【作者】 张媛；

【导师】 谢立信；

【作者基本信息】 武汉大学 ， 眼科学， 2021， 博士

【摘要】 背景:糖尿病是一类以血糖升高为特征的慢性代谢类疾病,也是全身大血管及微血管并发症的重要危险因素之一。在糖尿病患者中,约有47-64%的病人会并发糖尿病角膜病变(diabetic keratopathy,DK),DK的临床表现主要有干眼、角膜损伤修复延迟及神经营养性角膜溃疡等。目前,关于DK的研究,主要包括糖尿病角膜上皮病变和角膜神经病变两个方面,其中角膜神经来自于三叉神经节的眼部分支。微小RNAs(micro RNAs,miRNAs)是一类内源性单链非编码RNA,参与细胞增殖、分化和代谢等一系列重要的生命活动,且miRNA与DK的相关性也逐渐清晰。然而,DK的发病机制尚未完全阐明,miRNAs在DK中作用的研究亦相对缺乏。基于此,本研究运用了高通量测序手段,分析对比一型糖尿病及正常小鼠中,再生角膜上皮及三叉神经节组织差异表达的miRNAs和信使RNAs(messenger RNAs,mRNAs)。经过 miRNAs 和 mRNA 的关联分析,筛选并确定了 miR-223-5p/造血前列腺素 D 合成酶(hematopoietic prostaglandin D synthase,Hpgds)通路为下一步研究对象,并进一步研究miR-223-5p/Hpgds通路对糖尿病角膜上皮及神经再生的调控作用。第一部分糖尿病角膜上皮及神经再生中差异miRNA与mRNA的关联分析目的:运用高通量测序手段及生物信息学技术,筛选参与糖尿病角膜上皮及神经再生的miRNA/mRNA通路。方法:1.给予C57BL/6小鼠腹腔注射链脲佐菌素(streptozocin,STZ)构建一型糖尿病小鼠模型。检测正常与糖尿病小鼠血糖、体重及角膜敏感度指标。建立小鼠角膜上皮损伤模型,通过荧光素钠染色观察两组小鼠在0h、12h、24h和36h的角膜上皮修复情况。2.收集正常组和糖尿病组小鼠的三叉神经节组织和修复24h后的角膜上皮组织,利用高通量测序手段分析筛选差异表达的miRNAs(differentially expressed miRNAs,DEmiRNAs)和差异表达的mRNAs(differentially expressed mRNAs,DEmRNAs)。3.对正常组和糖尿病组小鼠的三叉神经节及再生角膜上皮组织中的DEmRNAs进行GO富集分析。4.通过靶基因预测分析,研究在糖尿病小鼠角膜上皮及神经再生中DEmiRNAs与DEmRNAs的关联性,构建miRNA/mRNA调控网络。5.用qRT-PCR对miRNA/mRNA调控网络中相对可信的miRNA/mRNA通路进行初步验证。结果:1.与年龄匹配的正常小鼠相比,我们构建的糖尿病小鼠模型具有血糖升高,体重下降,角膜敏感度降低,角膜损伤修复延迟等与DK相关的特征。2.通过高通量测序手段,发现与正常小鼠相比,糖尿病小鼠三叉神经节中有68个miRNAs和114个mRNAs发生表达变化。在再生角膜上皮中,77个miRNAs和186个mRNAs发生差异表达。文献调研表明,部分差异基因与糖尿病及其并发症密切相关。3.对参与糖尿病角膜神经病变及角膜上皮再生的DEmRNAs进行GO分析后发现,DEmRNAs能富集于许多与糖尿病相关的生物学过程中。4.TargetScan和miRDB数据库共同用来进行靶基因预测,结合评分筛选可信靶基因后发现,在糖尿病三叉神经节组织中,8个DEmiRNAs能靶向调控7个DEmRNAs,形成8条miRNA/mRNA通路。而再生的糖尿病角膜上皮中,12个miRNAs能靶向 9个DEmRNAs,形成 1 3条miRNA/mRNA通路。5.通过qRT-PCR验证,我们初步筛选出了,糖尿病三叉神经节组织中的miR-350-5p/主要尿蛋白20(major urinary protein 20,Mup20)、miR-592-5p/血管生成素样蛋白 7(angiopoietin-Like 7,Angptl7)、miR-8114/Hist1h2bc、miR-351-5p/超长链脂肪酸延伸酶6(elongation of very long-chain fatty acids family member 6,Elovl6)和miR-1291/Elovl6信号通路,以及再生的糖尿病角膜上皮中的miR-223-5p/Hpgds、miR-210-5p/间皮素(mesothelin,Msln)和miR-212-3p/高迁移率族AT Hook蛋白2(high-mobility group AT-hook 2 protein,Hmga2)信号通路。结论:我们通过高通量测序手段以及生物信息学分析,初步筛选出了参与糖尿病角膜上皮及神经再生的miRNA/mRNA信号通路,为下一步的机制研究提供了研究靶点。第二部分miR-223-5p靶向Hpgds参与调控糖尿病小鼠角膜上皮及神经再生目的:进一步探讨miR-223-5p/Hpgds信号通路对糖尿病小鼠角膜上皮及神经再生的影响。方法:1.将小鼠分为正常小鼠角膜上皮损伤组(NW组),糖尿病小鼠角膜上皮损伤组(DW组),糖尿病小鼠角膜上皮损伤给予miR-223-5p拮抗剂(antagomir)处理组(DW+miR-223-5p antagomir组),糖尿病小鼠角膜上皮损伤给予miRNA拮抗剂对照(miRNA antagomir negative control,miRNA antagomir NC)处理组(DW+miRNA antagomir NC组)。在角膜上皮损伤后的24小时,用qRT-PCR检测各组miR-223-5p的表达。用qRT-PCR及western blot检测各组Hpgds的表达。通过双荧光素酶报告基因实验进一步验证miR-223-5p对Hpgds的靶向调控作用。2.用荧光素钠染色法比较NW组,DW组,DW+miR-223-5p antagomir组和DW+miRNA antagomir NC组在角膜上皮损伤后0h、12h、24h和36h的角膜上皮缺损面积。3.比较 NW 组,DW 组,DW+miR-223-5p antagomir 组和 DW+miRNA antagomir NC组在角膜上皮损伤后第5天的角膜神经纤维密度和角膜敏感度。4.将小鼠分为DW组,DW+miR-223-5p antagomir组,糖尿病小鼠角膜上皮损伤给予miR-223-5p antagomir和Hpgds抑制剂(HQL-79)处理组(DW+miR-223-5p antagomir+HQL-79组),糖尿病小鼠角膜上皮损伤给予miR-223-5p antagomir和抑制剂溶剂(vehicle)处理组(DW+miR-223-5p antagomir+vehicle组)。在角膜上皮损伤后24小时,用western blot检测各组Hpgds蛋白的表达情况。用荧光素钠染色法检测在角膜上皮损伤后,各组不同时间点的角膜上皮缺损面积。5.