摘要
皮肤的创伤愈合过程分为炎症反应、组织增生和组织重塑三个阶段,包括血管通透性增加、炎症细胞浸润、局部生长因子的分泌、上皮再生和基质的沉积和重塑。在整个组织修复愈合过程中,涉及创面局部微环境中多种细胞与细胞外基质成分、多种蛋白分子之间的相互制约和相互促进作用。在这些过程中,有多种生长因子能在细胞浸润、增殖,细胞外基质的产生和降解等各个环节影响创伤愈合过程。如果创面过度愈合,则形成病理性瘢痕。
皮肤创伤后病理性瘢痕的形成是临床面临的难题之一,病理性瘢痕不仅带来外观上的畸形,严重者瘢痕大片增生孪缩可致功能障碍,并产生心理障碍。病理性瘢痕形成机制和防治的研究一直是整形外科的难题。尽管临床医生认知一疾病的历史非常久远,采取了手术、药物、放疗、激光、冷冻、加压等多种方法进行单一或联合治疗,效果仍不理想。一般认为,它是由于各种原因引起成纤维细胞异常增殖,胶原大量合成,导致细胞外基质成分过度沉积所致。
临床上对于瘢痕的防治,主要是通过阻断参与瘢痕组织形成过程中的细胞和细胞因子的作用,抑制细胞外基质的过度产生和积聚。在创伤过度愈合,形成增生性瘢痕或瘢痕疙瘩的过程中,转移生长因子-β1(TGF-β1)发挥着决定性的作用。TGF-β可以促进成纤维细胞的增殖、促进细胞外基质的产生,抑制胶原降解。而单核、巨噬细胞和成纤维细胞又能以自分泌的形式产生TGF-β1,维持着创伤局部TGF-β的浓度。在增生性瘢痕和瘢痕疙瘩成纤维细胞中均发现TGF-β1的高表达。在TGF-β1低表达的动物胚胎,其皮肤创伤后可以无瘢痕愈合,而外源性地加入TGF-β1却可以引起胎儿皮肤伤口的瘢痕形成。另外通过抑制TGF-β1的表达和信号转导也能够起到抑制瘢痕增生的作用。由此可见,TGF-β1的高表达是瘢痕增生的重要因素,阻断TGF-β1的生物学效应则可以作为抑制瘢痕增生的一个关键的切入点。
核心蛋白聚糖(decorin)是细胞外基质中一种富含亮氨酸的小分子蛋白多糖,由核心蛋白和一条糖胺聚糖(GAG)链构成,其核心蛋白分子量约为40KD,以10-12个富含亮氨酸重复序列的结构域为特征,decorin因可结合在胶原纤维表面(decorating collagen fibers in vivo)而命名。研究证实,decorin是正常真皮中含量最丰富的蛋白多糖,在非瘢痕愈合创面中,decorin表达早且丰富,在早期增生性瘢痕组织中decorin含量极低或无,而增生性瘢痕在成熟过程中又重新出现与正常真皮含量相当的decorin。在深二度烧伤创面愈合形成的瘢痕研究中,真皮网状层成纤维细胞合成decorin能力低于乳头层成纤维细胞,而成纤维细胞可能来源于组织损伤时真皮深部残留的成纤维细胞,这些研究均表明decorin减少可能与增生性瘢痕形成有关。
Decorin广泛存在于多种结缔组织中,具有多种生物学功能。现已证实decorin可与多种纤维胶原相互作用,尤其是与Ⅰ型胶原的相互作用研究最多。组织基质中decorin可与Ⅰ型胶原结合,结合位点在d带和e带,其GAG侧链垂直于胶原原纤维将相邻的原纤维连接起来。因此decorin可能通过GAG链间形成的抗平行的双螺旋结构,使胶原分子的侧向装配受限,从而调节胶原原纤维直径,并保证胶原原纤维准确装配,形成胶原纤维及胶原纤维束。而敲除decorin基因的小鼠皮肤胶原纤维厚度不均,皮肤易碎。Decorin还可调节细胞黏附,中和TGF-β1的活性。Decorin对TGF-β1的调节可能是因为细胞因子与其结合后妨碍了细胞因子与细胞表面受体的结合,对细胞外基质(ECM)中的生长因子起到隔绝作用。此外,decorin还具有抗细胞增殖等作用。在体外实验中,decorin可抑制TGF-β1刺激正常皮肤和增生性瘢痕成纤维细胞增殖与胶原合成的作用,进一步采用三维胶原凝胶网格培养证实decorin可能对增生性瘢痕的挛缩具有抑制作用,表明decorin在预防增生性瘢痕形成和促进瘢痕成熟中具有一定的作用。Decorin通过上述的一些途径和机制在抗纤维化中作用明确,一些国家已经尝试将提取和人工合成的decorin通过适当的途径用于动物和人体进行抗纤维化的治疗,国内也有用decorin干预肌腱、结膜瘢痕、肾脏纤维化的成功实验报道。
