【作者】 冯智；

【导师】 李世荣；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 增生性瘢痕(hypertrophic scar,HS)又称肥厚性瘢痕,是一种常见的病理性愈合现象,主要表现为创伤后局部组织的过度修复,常发生于深度烧伤或外伤后因各种原因延期愈合的创面。增生性瘢痕的危害主要在于,破坏人体组织器官的正常解剖结构,导致毁容及功能障碍,给创伤后的功能恢复和重建带来很大的障碍。目前增生性瘢痕的发病机理仍不完全清楚,可能与外伤、烧伤、手术、感染、异物、机体局部胶原代谢失调、遗传及局部免疫功能等多种因素有关。临床上常用治疗方法有:外科手术切除、物理治疗、硅凝胶治疗、放射治疗及基因治疗等,但是这些方法都有一定的创伤性,或是副作用太大,各有一定的局限性,不易广泛使用。临床上也曾采用一些药物用于增生性瘢痕的治疗,但由于效果不肯定,或毒副作用大,临床应用受到很大限制。所以寻找新的防治药物有重要意义。从组织病理学检查的结果看,增生性瘢痕和正常皮肤组织相比,最显著的改变是大量纤维化基质的堆积。在啮齿类动物,正常皮肤组织的胶原蛋白具有网状排列的模式,而增生性瘢痕组织中的胶原蛋白,则以几乎和基底膜成直角的方式大量成束状平行排列。而人类的胶原排列方式与此相比,胶原蛋白更多,胶原纤维更粗大,胶原排列更加有序,但这种胶原排列的方式却几乎和皮肤相平行。增生性瘢痕组织和正常皮肤相比,在结构上更多的区别包括不同比例的胶原类型和缺乏毛囊、汗腺等结构。在从肉芽组织向增生性瘢痕组织转化的过程中,胶原的重塑依赖于在低速率上持续的胶原合成代谢和分解代谢的改变。伤口或创面中胶原的讲解被几种称之为基质金属蛋白酶(matrix metalloproteinases,MMPs)的生物活性物质所控制,这些基质金属蛋白酶由巨噬细胞、上皮细胞、内皮细胞和成纤维细胞所分泌。伤口愈合的不同阶段,不同性质和数量组合的基质金属蛋白酶及其抑制物(tissue inhibitors of metalloproteinases,TIMPs)之间的组合与平衡发挥了重要的作用。增加的TIMPs和减少的MMPs的表达,暗示了过度纤维化或细胞外基质的过度沉积等疾病。经过近20年的研究发现,肝细胞生长因子具有多种生物学功能,例如,它是有丝分裂原(mitogenic)、促使细胞运动(motogenic)、抗凋亡(antiapoptotic)、抗纤维化(antifibrotic),并和形态发生有关(morphogenic)。因此,HGF在伤后和外伤后阻止纤维化并使伤口组织再生变得容易。有研究表明,在动物背部急性皮肤切割伤口使用HGF基因治疗,在切割伤缝合处,HGF基因显著抑制凋亡,产生极轻的纤维化,在愈合过程早期,断裂强度也下降。因此,可以推测HGF因为它的抗纤维化和抗炎症效应而减轻瘢痕化。但至目前为止,对于HGF在抗病理性瘢痕类纤维化疾病的研究方面,仍然缺乏比较细致的了解,例如在病理性瘢痕组织局部的蛋白表达水平、其对细胞外基质代谢的影响及其机制均缺乏足够的了解。对此,我们展开了以下实验研究,深入了解HGF/c-met对增生性瘢痕的抑制作用机制。方法:1.采集临床增生性瘢痕病理标本,进行免疫细胞化学染色,并行体外原代细胞培养,以多种方式研究HGF对体外培养的增生性瘢痕成纤维细胞胶原代谢的影响,2.具体的技术手段和检测指标有:(1)HGF/c-met在增生性瘢痕组织局部表达的对照检测:采用免疫组织化学染色的方法,结合计算机辅助图像分析技术,对所采集的增生性瘢痕和正常皮肤的临床标本进行检测;(2)体外成纤维细胞培养:采集成纤维细胞进行体外细胞培养,MTT法绘制细胞增殖曲线,并采用免疫细胞化学染色的技术对其进行细胞学鉴定;(3)对胶原蛋白表达的作用:3H-脯氨酸掺入胶原后检测胶原蛋白的总量变化;(4)对Ⅰ、Ⅲ型胶原和CTGF转录水平的测量:采用RT-PCR的方法检测Ⅰ、Ⅲ型胶原和CTGF mRNA的表达;(5)对MMP-1、MMP-2、MMP-9、TIMP-1、TIMP-3和c-jun的表达进行检测:采用免疫蛋白印记技术进行半定量检测; (6)探索HGF影响MMPs/TIMPs表达的信号通路:采用细胞信号转导阻滞技术,结合免疫蛋白印记的方法,对ERK 1/2信号转导途径进行检测。结果:1.在表皮层和真皮层的定位研究中,均能发现HGF/c-met蛋白的表达,但增生性瘢痕组织的阳性率较正常皮肤要高,且在真皮层中HGF的差异更加显著;2.体外培养的细胞镜下形态观察为梭形,胞浆丰富,胞体较大,波形蛋白免疫细胞化学染色呈阳性结果,MTT法绘制细胞自然增殖曲线表明细胞为稳定型成纤维细胞,可以满足后续的实验需要;3. 3H-脯氨酸掺入法的结果表明HGF处理细胞后胶原蛋白的含量变少;4. RT-PCR法检测结果表明HGF处理细胞后,Ⅰ、Ⅲ型胶原和CTGF mRNA转录水平下调;5. Westernblot法检测MMP-1、MMP-2、MMP-9、TIMP-1、TIMP-3的表达,结果表明HGF可以促进MMP-1、MMP-9的表达上调,而MMP-2、TIMP-1和TIMP-3的表达下调;6. PD98059阻断ERK 1/2信号转导实验结果表明,HGF通过ERK 1/2途径上调MMP-1的表达,下调TIMP-1的表达,并上调c-jun的表达。讨论:目前已知,HGF可在包括肝脏在内的多种组织细胞广泛表达,包括成纤维细胞、上皮细胞、内皮细胞、枯否式细胞以及某些肿瘤细胞[30-43]。皮肤组织中,HGF主要由表皮细胞和成纤维细胞合成、分泌,通过旁分泌和自分泌的方式作用于自身及临近的细胞[44]。癌基因c-met的产物是HGF的唯一受体,它同样在多种组织细胞内表达,包括上皮细胞、色素细胞、内皮细胞、小胶质细胞和神经元等。近年来,众多的动物实验表明,HGF是一种关键的抗纤维化疾病的内源性生长因子。本研究的结果表明,HGF/c-met在增生性瘢痕和正常皮肤组织中均有表达,阳性细胞主要位于表皮层;而相比较与正常皮肤组织,在增生性瘢痕组织真皮层成纤维细胞表达HGF上调,并可能由此诱导其受体c-met表达升高。这种真皮层HGF/c-met表达水平的改变,提示了其在增生性瘢痕的疾病转归过程中可能发挥了重要作用。增生性瘢痕的主要病理改变是细胞外基质(Extracullar matrix,ECM)的堆积。胶原(collagen)是ECM的主要组成成分,是ECM的骨架成分,主要由多种中胚层细胞分泌。正常生理状态下,胶原的合成和降解两种代谢维持着一种平衡,动态地调整组织形态结构。成纤维细胞是合成胶原的主要细胞,成纤维细胞的大量增殖或细胞合成与分泌胶原能力的增强,都可能导致胶原总量的增加。细胞外基质的过度沉积,是HS发生与形成的病理基础。胶原合成增加、胶原酶含量降低所导致的胶原代谢平衡失调,是瘢痕增生的重要因素,也是纤维化疾病发病的共同原因。3H-脯氨酸掺入实验是一种比较准确的测量胶原蛋白含量的方法,加入了HGF的细胞内其胶原蛋白含量明显减少,为了进一步的明确这种蛋白代谢的变化机制,我们从合成和分解两方面对胶原代谢进行了较深入的研究。经检测,Ⅰ、Ⅲ型胶原mRNA在HGF干预后转录水平下调,它和CTGF mRNA的下调是一致的,提示HGF对于CTGF表达具有一定的抑制效应,这可能是HGF抑制胶原蛋白合成的机制之一。