比较DW组,DW+miR-223-5p antagomir组,DW+miR-223-5p antagomir+HQL-79组和DW+miR-223-5p antagomir+vehicle组在角膜上皮损伤后第5天的角膜神经纤维密度和角膜敏感度。6.用免疫组化法检测在正常人及糖尿病患者角膜内HPGDS蛋白的表达情况。结果:1.在角膜上皮损伤后24小时,与NW组相比,DW组的miR-223-5p出现上调表达,结膜下注射miR-223-5p antagomir能抑制DW组中miR-223-5p的表达。与NW组相比,DW组Hpgds出现下调表达,DW+miR-223-5p antagomir组中Hpgds的表达量高于DW组。双荧光素酶报告基因实验表明,mmu-miR-223-5p能直接结合并抑制Hpgds基因的表达。2.荧光素钠染色结果表明,在角膜上皮损伤后的12h、24h和36h,DW组的角膜上皮缺损面积大于NW组,DW+miR-223-5p antagomir组的角膜上皮缺损面积小于DW组。3.在角膜上皮损伤后第5天,全角膜铺片神经染色结果显示,DW组小鼠的角膜神经纤维密度低于NW组,DW+miR-223-5p antagomir组小鼠的角膜神经纤维密度高于DW组。角膜知觉检测结果表明,DW组小鼠的角膜敏感度低于NW组,DW+miR-223-5p antagomir组小鼠的角膜敏感度高于DW组。4.western blot结果显示,在角膜上皮损伤后的24小时,DW+miR-223-5p antagomir 组角膜内的 Hpgds 表达量高于 DW 组,DW+miR-223-5p antagomir+HQL-79组角膜内Hpgds表达量低于DW+miR-223-5p antagomir组。在角膜上皮损伤后的12h、24h和36h,DW+miR-223-5p antagomir组的角膜上皮缺损面积均小于DW组,DW+miR-223-5p antagomir+HQL-79组的角膜上皮缺损区域大于DW+miR-223-5p antagomir 组。5.在角膜上皮损伤后的第5天,全角膜铺片神经染色结果表明,DW+miR-223-5p antagomir组的角膜神经纤维密度高于DW组,而DW+miR-223-5p antagomir+HQL-79 组的角膜神经纤维密度低于DW+miR-223-5p antagomir 组。角膜知觉检测结果表明,DW+miR-223-5p antagomir 组的角膜敏感度高于 DW 组,而 DW+miR-223-5p antagomir+HQL-79组的角膜敏感度低于DW+miR-223-5p antagomir组。6.免疫组化结果显示,在人角膜的上皮层表达有HPGDS,且与正常组相比,糖尿病患者的角膜内表达相对较少的HPGDS。结论:Hpgds基因是miR-223-5p的直接作用靶点。下调miR-223-5p的表达能促进糖尿病角膜上皮及神经再生,而抑制Hpgds蛋白的表达能逆转miR-223-5p拮抗剂的促进糖尿病角膜上皮及神经再生的作用。第三部分:miR-223-5p调控糖尿病小鼠角膜上皮及神经再生的机制探索目的:探讨miR-223-5p在糖尿病小鼠角膜上皮及神经再生过程中对炎症及再生相关通路的影响。方法:1.用免疫荧光染色和 qRT-PCR检测NW,DW 组,DW+miR-223-5p antagomir组和DW+miRNA antagomir NC组这四组小鼠角膜上皮损伤后24h,CD45在角膜中的表达情况。2.在角膜上皮损伤后24h,用qRT-PCR和ELISA法检测NW组,DW组,DW+miR-223-5p antagomir 组和 DW+miRNA antagomir NC 组角膜中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素(Interleukin,IL)-1β和 IL-6 的表达情况。3.用免疫荧光染色和western blot检测在角膜损伤后24h,NW组,DW组,DW+miR-223-5p antagomir 组和 DW+miRNA antagomir NC 组角膜中磷酸化蛋白激酶 B(phosphorylated protein kinase B,p-AKT)的表达情况。4.在角膜上皮损伤后24h,用免疫荧光染色和western blot检测NW组,DW组,DW+miR-223-5p antagomir 组和 DW+miRNA antagomir NC 组角膜中磷酸化信号转导和转录活化因子 3(phosphorylated signal transducer and activator of transcription-3,p-STAT3)的表达情况。结果:1.免疫荧光染色表明,在角膜上皮损伤修复过程中,与NW组相比,DW组中CD45的荧光强度明显升高。与DW组相比,DW+miR-223-5p antagomir组中CD45的荧光强度有所降低。qRT-PCR亦能在四组的角膜上皮中检测到相同的趋势。2.在角膜上皮损伤后的24小时,通过ELISA检测我们发现,DW组小鼠角膜内 TNF-α、IL-1β和 IL-6 的表达量高于 NW 组,而 DW+miR-223-5p antagomir组小鼠角膜内其表达量低于DW组。qRT-PCR结果亦表明下调miR-223-5p的表达,能减少糖尿病小鼠角膜损伤修复中过度表达的炎症因子。3.免疫荧光染色及western blot均显示在角膜上皮损伤后的24h,DW组角膜内p-AKT的表达低于NW组,抑制miR-223-5p能回复糖尿病小鼠角膜内p-AKT的表达。4.在角膜上皮损伤后的24h,免疫荧光染色及western blot均显示与NW组相比,DW组内p-STAT3的表达量有所减少,而结膜下注射miR-223-5p antagomir能上调糖尿病小鼠角膜内p-STAT3的表达。结论:miR-223-5p可能通过调控角膜组织的炎症反应及细胞增殖相关信号通路的活性,参与调控糖尿病角膜上皮与神经再生。更多还原

【Abstract】 Background:Diabetes mellitus is a chronic metabolic disease characterized by elevated blood glucose,which is one of the important risk factors for systemic macrovascular and microvascular disorders.47-64%diabetic patients could suffer from(diabetic keratopathy,DK).Patients with diabetes exhibit various types of DK,resulting in irreversible visual impairment such as dry eye,delayed wound healing of corneal injury and neurotrophic