【作者】 丁科；

【导师】 唐正严；

【作者基本信息】 中南大学 ， 临床医学（专业学位）， 2023， 博士

【摘要】 背景:尿道狭窄是泌尿外科常见病,主要治疗方式是外科治疗,但手术治疗具有难度高、易复发、并发症多等特点,因此寻找新的治疗手段是泌尿外科医师急需解决的问题。尿道瘢痕的形成是尿道狭窄发病的始动环节,其中以I型胶原蛋白(CollagenⅠ,COL-Ⅰ)为主的细胞外基质蛋白的沉积是其进展的主要原因。因此,探索胶原蛋白异常沉积的机制对于尿道狭窄的防治具有非常重要的意义。COL-Ⅰ翻译后羟基化修饰,尤其是赖氨酸羟基化修饰对于维护胶原蛋白结构稳定、促进胶原蛋白沉积有着重要作用。本课题旨在探索胶原蛋白赖氨酸羟基化在尿道纤维化中的作用,揭示其通过促进COL-Ⅰ沉积,增加细胞外基质刚度激活压电型机械敏感离子通道组件1(piezoelectric1,PIEZO1),进而通过钙离子-钙调蛋白依赖性蛋白激酶II(calcium-cam-dependent protein kinase II,CAMKII)CAMKII/缺氧诱导因子-1α(hypoxia-Inducible factor 1-alpha,HIF-1α)信号通路增加赖氨酸羟基化酶(procollagen-lysine,2-oxoglutarate5-dioxygenases,PLODs)的表达,使纤维化过程陷入恶性循环,推进纤维化-再纤维化的进行性发展。本课题将为尿道纤维化研究领域提供一个新的思路,为尿道狭窄的预防与治疗打下坚实的基础。方法:1、利用原子力显微镜(atomic force microscope,AFM)量化人体狭窄尿道组织与正常尿道组织基质刚度;采用液相色谱质谱/质谱联用(liquid chromatography tandem mass spectrometry/mass spectrometry,LC-MS/MS)技术对3例人体狭窄尿道组织和3例正常尿道组织胶原蛋白羟基化修饰水平进行检测分析。2、探究胶原蛋白翻译后修饰在尿道纤维化中的调控机制,收集3例人体狭窄尿道组织与3例人体正常尿道组织进行转录组学测序,发现人体狭窄尿道组织与正常尿道组织中的差异基因,采用生物信息学方法预测胶原蛋白赖氨酸羟基化修饰在尿道纤维化中的调控机制。3、组织免疫荧光及蛋白质免疫印迹法(western blot,WB)检测人体狭窄尿道组织和正常尿道组织中PIEZO1、PLODs、p CAMKII、HIF-1α以及纤维化指标COL-Ⅰ、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的表达情况。利用WB、细胞免疫荧光、膀胱尿道造影等方法在细胞和动物层面上验证PIEZO1是否通过上调CAMKII/HIF-1α信号通路增加胶原蛋白赖氨酸羟基化水平参与尿道纤维化。4、查阅文献寻找赖氨酸羟基化抑制剂,利用cck8实验筛选出赖氨酸羟基化抑制剂2,2’-联吡啶发挥作用的最佳有效浓度和作用时间。利用wb、苏木素-伊红染色(hematoxylin-eosin staining,H&E)和Masson染色以及膀胱尿道造影在细胞层面和动物层面验证2,2’-联吡啶延缓尿道纤维化进展的有效性。结果:1、AFM测量人体正常尿道组织基质刚度为4.2±1.92k Pa,狭窄尿道组织基质刚度为40.0±2.32k Pa。通过提取人体狭窄尿道组织与正常尿道组织的胶原蛋白进行LC-MS/MS检测,发现一共有1082个位点发生羟基化修饰,其中976个位点发生赖氨酸羟基化修饰,106个位点发生脯氨酸羟基化修饰,赖氨酸羟基化修饰占比90.2%。其中在有统计学差异的49个赖氨酸羟基化修饰位点中,有42个赖氨酸羟基化修饰位点表达增高,7个赖氨酸羟基化修饰位点表达降低。同时发现狭窄尿道组织中COL-Ⅰ的沉积较正常尿道组织显著增加。2、人体狭窄尿道组织与正常尿道组织转录组学共检测出1983种差异m RNA,其中546种m RNA表达上调,1437种m RNA表达下调,其中赖氨酸羟基化酶PLOD1、PLOD2、PLOD3的表达上升,除此之外PIEZO1、HIF-1α以及纤维化指标COL-Ⅰ、α-SMA的表达上升。通过生物信息学方法预测发现PIEZO1可能通过调控CAMKII/HIF-1α影响PLODs的表达,影响胶原蛋白的稳定性从而在尿道纤维化的进展中发挥作用。3、组织免疫荧光及免疫印迹(WB)发现人体和大鼠狭窄尿道组织中PLODs、PIEZO1、p CAMKII、HIF-1α以及纤维化指标COL-Ⅰ、α-SMA的表达较正常尿道组织高。利用人尿道成纤维细胞模型,发现基质刚度的升高可激活PIEZO1,活化的PIEZO1可磷酸化CAMKII调控HIF-1α表达,致使PLODs的表达增加,胶原蛋白稳定性增加。4、利用人尿道成纤维细胞模型,发现2,2’-联吡啶能抑制PLODs以及纤维化指标COL-Ⅰ、α-SMA的表达。在尿道狭窄大鼠模型中通过局部注射2,2’-联吡啶能延缓尿道狭窄的发生。结论:PIEZO1通过上调CAMKII/HIF-1α信号通路增加胶原蛋白赖氨酸羟基化水平促进尿道纤维化的进展,纤维化的进展促进基质刚度增加,正反馈调控PIEZO1的表达和激活。图38幅,表16个,参考文献120篇.更多还原

