【作者】 樊芳；

【导师】 段宣初；

【作者基本信息】 中南大学 ， 眼科学， 2011， 博士

【摘要】 研究背景青光眼滤过性手术(glaucoma filtering surgery, GFS)术后滤过道瘢痕化是导致手术失败的主要原因。一些抗代谢药物在手术中的局部应用虽然提高了手术的成功率,但是这类药物应用的时间难以个体化,常常因为放置时间过短而出现作用不明显或因为放置时间过长以及对眼部组织的广泛作用而导致一系列严重的并发症,对患者本来已经受损的视功能造成新的威胁。因此通过对滤过道的瘢痕形成机制的研究,寻找一种更加安全、特异、有效的方法是目前GFS术后抗瘢痕形成研究的热点和难点。由于GFS术后滤过道瘢痕化是一种局部组织的纤维增生性病变,而该病变的核心环节是由于人Tenon’s囊成纤维细胞(Human Tenon’s Fibroblasts,HTFs)的活化、增殖、转化并分泌众多促纤维化因子及合成大量的细胞外基质所导致的。因此阻止HTFs活化、增殖、转化是调控GFS术后滤过道瘢痕形成的关键。以往研究表明促使HTFs活化增殖最重要的诱导因子是转化生长因子-β(transforming growth factorsβ, TGF-β),它是伤口愈合的重要调节因子,在GFS术后的瘢痕形成中起重要的作用。但是TGF-β除了诱导HTFs活化外,还有其他诸如调节细胞分化、促进成骨和造血、促进血管生成等生物活性。由于其作用靶点繁多、生物学效应复杂,因此完全阻断TGF-β会有潜在抑制免疫、抑制炎症反应及阻碍伤口愈合的不利影响。因此目前研究集中于在TGF-β致瘢痕化作用的特异性信号传导通路上寻找更为特异、安全的治疗方法。在TGF-β促进瘢痕形成的的信号传导通路中,Smads蛋白家族被认为是起重要作用,因此常常作为靶位进行抗瘢痕研究。罗格列酮是一种人工合成的高选择性过氧化物酶体增生物激活受体-γ(peroxisome Proliferator-activated receptor-γ, PPAR-γ)的激动剂,是一种胰岛素增敏剂,目前常用于治疗2型糖尿病。近年来研究发现该药物还具有抗炎,调控免疫功能,抗纤维化的作用。本研究通过探讨罗格列酮对TGF-β1诱导的HTFs活化增殖的抑制作用及其相关机制,为GFS术后滤过道瘢痕化的预防及治疗提供新的思路。目的原代培养HTFs,研究TGF-β1及罗格列酮对体外培养的HTFs活化、增殖的影响,为罗格列酮抑制GFS术后滤过道瘢痕形成提供理论支持。方法1.在手术中取青光眼患者的Tenon’s囊组织,采用组织块培养法培养原代HTFs。2.建立TGF-β1诱导HTFs活化的细胞模型。研究TGF-β1作用的最适浓度。观察细胞形态变化,研究α平滑肌肌动蛋白(αsmooth muscle actin,α-SMA),结缔组织生长因子(connective tissue growthfactor, CTGF),Ⅰ型胶原蛋白(typeⅠCollagen, ColⅠ)以及细胞信号传导通路相关因子Smad2/3的表达。3.研究罗格列酮对HTFs活化的影响。以不同浓度的罗格列酮预处理细胞2h,再用最适浓度的TGF-β1诱导HTFs,摸索药物作用的最佳浓度,并研究细胞增殖、迁移能力的改变并进一步检测α-SMA, CTGF, ColⅠ, Smad2/3及PPAR-γ表达的改变。结果1.在体外成功地培养了人Tenon’s囊成纤维细胞,培养的细胞形态稳定,生长规律,冷冻和复苏对细胞的活性影响不大。2.建立了TGF-β1诱导HTFs活化的细胞模型。TGF-β1诱导HTFs后细胞形态发生改变,细胞免疫荧光证实细胞表达α-SMA蛋白,提示细胞活化；进一步研究表明α-SMA的表达在转录水平以及蛋白水平上均有上调,其表达峰值分别为24h和48h；CTGF的表达在转录水平以及蛋白水平上也有上调,其表达峰值分别为12h和24h；同时ColⅠ的的表达在转录水平以及蛋白水平上亦有上调；信号通路蛋白P-Smad2/3表达增加,峰值在24h。3.罗格列酮对TGF-β1诱导的细胞活化有抑制作用。5μMol/L、10μMol/L罗格列酮对HTFs无明显细胞毒性作用；可以抑制细胞的增殖迁移；在一定程度上可以抑制TGFβ1诱导的α-SMA, CTGF, ColⅠ的表达上调,同时抑制P-Smad2/3的表达,但对PPAR-γ的表达有促进作用。结论罗格列酮对体外培养HTFs没有明显毒性作用,可在一定程度上减弱了转化生长因子-β1对人Tenon’s囊成纤维细胞的活化作用,其机制可能是抑制了TGF/Smad信号传导通路的活化。更多还原