corneal ulcer.Currently,DK-associated studies mostly focused on dysfunctions of corneal epithelium and corneal nerves.microRNAs(miRNAs)are a class of endogenous single-stranded non-coding RNAs,which are involved in a series of important biological processes such as cell proliferation,differentiation and metabolism.Through the past few decades,researchers have gradually realized the correlation between miRNA and DK but the pathogenesis has not been fully elucidated.Here,we performed RNA sequencing to systematically screen out differentially expressed miRNAs and messenger RNAs(mRNAs)in regenerative corneal epithelium and trigeminal ganglia from diabetic and normal mice.Following,miRNA-mRNA regulatory networks were built,and miR-223-5p/hematopoietic prostaglandin D synthase(Hpgds)pathway was selected.The effects of miR-223-5p/Hpgds pathway on diabetic corneal epithelial and nerve regeneration were further investigated.Part one:Comprehensive analysis of differentially expressed miRNAs and mRNAs involved in diabetic corneal epithelial and nerve regenerationObjective:Though high-throughput sequencing and bioinformatics,we aimed to screen out potential miRNA/mRNA pathways regulating DK.Methods:1.C57BL/6 mice were intraperitoneally injected with streptozocin(STZ)to induce diabetes mellitus and blood glucose,body weight and corneal sensitivity were measured after 16 weeks.Based on this,corneal epithelial injury model was established and the corneal epithelial defect areas between diabetic mice and normal mice were observed by fluorescein sodium staining at 0h,12h,24h and 36h.2.We collected the trigeminal ganglions and regenerated corneal epithelium of normal and diabetic mice,and further analyzed the differentially expressed miRNAs(DEmiRNAs)and differentially expressed(DEmRNAs)by high-throughput sequencing.3.DEmRNAs were used to perform GO analysis to search biological processes involved in diabetic corneal epithelial wound healing and nerve regeneration.4.Target prediction of DEmiRNAs was performed to explore the relationship between DEmiRNAs and DEmRNAs and construct miRNA/mRNA regulatory network.5.The potential miRNA/mRNA pathways were verified by qRT-PCR.Results:1.Compared with normal group,diabetic mice showed elevated blood glucose,decreased body weight,less corneal sensitivity,and delayed corneal epithelial regeneration,indicating that STZ-induced diabetic mice possessed DK related characteristics.2.By high-throughput sequencing,we found that 68 miRNAs and 114 mRNAs were differentially expressed in diabetic trigeminal ganglion.77 miRNAs and 186 mRNAs were differentially expressed in the regenerated corneal epithelium of diabetic mice.Some of them have close relationships with diabetes.3.GO analysis revealed that DEmRNAs detected by our study participate in many biological processes related to diabetes.4.Target gene prediction was performed by TargetScan and miRDB database and we observed that 8 DEmiRNAs target 7 DEmRNAs to form 8 