傷修復(fù)。但引起糖尿病角膜SIRT1表達(dá)下調(diào)的原因尚不清楚。 MicroRNAs(miRNAs, miRs)是一類長度約為22個(gè)核苷酸(nucleotide, nt)的內(nèi)源性非編碼RNA,通過與其靶蛋白的3端非編碼區(qū)(Untranslated Regions, UTRs)區(qū)結(jié)合,完成轉(zhuǎn)錄后水平的基因調(diào)控。它參與了多個(gè)生物學(xué)進(jìn)程如發(fā)育、細(xì)胞死亡、細(xì)胞增殖、中樞神經(jīng)系統(tǒng)功能及腫瘤形成等。它能通過調(diào)控眾多靶基因從而發(fā)揮生物學(xué)效應(yīng)。目前對(duì)miRNAs的研究雖廣泛,但有關(guān)糖尿病角膜病變的miRNAs研究少且不深入。因此本研究選擇圍繞SIRT1這一糖尿病的重要靶點(diǎn),擬從表觀遺傳學(xué)的角度探討是否在角膜組織中存在某種或某些miRNAs,它能否通過影響SIRT1的表達(dá)進(jìn)而改變糖尿病角膜上皮的損傷修復(fù)能力。 本研究目的是篩選角膜上皮中可能調(diào)控SIRTl的miRNAs,探討所篩選出的miRNAs通過SIRT1在糖尿病角膜病變中的作用,以期探明其是否參與糖尿病角膜病變以及和提供新的治療靶點(diǎn)。 第一部分篩選microRNAs及對(duì)靶基因SIRT1的作用研究 目的：篩選糖尿病角膜病變中調(diào)控Sirt1表達(dá)的microRNA (miRNA) 方法：采用生物信息學(xué)的方法預(yù)測調(diào)控Sirtl的可能miRNAs,通過實(shí)時(shí)定量PcR(Quantitative ReaI time PCR,qRT-PCR)的方法在糖尿病小鼠角膜上皮組織進(jìn)行驗(yàn)證；采用miRNA的模擬物或抑制劑轉(zhuǎn)染正常小鼠角膜緣上皮干／祖細(xì)胞系(TKE2)細(xì)胞,從而實(shí)現(xiàn)miRNA在TKE2細(xì)胞的過量或者下降的表達(dá) 結(jié)果：從生物信息學(xué)結(jié)果中挑選出9個(gè)miRNAs用于其后的qRT-PCR的驗(yàn)證,其中miR-204-5p用于后續(xù)的研究,與非糖尿病的對(duì)照小鼠角膜上皮組織相比,其表達(dá)是糖尿病角膜上皮組織的5.16倍。正常TKE2轉(zhuǎn)染miR-204-5p的模擬物或抑制物后,SIRT1表達(dá)顯著下調(diào)或上調(diào)。雙熒光素酶報(bào)告基因分析結(jié)果顯示miR-204-5p的靶基因是Sirt1。結(jié)論：在糖尿病角膜中miR-204-5p表達(dá)顯著上調(diào)。MiR-204-5p能負(fù)性調(diào)控SIRT1,其高表達(dá)是導(dǎo)致糖尿病角膜上皮中Sirtl低表達(dá)的原因之一。 第二部分調(diào)控microRNA-204-5p通過SIRT1促進(jìn)高糖環(huán)境下小鼠角膜緣上皮干／祖細(xì)胞系(TKE2)細(xì)胞周期循環(huán) 目的：探究miR-204-5p通過SIRTl是否能影響因高糖而被阻滯TKE2細(xì)胞周期循環(huán)。 方法：MiR-204-5p的抑制劑轉(zhuǎn)染高糖環(huán)境下的TKE2細(xì)胞：q RT-PCR/Western blot檢測SIRT1,細(xì)胞周期相關(guān)蛋白cyclin D1.p21.p16的表達(dá)；MTT法檢測細(xì)胞增殖變化；PI染色法流失細(xì)胞儀檢測細(xì)胞周期變化。 結(jié)果：TKE2細(xì)胞轉(zhuǎn)染miR-204-5p的抑制物后顯著促進(jìn)了高糖環(huán)境下細(xì)胞生長與細(xì)胞周期的循環(huán),Sirtl與Cyclin D1的表達(dá)上調(diào),而p16的表達(dá)下調(diào)。 結(jié)論：抑制miR-204-5p的表達(dá),可通過對(duì)Sirtl的調(diào)控,促進(jìn)高糖環(huán)境下被阻滯的角膜上皮細(xì)胞周期循環(huán) 第三部分調(diào)控microRNA-204-5p通過SIRT1促進(jìn)糖尿病小鼠C57BL/6J-INS2AKita (INS2Akita/+)角膜上皮損傷修復(fù)究 目的：探究在糖尿病角膜中miR-204-5p通過SIRTl是否能影響角膜上皮損傷修復(fù)。 方法：建立機(jī)械性角膜上皮損傷小鼠動(dòng)物模型,結(jié)膜下注射miR-204-5p,觀察建模成功后0-72小時(shí)內(nèi)角膜上皮缺損面積,用于評(píng)價(jià)該miRNA對(duì)糖尿病角膜上皮損傷修復(fù)的作用。qRT-PCR/Western blot檢測角膜中SIRT1,細(xì)胞周期相關(guān)蛋白cyclin D1、 p21、 p16的表達(dá)。 結(jié)果：對(duì)Ins2AKita/+小鼠結(jié)膜下注射miR-204-5p的模擬物后,顯著促進(jìn)了角膜上皮的損傷修復(fù),該過程中SIRTl的表達(dá)增加,激活Cyclin D1/CDK1途徑,Cyclin D1的表達(dá)增加而p16的表達(dá)下降。 結(jié)論：抑制miR-204-5p的表達(dá),能通過Sirt1促進(jìn)糖尿病角膜上皮的損傷修復(fù)。
[Abstract]:Diabetes Mellitus ( DM ) is a group of metabolic diseases characterized by elevated levels of chronic blood glucose , which are caused by insulin secretion and / or deficiency of action . In the field of ophthalmology , diabetic keratopathy is more common in diabetic patients . In the field of ophthalmology , diabetic keratopathy is more common in diabetic patients .