寻找和建立有效的瘢痕动物模型是瘢痕研究的基础,兔耳腹创面愈合后形成瘢痕,已得到整形界的认可。目前国内尚无关于通过兔耳瘢痕动物模型,观察外源性decorin对增生性瘢痕作用的报道。本实验以兔耳增生性瘢痕为模型,给予外源性decorin,观察其能否促进创面愈合,预防瘢痕增生或促进瘢痕成熟,以期为今后应用于临床提供理论依据。本研究将分为二部分,第一部分建立兔耳增生性瘢痕模型,在瘢痕形成早期,注射外源性decorin,观察decorin是否可减轻瘢痕增生,促进瘢痕软化。第二部分则分别在兔耳创面愈合早期和后期,给予外源性decorin,观察decorin是否可促进兔耳创面的愈合和抑制瘢痕形成,并比较不同时期应用后效果是否有差异性。
第一部分瘢痕形成早期核心蛋白聚糖decorin对兔耳增生性瘢痕的抑制作用研究
目的：建立兔耳瘢痕增生模型,于瘢痕形成后向瘢痕内注射核心蛋白聚糖decorin,观察decorin对瘢痕增生是否有抑制作用,分析组织内Ⅰ型胶原和TGF-β1蛋白合成和基因表达变化。
方法12只新西兰大白兔,随机分成3组,实验组(DCN组)、对照组(PBS组)和空白对照组,每组4只。于耳腹做直径8mm圆形创面,建立增生性瘢痕动物模型。观察每组创面愈合和瘢痕形成情况,比较各组平均创面愈合时间和瘢痕形成率。术后第20天和第25天实验组行瘢痕内注射10μg/ml核心蛋白聚糖decorin 2次,对照组注射PBS溶液2次,空白对照组不做任何处理。至术后30天(末次注射后5d),每组切取标本30份。15份行HE染色和Ⅰ型胶原和TGF-β1免疫组织化学染色,比较各组瘢痕增生指数(SEI),Ⅰ型胶原蛋白和TGF-β1阳性染色平均吸光度差异。另15份标本提取组织RNA, real-time PCR检测各组增生性瘢痕中Ⅰ型前胶原和TGF-β1 mRNA表达2-△Ct值。
结果除感染创面外,术后15-17天大部分创面兔耳已愈合,伤后20天左右瘢痕逐渐形成。增生瘢痕呈红色或淡红色,中央开始出现“小丘”样凸起,高于皮面,瘢痕质硬。观察术后三组创面平均愈合天数：DCN注射组15.794±1.393 d,PBS组15.602±1.389d,对照组15.772±1.389d；术后20天瘢痕形成率为DCN组80.492±3.731%,PBS组为79.659±3.222%,对照组为80.038±3.624%,采用单因素方差分析比较,三组创面平均愈合时间(F=0.253,P=0.776)和瘢痕形成率(F=0.056,P=0.946)差异均无显著性。
术后30天(末次注射后5天)时,三组兔耳增生瘢痕差别明显。PBS组和对照组增生瘢痕明显高于周围正常皮肤,中央呈“小丘”样凸起明显,局部质地较硬,部分增生呈鱼钩状弯曲,用手无法使其变形。DCN组瘢痕平坦,质地大部较软,只有中央增生部分质地较韧,高出皮面,瘢痕表面呈淡粉红色,周围与正常皮肤接近。
显微镜下可见,PBS注射组和对照组兔耳增生瘢痕表皮层和真皮层均明显增厚。真皮层内可见大量排列无序的成纤维细胞,细胞核深染。成纤维细胞在真皮浅层排列混乱,呈漩涡或结节状,在真皮深层排列规整。基质中大量胶原沉积,排列呈结节或漩涡状结构,并可见增生的毛细血管、血管内红细胞,及炎症细胞浸润。DCN组真皮层内成纤维细胞排列较规则,胞核淡染,胶原形态纤细,排列规整。毛细血管管腔部分闭塞,炎症细胞浸润不明显。单因素方差分析术后30天,三组瘢痕增生指数差异具有统计学意义(F=53.198,P=0.000),DCN注射组瘢痕增生指数(1.716±0.290)显著低于PBS组(2.785±0.435)和对照组(2.982±0.475),差异有统计学意义(P=0.000),PBS组和对照组之间无显著差异(P>0.05)。
