【作者】 沈剑；

【导师】 徐靖宏；

【作者基本信息】 浙江大学 ， 外科学（整形外科）， 2019， 硕士

【摘要】 研究背景瘢痕疙瘩是一种临床常见的皮肤创口病理性纤维化愈合疾病,其本质是成纤维细胞过度增殖以及细胞外基质(尤其是胶原蛋白和粘多糖)过度合成和沉积,主要临床表现为超出创口边缘的持续性瘤样增生,且常常侵犯邻近皮肤,并无法自行好转,仅靠手术切除复发概率非常高,形成局部的畸形,甚至对功能产生一定的影响,常常给病人带来困扰和自卑。目前尚缺乏有效的治疗手段,是整形外科亟需解决而尚未解决的难题之一。目前认为瘢痕疙瘩组织内缺氧微环境及慢性炎症与疾病的发生发展密切相关,但是其明确的发病及发展机制尚未完全明了。目前国内外学者仍然持续不断努力探索瘢痕疙瘩发病及发展的机制及有效的治疗靶点。瘢痕疙瘩多发生于累及真皮深层的外伤、手术或感染之后,由于组织创伤时皮肤血管网破坏、炎症及修复过程中细胞高代谢状态使耗氧量增加等多方面因素导致组织内常呈缺氧状态,而缺氧微环境在促进瘢痕疙瘩成纤维细胞异常增殖和胶原合成起着重要的作用。缺氧诱导因子-1α(HIF-1α)是调节缺氧应激反应并使细胞适应低氧环境的核心因子,研究报道瘢痕疙瘩中的HIF-1α上调显著促进结缔组织生长因子(CTGF)表达,诱导ECM合成,并在瘢痕疙瘩形成中起到重要作用。此外,我们前期的研究表明,缺氧条件下HIF-1α可以激活TGF-β/Smad信号,并增加瘢痕疙瘩成纤维细胞增殖及胶原沉积。Parkin是调节有丝分裂、线粒体稳态、抗氧化应激的重要因子。同时,Parkin可作为一种E3泛素化酶,控制多种蛋白底物的降解,如Cdcrel-1、Pael受体、α-突触核蛋白和Synphilin-1,达到预防帕金森病(PD)发生的作用。此外,一些研究表明,Parkin可能是一种肿瘤抑制因子。缺氧诱导因子HIF-1α可以促进肿瘤生长和转移,Parkin可以通过促进HIF-1α泛素化降解,从而抑制肿瘤转移。瘢痕疙瘩是一种类似肿瘤的疾病,其特征是组织内呈现高HIF-1α表达水平。然而,Parkin是否与瘢痕疙瘩进展的发病机制有关仍未明确。所以,本课题根据瘢痕疙瘩的生物学特性与恶性肿瘤类似的特点,借鉴Parkin在肿瘤学领域的研究成果并结合我们前期研究结果,在此基础上本论文进一步深入研究,从组织及细胞水平检测瘢痕疙瘩Parkin与HIF-1α表达的关联,并在细胞水平明确Parkin对瘢痕疙瘩成纤维细胞增殖能力的作用,阐明Parkin及其调控的HIF-1α/TGF-β/Smad通路在瘢痕疙瘩形成中的作用和分子机制,以期推动对瘢痕疙瘩形成机制的了解,为瘢痕疙瘩的防治提供一种新思路、新靶点。研究目的1.明确瘢痕疙瘩组织、瘢痕疙瘩成纤维细胞中Parkin、HIF-1α等效应分子的表达情况。2.明确瘢痕疙瘩成纤维细胞中Parkin、HIF-1α之间的相互作用及关系。3.明确缺氧条件下瘢痕疙瘩成纤维细胞Parkin的表达改变及其对其细胞增殖和凋亡的影响;明确Parkin对瘢痕疙瘩成纤维细胞的异常增殖有着重要的调控作用。4.明确Parkin通过调控HIF-1α/TGF-β/Smad通路影响瘢痕疙瘩生长的的作用及机制。研究方法1.第一部分Western Blot及免疫组化检测瘢痕疙瘩组织与正常皮肤组织中Parkin、HIF-1α等蛋白的差异性表达;人瘢痕疙瘩、正常皮肤成纤维细胞的体外培养,RT-PCR和Western Blot检测瘢痕疙瘩成纤维细胞(KFs)与正常皮肤组织成纤维细胞(NFs)中Parkin、HIF-1α等效应因子的表达差异。通过Western Blot、Co-IP检测Parkin与HIF-1α的相互作用关系。2.第二部分培养细胞的供应气体是由1%氧气、5%二氧化碳、94%氮气混合气体组成,以此构建缺氧模型,采用RNA干扰技术沉默Parkin及HIF-1α的表达,不同条件处理细胞后将瘢痕疙瘩成纤维细胞(KFs)分为si-cont组、si-HIF-1α组、si-Parkin组、si-HIF-1α+Parkin组,分别通过CCK8试验、流式细胞仪检测上述四组细胞的增殖及凋亡情况。通过Western Blot、Co-IP分别检测KFs在常氧、缺氧条件下,si-cont组、si-Parkin 组、si-HIF-1 α 组及 vec 组、Flg-Parkin 组中 Parkin、HIF-1 α、TGF-β1、p-Smad2/3等蛋白的表达情况及差异,此外在缺氧条件下,通过CCK8试验检测si-cont组、si-Parkin 组、si-Parkin+TGF-β 1 组 KFs 增殖的情况。通过Western Blot、Q-PCR检测二甲双胍处理后KFs中Parkin的表达;通过Western Blot分别检测在常氧及缺氧条件下,二甲双胍处理后KFs中Parkin、HIF-1α、TGF-β1、p-Smad2/3等蛋白的表达情况及差异。通过Co-IP检测二甲双胍处理后KFs中Parkin与HIF-1α的相互作用情况。研究结果1.第一部分 Parkin蛋白在瘢痕疙瘩组织及细胞水平表达下调,其表达水平与HIF-1α呈负相关;另外Parkin可以降低KFs中HIF-1α蛋白表达水平,KFs中HIF-1α与Parkin存在相互作用,HIF-1α在翻译后水平上受到Parkin调节。2.第二部分 Parkin在缺氧条件下抑制KFs异常增殖和诱导细胞凋亡;在缺氧条件下Parkin可以抑制HIF-1 α/TGF-β/Smad信号通路进而抑制KFs增殖;在HIF-1α敲低KFs中,Parkin的沉默不能促进HIF-1α/TGF-β/Smad信号传导;二甲双胍可以适度上调在常氧KFs中Parkin的表达,在缺氧条件下则显著增加了 Parkin的表达并降低了 HIF-1α的表达,二甲双胍增强了 Parkin与HIF-1α之间的相互作用。结论1.瘢痕疙瘩中Parkin蛋白表达下调,其表达与HIF-1α负相关,Parkin通过翻译后水平调节HIF-1α表达,HIF-1α可能可以作为Parkin的底物被泛素化降解。2.Parkin在缺氧条件下调控KFs细胞增殖和凋亡,瘢痕疙瘩中的Parkin下调可能是在缺氧条件下维持KFs增殖能力和保护细胞免受凋亡的关键机制。3.Parkin参与KFs中HIF-1α的降解,在缺氧条件下中Parkin可以抑制HIF-1α/TGF-β/Smad信号通路进而抑制KFs增殖。4.二甲双胍可以诱导KFs中Parkin的表达,增加HIF-1α和Parkin之间的相互作用。更多还原

【Abstract】 BackgroundKeloid is a common clinical disease which occurred in the wounded skin healed with the pathological fibrosis.The essence of keloid is excessive proliferation of fibroblasts and excessive synthesis and deposition of extracellular matrix(especially collagen and mucopolysaccharide.The main clinical manifestations of keloid are persistent neoplastic hyperplasia beyond the edge of the original wound,often invading adjacent tissues,and unable to degenerate spontaneously.Simple surgical excision is also prone to recurrence,often resulting in appearance deformity and dysfunction.Treatment is