【Abstract】 BACKGROUNDThe first irreversible blind leading disease in the world is glaucoma. In our country, glaucoma filtering surgery (GFS) is still one of the principal means for treating advanced glaucoma. A main problem threatening long-term success rate of GFS is postoperative scarring process. Though antimetabolite drugs could effectively inhibit the formation of scarring, they also related with serious complications such as wound leakage, infection, hypotony maculopathy, even endophthalmitis which limit their application. Antifibrotic therapeutic methods with safe and effective effects are desirable.The activation of Human Tenons fibroblasts (HTFs) is essential for postoperative repairing, however continuous presence of this active state is related to increased deposition of extra cellular matrix (ECM) proteins which lead to postoperative scarring. This process is regulated by various inflammatory factors, among which transforming growth factor (TGF)-βhas been recognized as a key cytokine. However besides inducing HTFs activation TGF-βtill has other biological activity such as regulating cell differentiation, promoting osteogenesis and hematopoiesis, and inducing angiogenesis. For its complicated biological effects completely blocking TGF-βmay potentially suppress immune and block wound healing. So we committed to find a even more specific and safe method to regulate fibrosis after GFS. TGF-βexerts its multiple biologic actions by activating several intracellular signal transduction systems, and Smads family of proteins has been recently identified as a predominant signal transducer of TGF-β.Rosiglitazone one of the specific thiazolidinediones (TZDs) is a synthetic high-affinity ligands for peroxisome proliferator-activated receptor (PPAR)-γa member of the nuclear hormone receptor super family of ligand-activated transcription factors. In clinic it widely used for treating of non-insulin-dependent diabetes mellitus. Besides it effects on metabolism, current studies have begun to indicate additional important roles for PPAR-γagonists in the regulation of inflammation, connective tissue remodeling, and organ fibrosis.In the present study we provided novel data demonstrating the ability of rosiglitazone to block TGF-βmediated profibrotic effects in HTFs. These activities support the concept that rosiglitazone may have exciting potential method for counteract postoperative scarring after GFS.PURPOSETo culture human Tenon’s fibroblasts. To investigate the inhibition role of the synthetic peroxisome proliferators-activated receptor (PPAR)-γagonist rosiglitazone on TGF-βinduced human Tenon’s fibroblasts activation, and to assess the possible mechanism of it.METHODS1. Tenon’s biopsy samples were obtained during standard Glaucoma Filtering Surgery and cultured in vitro.2. HTFs were treated with various concentration of TGF-β. The optimization concentration was assessed by Cell count kit-8. The morphologic changes of HTFs were observed. The expression of Alpha smooth muscle action (a-SMA), connective tissue growth factor (CTGF), typeⅠCollagen (ColⅠ), were detected with RT-PCR, Western-Blot,α-SMA was also examined with immunofluorescence. Smad2/3, was assessed by Western-Blot.3. HTFs were pretreated with different concentrations of rosiglitazone (0.1umol/1-3μumol/1) and then activated with 5ng/mL TGF-β1. Cell count kit-8 was accessed for cell viability and proliferation, wound closure assay was performed to assess cell migration. The expression of Alpha smooth muscle action (a-SMA), connective tissue growth factor (CTGF), typeⅠCollagen (ColⅠ), were detected with RT-PCR, Western-Blot, a-SMA was also examined with immunofluorescence. PPAR-y, P-Smad2/3 were assessed by Western-Blot.RESULTS1. HTFs were successfully cultured in vitro. They had normal morphosis and stable growth characteristic, before and after freezing and resusxitation.2. After induced by TGF-β1 the mRNA and protein expression of a-SMA were up-regulation, with a peak at 24 and 48 hours. The mRNA and protein expression of CTGF were up-regulation, with a peak at 12 and 24 hours. The expression of ColⅠand P-Smad2/3 were also up-regulated.3. Rosiglitazone could attenuate TGF-β1 induced up-regulation of a-SMA, CTGF and ColⅠ. It could availably increase the expression of PPAR-γ, and effectively attenuated the phosphorylation of Smad2/3.CONCLUSIONSRosiglitazone can effectively attenuate TGF-β1 induced activation of HTFs, without obvious toxic effect. The possible mechanism might be rosiglitazone interfering the TGF-β/Smad pathway.更多还原