免疫组化染色结果显示,Ⅰ型胶原蛋白阳性表达信号呈现棕黄色染色,主要位于细胞外基质中,成纤维细胞胞浆也可表达。PBS组和对照组Ⅰ型胶原染色分布密集,粗大呈束状,成纤维细胞胞浆内着色较深,呈深棕黄色染色。DCN组细胞外基质中可见略加深染不规则的纤维网状条索,分布稀疏,纤维较纤细。β1阳性表达信号主要定位于成纤维细胞胞浆中,细胞外基质轻微淡染。PBS组和对照组中,成纤维细胞胞浆内可见密集的棕黄色颗粒,或深染的棕黄色团块,质地不均。DCN注射组中,单位面积内阳性细胞个数明显减少,胞浆着色不均,大部分阳性细胞胞浆淡染。计算机图像分析系统计算阳性信号的吸光度,表示Ⅰ型胶原蛋白和TGF-β1蛋白表达水平,结果显示,三组Ⅰ型胶原(F=25.690,P=0.000)和TGF-β1(F=31.455, P=0.000)平均吸光度差异均具有显著性。DCN组Ⅰ型胶原和TGF-β1吸光度均显著低于PBS组(P均=0.000)和对照组(P均=0.000),差异具有显著统计学意义(P<0.05)。PBS组和对照组之间Ⅰ型胶原和TGF-β1平均吸光度无统计学差异(P>0.05)。
Real-time PCR以2-△Ct值表示Ⅰ型前胶原和TGF-β1mRNA相对表达量。术后30天DCN组中Ⅰ型前胶原和TGF-β1mRNA相对表达量均低于PBS组和对照组(P<0.05),差异具有显著统计学意义,PBS组和对照组之间无差异(P>0.05)。
结论本部分实验以兔耳增生性瘢痕为模型,于瘢痕形成早期行瘢痕内注射decorin,证实外源性decorin可以减少细胞外基质中Ⅰ型胶原和TGF-β1的合成和mRNA的表达,抑制瘢痕增生,促进瘢痕成熟软化。
第二部分创面愈合期核心蛋白聚糖decorin对兔耳创面愈合和瘢痕形成的影响研究
目的：建立兔耳瘢痕模型,分别在创面愈合早期和愈合后期应用外源性decorin,比较兔耳创面愈合、瘢痕形成和增生有无差异。
方法：选择15只新西兰大白兔,于耳腹做直径8mm圆形创面,每只12个创面,共180个创面。随机分成3组,早期治疗组(E-DCN组)、晚期治疗组(L-DCN组)和空白对照组,每组5只。早期治疗组(E-DCN组)术后第1、3、5天创面外涂和创周注射10μg/ml核心蛋白聚糖decorin;晚期治疗组于术后第9、11、13天创面及创周注射10μg/ml核心蛋白聚糖decorin;空白对照组创面暴露,自然愈合。记录创面愈合时间及瘢痕形成情况,比较各组创面平均愈合时间和瘢痕形成率。于术后第7天和术后30天分别切取标本20份,10份行HE染色和Ⅰ型胶原和TGF-β1免疫组织化学染色,比较瘢痕增生指数(SEI)(只用于术后30天标本)、Ⅰ型胶原和TGF-β1表达平均吸光度。另10份标本提取组织RNA, real-time PCR检测各组组织中Ⅰ型前胶原和TGF-β1 mRNA表达2-Act值。
结果：术后观察三组创面平均愈合时间具有统计学差异(F=3.219,P=0.044),早期治疗组为16.188±1.856d,较晚期治疗组(15.338±1.618d)和对照组延长(15.388±1.569d),差异具有统计学意义(P<0.05)。晚期治疗组和对照组无统计学差异(P>0.05)。术后20天各组瘢痕形成率早期治疗组为80±6.847%,晚期治疗组为85±5.590%,对照组为82.5±6.847%,三组间瘢痕形成率分别为无统计学差异(F=0.056,P=0.946)。
显微镜下显示,术后30天早期治疗组和对照组瘢痕组织结构无明显的组织学差异。表皮明显增厚,真皮层次紊乱,胶原纤维粗大,排列紊乱,可见漩涡状胶原结节,成纤维细胞数量较多,体积较大,微血管分布丰富,其间可见较多的炎细胞浸润。晚期治疗组瘢痕组织表皮略微增厚,真皮层较正常变厚,胶原纤维排列略有序,成纤维细胞数增加,更接近正常皮肤结构。瘢痕组织中微血管分布相对较少。测术后30天瘢痕增生指数,晚期治疗组SEI为1.951±0.312,低于早期治疗组(2.770±0.519)和对照组(2.858±0.38),差异具有统计学意义(P均=0.000),早期治疗组和对照组之间无统计学差异(P>0.05)。