miRNA/mRNA pathways in diabetic corneal neuropathy.12 miRNAs can target 9 mRNAs,forming 13 miRNA/mRNA pathways during diabetic corneal epithelial regeneration.5.We preliminarily screened out the miR-350-5p/major urinary protein 20(Mup20),miR-592-5p/angiopoietin-Like7(Angptl7),miR-8114/Hist1h2bc,miR-351-5p/Elongation of very long-chain fatty acids family member 6(Elovl6)and miR-1291/Elovl6 signaling pathways involved in diabetic corneal neuropathy,as well as the miR-223-5p/Hpgds,miR-210-5p/mesothelin(Msln)and miR-212-3p/high-mobility group AT-hook 2 protein(Hmga2)signaling pathways involved in diabetic corneal epithelial regeneration by qRT-PCR.Conclusion:We screened out miRNA/mRNA signaling pathways involved in diabetic corneal epithelial regeneration and diabetic corneal neuropathy,providing some targets for further mechanism researches.Part two:miR-223-5p regulates the diabetic corneal epithelial and nerve regeneration via targeting HpgdsObjective:We aimed to further investigate the effects of miR-223-5p/Hpgds signaling pathway on diabetic corneal epithelial and nerve regeneration.Methods:1.Mice were divided into four groups,including normal mice with corneal epithelial injury(NW group),diabetic mice with corneal epithelial injury(DW group),diabetic mice with corneal epithelial injury treated with miR-223-5p antagomir(DW+miR-223-5p antagomir group)and diabetic mice with corneal epithelial injury treated with miRNA antagomir negative control(DW+miRNA antagomir NC group).The expression of miR-223-5p was detected by qRT-PCR in each group at 24h post-wounding.The expression of Hpgds in each group was detected at 24h post-wounding by qRT-PCR and western blot.By dual luciferase reporting assay,we revealed that Hpgds is directly targeted by miR-223-5p.2.Corneal epithelial defect area in the NW group,DW group,DW+miR-223-5p antagomir group and DW+miRNA antagomir NC group was detected by sodium fluorescein staining at 0h,12h,24h and 36h after wounding.3.The corneal nerve fiber density and corneal sensitivity of the NW group,DW group,DW+miR-223-5p antagomir group and DW+miRNA antagomir NC group were compared on the 5th day after corneal epithelial injury.4.Mice were divided into four groups,including DW group,DW+miR-223-5p antagomir group,diabetic mice with corneal epithelial injury treated with miR-223-5p antagomir and Hpgds inhibitor(HQL-79)(DW+miR-223-5p antagomir+HQL-79 group)as well as diabetic mice with corneal epithelial injury treated with miR-223-5p antagomir and inhibitor control(vehicle)(DW+miR-223-5p antagomir+vehicle group).The expression of Hpgds in each group was detected at 24h post-wounding by western blot.Corneal epithelial defect area was detected by sodium fluorescein staining at different time points after wounding.5.The corneal nerve fiber density and corneal sensitivity of DW group,DW+miR-223-5p antagomir group,DW+miR-223-5p antagomir+HQL-79 group as well as DW+miR-223-5p antagomir+vehicle group were compared on