The silencing signal regulating factor 1 ( SlRT1 ) is a new target for the treatment of diabetes , which is a new target for the treatment of diabetes mellitus .

MicroRNAs ( miRs ) are endogenous non - coding RNAs with a length of about 22 nucleotides ( nucleotides , nt ) , which are combined with the 3 - terminal non - coding region of their target protein to complete the gene regulation at post - transcriptional level . It has been involved in many biological processes such as development , cell death , cell proliferation , central nervous system function and tumor formation .

The aim of this study was to screen the possible regulation of SIRTl in corneal epithelium and investigate the role of SIRT1 in diabetic keratopathy , with a view to exploring whether it was involved in diabetic keratopathy and providing a new treatment target .

The first part of screening microRNAs and the effect on target gene SIRT1

Objective : To screen the microRNA ( miRNA ) regulating Sirt1 expression in diabetic keratopathy .

Methods : Using bioinformatics methods to predict the possible expression of Sirtl , we used real - time quantitative PCR ( qRT - PCR ) to verify the corneal epithelial tissue of diabetic mice .
miRNA - based mimics or inhibitors were used to transfected normal mouse limbal epithelial stem / progenitor cell line ( TKE2 ) cells , thereby enabling overexpression or decreased expression of miRNA in TKE2 cells

Results : The expression of miR - 204 - 5p was 5 . 16 times that of non - diabetic control mice . The expression of miR - 204 - 5p was significantly downregulated or up - regulated after normal TKE2 transfection of miR - 204 - 5p . Conclusion : The expression of miR - 204 - 5p in diabetic cornea is significantly up - regulated . Conclusion : The high expression of miR - 204 - 5p is one of the causes of low expression of Sirtl in diabetic corneal epithelium .

The second part regulates microRNA - 204 - 5p to promote cell cycle circulation of mouse corneal epithelial stem / progenitor cell line ( TKE2 ) in high - sugar environment through SIRT1 .

Objective : To investigate the effect of miR - 204 - 5p on cell cycle of TKE2 blocked by high glucose .

Methods : The inhibitors of MiR - 204 - 5p were transfected into TKE2 cells in high - sugar environment : q - RT - PCR / Western blot was used to detect the expression of cyclin D1.p21 . p16 .
MTT assay was used to detect cell proliferation .
Cell cycle changes were detected by PI staining .

Results : After transfection of the inhibitor of miR - 204 - 5p by TKE2 cells , the cycle of cell growth and cell cycle in high glucose environment was significantly promoted , and the expression of Sirtl and Cyclin D1 was up - regulated , while the expression of p16 was down regulated .

Conclusion : The expression of miR - 204 - 5p can be inhibited and the cycle of corneal epithelial cells blocked in high glucose environment can be promoted by regulating Sirtl .

The third part regulates microRNA - 204 - 5p to promote the repair of corneal epithelial damage in diabetic mice C57BL / 6 J - INS2AKita ( INS2Akita / + ) by SIRT1 .

Objective : To investigate whether miR - 204 - 5p can influence corneal epithelial damage repair by SIRTl in diabetic cornea .

Methods : To establish an animal model of mechanical corneal epithelium injury in mice . miR - 204 - 5p was injected into the conjunctiva , and the area of corneal epithelium defect was observed within 0 - 72 hours after successful modeling . The expression of SIRT1 , cyclin D1 , p21 and p16 in the cornea was detected by qRT - PCR / Western blot .

Results : After injection of miR - 204 - 5p in the conjunctiva of Ins2AKita / + mice , the repair of corneal epithelium was significantly promoted , the expression of SIRTl increased , the expression of Cyclin D1 / CDK1 and Cyclin D1 increased , and the expression of p16 was decreased .

Conclusion : Inhibition of miR - 204 - 5p expression can promote the repair of diabetic corneal epithelium by Sirt1 .

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R587.2;R772.2

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1 黃劍，，蔡愛玲，李社會(huì);風(fēng)濕熱引起角膜病變一例[J];眼科研究;1995年02期

2 崔巍,高偉,賀q

本文編號(hào)：1782977