免疫组化染色结果显示,术后第7天三组肉芽组织中Ⅰ型胶原(F=3.395,P=0.048)和TGF-β1(F=9.582, P=0.001)平均吸光度差异均具有统计学意义。早期治疗组Ⅰ型胶原和TGF-β1吸光度均低于晚期治疗组(P=0.025,P=0.001)和对照组(P=0.044,P=0.001),晚期治疗组和对照组之间无统计学差异(P>0.05)。术后第30天三组瘢痕组织中晚期治疗组Ⅰ型胶原和TGF-β1吸光度则均低于早期治疗组和对照组,差异具有统计学意义(P<0.05),早期治疗组和对照组之间无统计学差异(P>0.05)。
以2-△Ct值表示Ⅰ型前胶原和TGF-β1 mRNA表达量,术后第7天早期治疗组中Ⅰ型前胶原mRNA水平为1.196±0.310,较晚期治疗组(1.6199±0.380)和对照组(1.747±0.486)显著减少(P<0.05),晚期治疗组和对照组相比无显著差异(P>0.05)。至术后30天,三组Ⅰ型前胶原mRNA表达2-△Ct值均较术后7天显著增加(P<0.05),早期治疗组升高达3.926±0.969,晚期治疗组为2.677±0.519,对照组为4.012±0.797。术后30天组间比较,晚期治疗组Ⅰ型前胶原mRNA表达水平显著低于早期治疗组和对照组(P<0.05),早期治疗组和对照组间无统计学差异(P>0.05)。
术后第7天早期治疗组中TGF-β1 mRNA水平为0.025±0.087,较晚期治疗组(0.067±0.022)和对照组(0.061±0.020)显著减少(P<0.05),晚期治疗组和对照组相比无显著差异(P>0.05)。术后30天,早期治疗组TGF-β1 mRNA表达2-△Ct值较术后7天显著增加,为0.080±0.029,晚期治疗组TGF-β1 mRNA水平则下降为0.038±0.012,差异均具有统计学意义。术后30天对照组TGF-β1mRNA为0.074±0.032,虽与术后7天相比有增加,但无统计学意义(P>0.05)。术后30天组间比较,晚期治疗组表达水平低于早期治疗组(P=0.004)和对照组(P=0.014),差异有显著性。早期治疗组和对照组之间无统计学差异(P>0.05)。
结论：外源性decorin应用于兔耳创面愈合的不同时期,均可减少组织中Ⅰ型胶原和TGF-β1的蛋白表达和基因表达水平。
兔耳创面愈合早期(创面上皮开始形成之前)给予外源性decorin延长创面愈合时间,没有进一步抑制瘢痕形成和增生的作用。兔耳创面愈合晚期(创面上皮开始形成之后)给予外源性decorin对创面愈合时间和瘢痕形成率无影响,可抑制创面愈合后瘢痕进一步增生。
在以拮抗TGF-β1活性为主要目标的创面治疗中,应在创面开始上皮化后再给予干预,可能会取得较好的抗瘢痕形成作用。否则,过早抑制TGF-β1的活性,可能会影响创面的愈合进程,达不到抑制瘢痕增生的作用。
Cutaneous wound healing consists of three phases:inflammatory, tissue formation and tissue remodeling. This complicated process involves increased permeability of blood vessels. inflammatory cell migration to wound site and production of growth factors in a wound, re-epithelialization, and matrix deposition and remodeling mediated by fibroblasts. When successful, wound healing restores normal function with a well organized minimal scar. But when the controlling mechanisms are abnormal, hypertrophic scar formation, characterized by excessive connective tissue formation, can occur.