the 5th day after corneal epithelial injury.6.The expression of HPGDS in the cornea of normal subjects and diabetic patients was detected by immunohistochemistry.Results:1.At 24h after the corneal epithelium scraping,miR-223-5p expression was upregulated in the DW group compared with the NW group and subconjunctival injection of miR-223-5p antagomir inhibited the expression of miR-223-5p in the corneal epithelium of diabetic mice.At 24h after the corneal epithelium scraping,the expression of Hpgds was down-regulated in the DW group compared with the NW group,and the expression of Hpgds in the DW+miR-223-5p antagomir group was higher than that in the DW group.Dual luciferase reporter assay showed that mmu-miR-223-5p could directly bind and inhibit the expression of m-Hpgds gene.2.At 12h,24h and 36h after wounding,the corneal epithelial defect area in the DW group was larger than that in the NW group,and the corneal epithelial defect area in the DW+miR-223-5p antagomir group was smaller than that in the DW group.3.On the 5th day after corneal epithelial injury,the results of corneal whole-mount staining revealed that the corneal nerve fiber density of the DW group was lower than that of the NW group,and the corneal nerve fiber density of the DW+miR-223-5p antagomir group was higher than that of the DW group.The corneal sensitivity of DW group was less than that of NW group,and the corneal sensitivity of DW+miR-223-5p antagomir group was more than that of DW group.4.The protein expression level of Hpgds in the DW+miR-223-5p antagomir group was higher than that in the DW group,and the protein expression level of Hpgds in the DW+miR-223-5p antagomir+HQL-79 group was lower than that in the DW+miR-223-5p antagomir group at 24h after the corneal epithelium scraping.At 12h,24h and 36h after corneal epithelial injury,the area of corneal epithelial defect in the DW+miR-223-5p antagomir group was smaller than that in the DW group,and the area of corneal epithelial defect in the DW+miR-223-5p antagomir+HQL-79 group was larger than that in the DW+miR-223-5p antagomir group.5.On the 5th day after corneal epithelial injury,the results of corneal whole-mount staining revealed that the corneal nerve fiber density in the DW+miR-223-5p antagomir group was higher than that in the DW group,while the corneal nerve fiber density in the DW+miR-223-5p antagomir+HQL-79 group was lower than that in the DW+miR-223-5p antagomir group.Corneal sensitivity measurement results showed that the corneal sensitivity of the DW+miR-223-5p antagomir group was more than that of the DW group,while the corneal sensitivity of the DW+miR-223-5p antagomir+HQL-79 group was less than that of the DW+miR-223-5p antagomir group.6.Immunohistochemical results revealed that HPGDS were highly expressed in the epithelium of human cornea,and there was a relatively low expression of HPGDS in the cornea of diabetic patients compared with the normal group.Conclusion:Hpgds is a direct