one of the most urgent and unsolved problems in plastic surgery.At present,hypoxic microenvironment and chronic inflammation in keloid tissues are closely related to the occurrence and development of the disease,but its clear pathogenesis and development mechanism are not yet fully understood.At present,scholars at home and abroad continue to make efforts to explore the pathogenesis and development of keloid and effective treatment targets.Keloid often occurs after trauma,operation or infection involving deep dermis.Oxygen consumption increases due to the destruction of skin vascular network,inflammation and hypermetabolism of cells in the process of tissue trauma.Hypoxic microenvironment plays an important role in promoting abnormal proliferation of keloid fibroblasts and collagen synthesis.Hypoxia inducible factor-lalpha(HIF-la)is the core factor regulating hypoxic stress response and adapting cells to hypoxic environment.It has been reported that the up-regulation of HIF-la in keloid significantly promotes the expression of connective tissue growth factor(CTGF),induces the synthesis of ECM,and plays a key role in keloid formation.In addition,our previous studies have shown that HIF-la can activate TGF-beta 1/Smad signal under hypoxia,and increase fibroblast proliferation and collagen deposition in keloid.Parkin is a key factor in regulating mitochondrial homeostasis,antioxidant stress,mitosis and preventing Parkinson’s disease(PD).As an E3 ubiquitin ligase,Parkin also controls the conversion of several substrates,such as cdcrel-1,Pael receptor,alpha-synuclein and synphilin-1,to promote their role in the prevention of PD.In addition,some studies suggest that Parkin may be a novel tumor suppressor.HIF-la is a factor that promotes the growth and metastasis of tumors.Parkin can ubiquitinate and degrade HIF-la to inhibit cancer metastasis.Keloid is a cancer-like disease characterized by high levels of HIF-la.However,whether Parkin is related to the pathogenesis of keloid progression remains unclear.Therefore,according to the biological characteristics of keloid and the similar characteristics of malignant tumors,drawing on Parkin’s research results in the field of oncology and combining with our previous research results,this paper further studies the relationship between the expression of HIF-la and Parkin in keloid at the tissue and cell level,and clarifies the fibrosis of Parkin in keloid at the cell level.The role of Parkin and its regulatory HIF-la/TGF-beta 1/Smad pathway in keloid formation and its molecular mechanism were elucidated in order to promote the understanding of keloid formation mechanism and provide a new idea and target for keloid prevention and treatment.Objective1.To clear and definite the expression of Parkin and HIF-la in keloid tissue and keloid fibroblasts.2.To clear and definite the interaction and relationship between Parkin and HIF-la in keloid fibroblasts.3.To clear and definite the expression of Parkin in keloid fibroblasts under hypoxia and its effect on cell proliferation and apoptosis,and to clarify that Parkin plays an important role in regulating the abnormal proliferation of keloid fibroblasts.4.To clear and definite the effect and mechanism of Parkin on keloid growth by regulating HIF-1α/TGF-beta/Smad pathway.Methods1.Part I Western Blot and immunohistochemistry