胶原的分解代谢是由MMP/TIMP表达比例平衡的结果来决定的。本研究的结果表明,HGF可以改善人增生性瘢痕成纤维细胞MMP/TIMP表达的比例,从而促进胶原分解代谢;在这种生物学效应中,ERK信号转导通路和c-jun转录因子发挥了重要的作用。结论:1.HGF/c-met蛋白在增生性瘢痕组织中表达明显升高,表明其在增生性瘢痕的病理生理转归中具有重要的生物学意义;2. HGF可以减少人增生性瘢痕成纤维细胞胶原蛋白总量,抑制纤维化的发生;4. HGF可以在转录水平抑制胶原的合成;能促进胶原蛋白合成代谢的CTGF mRNA在转录水平同样被HGF明显下调;4. HGF可以改善人增生性瘢痕成纤维细胞MMP/TIMP的比例,提示这是HGF调节人增生瘢痕成纤维细胞胶原代谢的重要方式。5. HGF可以促进c-jun蛋白的表达,这可能是HGF抑制纤维化病程进展的另一重要机制;6.在人增生性瘢痕成纤维细胞中,HGF改善MMP/TIMP比例的重要机制是通过改变细胞信号分子ERK 1/2的磷酸化水平来实现的,而ERK 1/2的磷酸化水平还影响到了c-jun蛋白的表达,从而表明HGF抑制胶原蛋白合成、促进胶原蛋白分解的机制是互相影响的。更多还原

【Abstract】 Hypertrophic scar (HS) is one of the major unsettled clinical problems that due to abnormal proliferation of fibroblast and excessive deposition of extracellular matrix. The scars most commonly occur when epithelialization has been delayed during, foe example, the healing of deep dermal burn wounds. The HS are thick and raised and often darker in color than surrounding skin, moreover, that are frequently associated with pruritus and pain. Therefore, it is significant that exploring the mechanism of HS formation to look for effective prevention and treatment. Many published reports documented the mechanism and advocated a variety of therapies; however, few studies provide a coherent therapeutic plan.Collagen remodeling during the transition from granulation tissue to hypertrophic scar is dependent on continued synthesis and catabolism of collagen at a low rate. The degradation of collagen in the wound is controlled by several proteolytic enzymes termed matrix metalloproteinases (MMPs), which are secreted by macrophages, epidermal cells, and endothelial cells, as well as fibroblasts. The various phases of wound repair rely on distinct combinations of matrix metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). When MMPs gene expression was examined in fetal scarless and scarring rat skin wounds, an increase in expression occurred. However, the magnitude of this increase was greater in scarless fetal wounds than in scarring wounds. Moreover, expression of the tissue inhibitor of metalloproteinases (TIMPs) was higher in the scarred wounds, suggesting that the MMP/TIMP activity ratio maybe higher in scarless wounds. The production of MMPs and their associated inhibitors produces a delicate balance between matrix deposition and degradation. Subtle differences in MMP activity in fetal wounds may therefore tip this balance, allowing more rapid ECM turnover, which maybe necessary for scarless repair. Increased TIMP and decreased MMP expression has been implicated in diseases of excessive fibrosis and extracellular matrix deposition, such as scleroderma.Hepatocyte growth factor (HGF) can be produced by various cells, including fibroblasts, epithelial and endothelial cells, Kupffer’s cells and fat-storing cells in the liver, and malignant cells such as lung and pancreatic carcinoma and leukaemic cell lines. Although earlier studies implicated HGF in embryo development and in promoting tissue regeneration after acute injury, evidence is now emerging that