target of miR-223-5p.Downregulation of miR-223-5p expression can promote diabetic corneal epithelial and nerve regeneration,while inhibition of Hpgds protein expression can reverse the promoting effects of miR-223-5p antagomir on diabetic corneal epithelial and nerve regeneration.Part three:Mechanisms of miR-223-5p regulating diabetic corneal epithelial and nerve regenerationObjective:We aimed to investigate the effect of miR-223-5p on inflammatory response and regeneration-related signalings in diabetic corneal epithelial and nerve regenerationMethods:1.Immunofluorescence staining and qRT-PCR were used to detect the expression of CD45 in the cornea of NW group,DW group,DW+miR-223-5p antagomir group and DW+miRNA antagomir NC group at 24h after the corneal epithelium scraping.2.The expressions of TNF-α,IL-1β and IL-6 in the cornea of NW group,DW group,DW+miR-223-5p antagomir group and DW+miRNA antagomir NC group were detected by qRT-PCR and ELISA at 24h after corneal epithelial injury.3.The expression of p-AKT in the cornea of NW group,DW group,DW+miR-223-5p antagomir group and DW+miRNA antagomir NC group were detected by immunofluorescence staining and western blot at 24h post-wounding.4.Immunofluorescence staining and western blot were utilized to explore the expression of p-STAT3 in the cornea of NW group,DW group,DW+miR-223-5p antagomir group and DW+miRNA antagomir NC group at 24h after corneal epithelial injury.Results:1.Immunofluorescence staining showed that compared with the NW group,the fluorescence staining of CD45 was stronger in the DW group at 24h after corneal epithelial injury.Compared with the DW group,CD45 fluorescence staining was weaker in the DW+miR-223-5p antagomir group.Similar trends of CD45 were also detected in the corneal epithelium of these four groups by qRT-PCR.2.At 24h after corneal epithelial injury,ELISA results revealed that the expression levels of TNF-α、IL-1β and IL-6 in cornea of DW group were higher than that of NW group,while the expression levels of TNF-α、IL-1β and IL-6 in DW+miR-223-5p antagomir group were lower than that of DW group.qRT-PCR results also showed that inhibition of miR-223-5p expression could reduce overexpressed inflammatory cytokines in diabetic regenerated corneal epithelium.3.Immunofluorescence staining and western blot showed that at 24h after corneal debridement,the expression of p-AKT in the cornea of DW group was lower than that of NW group,and inhibiting miR-223-5p expression could reactivate the expression of p-AKT in the cornea of diabetic mice.4.At 24h post-wounding,immunofluorescence staining and western blot revealed that p-STAT3 was down-regulated in DW group compared with NW group,and subconjunctival injection of miR-223-5p antagomir could induce the expression of p-STAT3 in the cornea of diabetic mice.Conclusion:miR-223-5p can regulate inflammatory process and proliferation-related signaling pathways in diabetic corneal epithelial and nerve regeneration.更多还原