were used to detect the expression of Parkin and HIF-1α in keloid tissue and normal skin tissue.Human keloid and normal skin fibroblasts were cultured in vitro.The expression of Parkin and HIF-la in keloid fibroblasts(KFs)and normal skin fibroblasts(NFs)was detected by Q-PCR and Western Blot.Different.The interaction between Parkin and HIF-la was detected by Western Blot and Co-IP.2.In the second part,the hypoxia model was constructed by using 1%oxygen,5%carbon dioxide and 94%nitrogen as the gas supply to the cultured cells.The expression of Parkin and HIF-1α was silenced by RNA interference technology.The keloid fibroblasts(KFs)were divided into si-cont group,si-HIF-la group,si-Parkin group,si-HIF-1α+Parkin group after different treatment conditions.CCK8 test,CCK8 test and HIF-1α+Parkin group were used respectively.The proliferation and apoptosis of these four groups were detected by flow cytometry.Western Blot and Co-IP were used to detect the expression and difference of Parkin,HIF-1α,TGF-beta 1,p-Smad2/3 proteins in si-cont group,si-Parkin group,si-HIF-la group and VEC group,Flg-Parkin group under normal and hypoxic conditions respectively.In addition,the proliferation of KFs in Parkin+TGF-beta 1 group,si-Parkin group and si-cont group was detected by CCK8 test.Western Blot and Q-PCR were used to detect the expression of Parkin in KFs treated with metformin,and Western Blot was used to detect the expression and difference of Parkin,HIF-la,TGF-beta 1 and p-Smad2/3 in KFs treated with metformin under normal and hypoxic conditions respectively.The interaction between Parkin and HIF-1α in KFs treated with metformin was detected by Co-IP.Research Results1.Part I:The expression of Parkin protein was down-regulated in keloid tissues and cells,and its expression level was negatively correlated with HIF-la.In addition,Parkin could reduce the expression level of HIF-la in KFs.There was interaction between HIF-1α and Parkin in KFs,and HIF-1α was regulated by Parkin at post-translational level.2.Part II Parkin inhibits the abnormal proliferation of KFs and induces apoptosis under hypoxia;Parkin inhibits the HIF-la/TGF-beta 1/Smad signaling pathway and KFs proliferation under hypoxia;in HIF-la knockdown of KFs,Parkin silence does not promote HIF-la/TGF-beta 1/Smad signal transduction;metformin can moderately up-regulate the expression of Parkin in normoxic KFs,while in hypoxic condition,Parkin can inhibit the proliferation of KFs.The expression of Parkin was significantly increased and HIF-1α was decreased.Metformin enhanced the interaction between Parkin and HIF-1α.Conclusion1.The expression of Parkin protein in keloid is down-regulated,which is negatively correlated with HIF-1α.Parkin regulates the expression of HIF-1α at post-translational level.HIF-1α can be degraded by ubiquitination as the substrate of Parkin.2.Parkin regulates the proliferation and apoptosis of KFs cells under hypoxia.The down-regulation of Parkin in keloids may be the key mechanism to maintain the proliferation ability of KFs and protect cells from apoptosis under hypoxia.3.Parkin participates in the degradation of HIF-1α in KFs.Parkin can inhibit HIF-1α/TGF-beta 1/Smad signaling pathway and KFs proliferation under hypoxia.4.Metformin can induce Parkin expression in KFs and increase the interaction between HIF-la and Parkin.更多还原