The post-traumatic hypertrophic scar is one of the clinical problems faced by the current lack of effective means of prevention and treatment. Hypertrophic Scar forming mechanism is not clear, generally thought, it is because of various fators causes abnormal fibroblast proliferation and collagen synthesis of substantial, resulting in excessive collagen-induced extracellular matrix deposition.
To prevention and treatment for the scar, researchers mainly focuse on blocking the participating process of cells and the role of cytokines, inhibition of excessive extracellular matrix production and accumulation. Of many kinds of growth factors that influence wound repair and induce scar formation, TGF-β1 appears to play the most important role. TGF-β1 can promote fibroblast proliferation, promote extracellular matrix production, and inhibit collagen degradation. In addition, monocytes, macrophages and fibroblasts perpetuate the high concentration of TGF-β1 at the wound site by expressing TGF-β1 in an autocrine manner. TGF-β1 also is over-expressed in cultured fibroblasts derived from hypertrophic scars and keloids. In contrast, fetal skin of animals, which has very low levels of TGF-βq expression, can heal without scar formation after injury. Whereas exogenously added TGF-β1 can induce scar formation in fetus. All these facts indicate that TGF-β1 plays essential role in scar development. In addition, scar formation can decrease by inhibiting the expression and signal transduction of TGF-β1. Thus, a strategy for inhibiting hypertrophic scarring is to block the bioactivity of TGF-β1.