【Abstract】 BackgroundUrethral stricture is a common condition in urology,and the main treatment method is surgery.However,surgical treatment is characterized by its high difficulty,high recurrence rate,and multiple complications.Therefore,finding new treatment methods is an urgent issue for urologists.The formation of urethral scar is the initial step in the development of urethral stricture,and the deposition of extracellular matrix proteins,mainly type I collagen(COL-I),is the main cause of its progression.Therefore,exploring the mechanism of abnormal deposition of collagen is of great significance for the prevention and treatment of urethral stricture.Post-translational hydroxylation modification of COL-I,especially lysine hydroxylation modification,plays an important role in maintaining the stability of collagen structure and promoting collagen deposition.This study aims to explore the role of lysine hydroxylation of collagen in urethral fibrosis and to reveal its mechanism of promoting fibrosis by increasing the deposition of COL-I,activating the piezoelectric mechanosensitive ion channel component 1(PIEZO1)through increased extracellular matrix stiffness,and subsequently increasing the expression of lysine hydroxylase(procollagen-lysine,2-oxoglutarate 5-dioxygenases,PLODs)through the calcium-cam-dependent protein kinase II(CAMKII)/hypoxia-inducible factor 1-alpha(HIF-1α)signaling pathway,leading to a vicious cycle in the fibrosis process and promoting the progressive development of fibrosis-re-fibrosis.This study will provide a new perspective for the research field of urethral fibrosis and lay a solid foundation for the prevention and treatment of urethral stricture.Method1.Using atomic force microscopy(AFM),the stiffness of narrowed urethral tissue and normal urethral tissue matrix was quantified.Liquid chromatography tandem mass spectrometry/mass spectrometry(LCMS/MS)was used to detect and analyze the level of collagen hydroxylation modification in 3 cases of narrowed urethral tissue and 3 cases of normal urethral tissue.2.The regulatory mechanism of post-translational modification of collagen in urethral fibrosis was investigated.Transcriptional sequencing was performed on 3 cases of narrowed urethral tissue and 3 cases of normal urethral tissue to identify differential genes between the two tissues.Bioinformatics methods were used to predict the regulatory mechanism of collagen lysine hydroxylation modification in urethral fibrosis.3.Tissue immunofluorescence and western blotting were used to detect the expression levels of PIEZO1,PLODs,p CAMKII,HIF-1α,and fibrosis markers COL-I and α-smooth muscle actin(α-SMA)in narrowed and normal urethral tissue.Western blotting,cell immunofluorescence,bladder urethral imaging,and other methods were used to validate whether PIEZO1 increased collagen lysine hydroxylation levels by up-regulating the CAMKII/HIF-1α signaling pathway and participated in urethral fibrosis.4.Literature was searched for lysine hydroxylation inhibitors.CCK8 experiments were used to screen for the optimal effective concentration and time of action of the lysine hydroxylation inhibitor 2,2’-dipyridine.Western blotting,hematoxylin-eosin staining,Masson staining,and bladder urethral imaging were used at the cellular and animal levels to validate the effectiveness of 2,2’-dipyridine in delaying urethral fibrosis progression.Results1.AFM measurements showed that the stiffness of normal human urethral tissue matrix was 4.2±1.92 k Pa,while the stiffness of narrowed urethral tissue matrix was 40.0±2.32 k Pa.LC-MS/MS detection of collagen extracted from narrowed and normal urethral tissue revealed a total of 1082 hydroxylation modification sites,among which 976 sites were hydroxylated on lysine residues and 106 sites were hydroxylated on proline residues.Lysine hydroxylation accounted for 90.2% of all modifications.Among the 49 lysine hydroxylation sites with statistically significant differences,42 sites showed increased expression while 7 sites showed decreased expression.Furthermore,the deposition of COL-I in the narrowed urethral tissue was significantly increased compared to normal urethral tissue.2.Transcriptomic analysis of human narrowed and normal urethral tissue identified 1983 differentially expressed m RNAs,with 546 m RNAs upregulated and 1437 m RNAs downregulated.The expression of lysyl hydroxylases PLOD1,PLOD2,and PLOD3 was upregulated,along with PIEZO1,HIF-1α,and fibrosis markers COL-I and α-SMA.Bioinformatic prediction suggested that PIEZO1 may regulate the expression of PLODs by modulating CAMKII/HIF-1α,affecting the stability of collagen and playing a role in the progression of urethral fibrosis.3.Immunofluorescence and Western blot analysis revealed higher expression levels of PLODs,PIEZO1,p CAMKII,HIF-1α,as well as fibrotic markers COL-Ⅰ and α-SMA in narrowed urethral tissue from both humans and rats compared to normal urethral tissue.Using a human urethral fibroblast model,it was found that increased matrix stiffness could activate PIEZO1,and the activated PIEZO1 could phosphorylate CAMKII to regulate HIF-1α expression,leading to increased expression of PLODs and enhanced stability of collagen.4.Using a human urethral fibroblast model,it was discovered that 2,2’-dipyridine could inhibit the expression of PLODs and fibrotic markers COL-Ⅰ and α-SMA.In a rat model of urethral stenosis,local injection of2,2’-dipyridine could delay the occurrence of urethral narrowing.ConcussionPIEZO1 promotes the progression of urethral fibrosis by upregulating the CAMKII/HIF-1α signaling pathway to increase collagen lysine hydroxylation levels.The progression of fibrosis promotes an increase in matrix stiffness,which positively regulates the expression and activation of PIEZO1.更多还原