HGF is also an intrinsic antifibrotic factor that plays a critical role in preventing tissue fibrosis in various animal models. Over the past several years, progress has been made in identifying the cellular targets of HGF and in unraveling the molecular mechanisms that underlie its action in tissue fibrosis. One of the anti-fibrogenic effects of HGF is thought to be expressed by the induction of matrix metalloproteinases (MMPs)In this study, it was hypothesised by the author that HGF, a soluble cytokine, could paly an important role to ease hypertrophic scar. But it is not clear that wheather HGF and its receptor c-met, a receptor tyrosine kinases (RTK), expression differs between hypertrophic scars and normal skins. Moreover, the exprssion of MMP-1, MMP-9, TIMP-1, and TIMP-2 of fibroblasts in vitro in adition with/without HGF were investigated to verify the hypothesis.Method: Hypertrophic scar tissues were obtained from 5 asian patients who had deeply burns on the limb or face. Immunohistochemistry procedure was used to investgae the localization an expressiong of HGF/c-met. HS fibroblasts were isolated and cultured by routine method. After identifaction, the 3-7 generation cells could be used. The concentrations of HGF for experimental group were: 25ug/ml, 50 ug/ml and 75 ng/ml.Detecting indexes:1. Immunohistochemistry and computer-assist image analysis technology were used to detect localization an expressiong of HGF/c-met.2. Detecting the cell proliferation curve using the method of MTT.3. immunocytochemistry identified the cultured cell with vimentin monoclone antibody.4. 3H-Proline dope procedure was used to detect the quantum variation of colleagen.5. RT-PCR to checked the transcription of mRNA ofⅠ,Ⅲtype collagen and CTGF 6. Westernblot detected MMP-1,MMP-2, MMP-9, TIMP-1, TIMP-3, c-jun.Result:1. In both hypertrophic scar and normal skin groups, intense HGF-positive and c-met-positive cells locating at the epidermis were oberved. There was an increase in the number of HGF-positive and c-met-positive cells in granulation tissue at the dermis in the hypertrophic scar tissue groups but not in the control group. The HGF-positive cells in the tissues detected in both groups were probably mainly fibroblasts as judged by their morphology.2. The result of MTT with immunocytochemistry showed that the culltured HSFb met the expriment requirment.3. By administration of HGF, collagen synthesis of experimental HSFb decreased with the relationship with the dose.4. HGF could inhibit the transcription of mRNA ofⅠtype collagen,Ⅲtype collagen and CTGF.7. HGF could enhance the expression of MMP-1, MMP-9 and c-jun.8. HGF could inhibit the MMP-2, TIMP-1, TIMP-3expression of HSFb.9. HGF could acive the ERK 1/2 signal transduction pathway.Conclusion:HGF expression up-regulated in hypertrophic scar fibroblasts could change the colleagen metabolism via inhibiting the expression of colleagen gene, improving the MMP/TIMP. ERK 1/2 signal transduction pathway was activated by HGF to produce the complicate effect.更多还原