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μ悄虿〖捌洳l(fā)癥防治予以重視。在眼科領(lǐng)域,糖尿病角膜病變在糖尿病患者比較常見,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)病機制尚不明確,其發(fā)生的機制以及治療對策仍是臨床中亟待解決的科學(xué)問題。 沉默信號調(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。已有研究證實SIRT1在糖尿病角膜中表達顯著下調(diào),過量表達SIRT1后可以顯著促進糖尿病小鼠角膜上皮的損傷修復(fù)。但引起糖尿病角膜SIRT1表達下調(diào)的原因尚不清楚。 MicroRNAs(miRNAs, miRs)是一類長度約為22個核苷酸(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。它參與了多個生物學(xué)進程如發(fā)育、細胞死亡、細胞增殖、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,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的表達進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。 第一部分篩選microRNAs及對靶基因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,通過實時定量PcR(Quantitative ReaI time PCR,qRT-PCR)的方法在糖尿病小鼠角膜上皮組織進行驗證；采用miRNA的模擬物或抑制劑轉(zhuǎn)染正常小鼠角膜緣上皮干／祖細胞系(TKE2)細胞,從而實現(xiàn)miRNA在TKE2細胞的過量或者下降的表達 結(jié)果：從生物信息學(xué)結(jié)果中挑選出9個miRNAs用于其后的qRT-PCR的驗證,其中miR-204-5p用于后續(xù)的研究,與非糖尿病的對照小鼠角膜上皮組織相比,其表達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表達顯著下調(diào)或上調(diào)。雙熒光素酶報告基因分析結(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表達顯著上調(diào)。MiR-204-5p能負性調(diào)控SIRT1,其高表達是導(dǎo)致糖尿病角膜上皮中Sirtl低表達的原因之一。 第二部分調(diào)控microRNA-204-5p通過SIRT1促進高糖環(huán)境下小鼠角膜緣上皮干／祖細胞系(TKE2)細胞周期循環(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細胞：q RT-PCR/Western blot檢測SIRT1,細胞周期相關(guān)蛋白cyclin D1.p21.p16的表達；MTT法檢測細胞增殖變化；PI染色法流失細胞儀檢測細胞周期變化。 結(jié)果：TKE2細胞轉(zhuǎn)染miR-204-5p的抑制物后顯著促進了高糖環(huán)境下細胞生長與細胞周期的循環(huán),Sirtl與Cyclin D1的表達上調(diào),而p16的表達下調(diào)。 結(jié)論：抑制miR-204-5p的表達,可通過對Sirtl的調(diào)控,促進高糖環(huán)境下被阻滯的角膜上皮細胞周期循環(huán) 第三部分調(diào)控microRNA-204-5p通過SIRT1促進糖尿病小鼠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：建立機械性角膜上皮損傷小鼠動物模型,結(jié)膜下注射miR-204-5p,觀察建模成功后0-72小時內(nèi)角膜上皮缺損面積,用于評價該miRNA對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,細胞周期相關(guān)蛋白cyclin D1、 p21、 p16的表達。 結(jié)果：對Ins2AKita/+小鼠結(jié)膜下注射miR-204-5p的模擬物后,顯著促進了角膜上皮的損傷修復(fù),該過程中SIRTl的表達增加,激活Cyclin D1/CDK1途徑,Cyclin D1的表達增加而p16的表達下降。 結(jié)論：抑制miR-204-5p的表達,能通過Sirt1促進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)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R587.2;R772.2

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1 黃劍，，蔡愛玲，李社會;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

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