Decorin belongs to a family of small leucine-rich proteoglycans and is found in the extracellular matrix, consisting of a 40KD core protein and a single glycosaminoglycan chain. The core protein consists of 10 to 12 repeats of a leucine-rich sequence of 24 amino acids. Decorin is one of the most abundant proteoglycans in normal dermis. But hypertrophic scar in the early stage contains reduced amounts of decorin, Interestingly, the content of decorin in mature scar is similar to that in normal skin. Fibroblasts isolated from reticular dermis reportedly synthesize less decorin than cells from papillary dermis, while fibroblasts responsible for healing may come from deep dermal survival fibroblasts in scar-healing wound. Decorin appears to be expressed early and abundantly in normally healing wounds. These data suggest that reduction of decorin is possibly related to hypertrophic scar formation. Decorin is present in a variety of connective tissues, and is involved in important biological functions. As named for its "decorating" association with collagen fibrils, decorin controls the morphology of collagen fbrils, as demonstrated in mouse knockouts by non-uniform fibril thickness and skin fragility. It also modulates cell adhesion and neutralize TGF-β1 activity.
Find and establish an effective animal model is the basis of scar research. Many researchers had established a reproducible in vivo rabbit dermal ear ulcer model that produces hypertrophic scars mimicking the human condition. This model has been successfully used for investigation and modulation of wound healing and hypertrophic scar formation. For example, topical application of wounds with TGF-β1 improves wound healing, without altering scar prominence. In contrast, the targeted inhibition of collagen deposition with a prolyl hydroxylase inhibitor decreases scar hypertrophy, without delaying wound healing.
At present, there is no report about the effect of decorin on the hypertrophic scars of the rabbit ear scar model. The purpose of this study was to examine the effect of decorin on wound healing and hypertrophic scar formation in the rabbit dermal wound model. This study will be divided into two parts. In the first part decorin was injected into the hypetrophic scar of rabbit ear right after scar formation, to examine that if decorin could reduce scar hypertrophy and promote scar softening. In the second part, decorin would be ocured on the rabbit dermal wound model in the early wound healing stage and late wound healing stage respectively, to observe that if decorin could promote rabbit ear wound healing and inhibit scar formation. We hope that the findings of this study have clinical implications for patients undergoing procedures in plastic surgery in the future.
Part I The inhibition of decorin on the rabbit ear scar model in the early scar formation stage
Objective To observe the effect of decorin on inhibiting hypertrophic scar by establishing hypertrophic scar model on the ventral side of rabbit ears.
Methods Full-thickness dermal wounds, 8mm in diameter, were made over the ventral side of ears in 12 adult New Zealand white rabbits. Then the rabbits were divided into 3 groups:DCN group is experimental group (decorin, 10μg/ml) and PBS group(PBS) and control group. Observe wound healing and scar formation in each case and compare average wound healing time and scar formation rate of three groups. On the days 20 and 25 after operation, the hypertrophic scar were injected with decorin and PBS intralesionally.30 Samples were harvested 5 days after the last injection (30s day). Half of the specimens were stored for sirrus red staining and type I collagen and TGF-β1 monocolonal antibody immunohistochemistry staing, to measure the Scar Elevation Index (SEI) and quantify the expression level of type I collagen and TGF-β1 protein. The other half of were extracted total RNA and quantified the mRNA expression level of procollagen I and TGF-β1 by real-time polymerase chain reaction.
Results:In addition to several infected wounds, the majority of the rabbit ear dermal wounds healed on days 15-17 after wounding. The healing wounds were red or light red, hard and "hill" like scar in the central of the wounds. The average healing wounds time of DCN group was 15.794±1.393 d, that of PBS group and the control group were15.602±1.389d and 15.722±1.389d respectively. Scar formation rate on post-wounding 20 days of DCN group was 80.492±3.731%, PBS group and control group were 79.659±3.222% and 80.038±3.624% respectively. There were no significantly difference among three groups.
On the post-wounding days 30 the DCN group had their scars appeared to be flatter, softer and lighter in color, compared with PBS group and control group. Under the microscope, the fibroblasts and more slender collagen of DCN group scar, arranged in rules, without obviously swirling structure. PBS injection group and control group had hypertrophic scars, hook-shaped and hard to be bent. Under the microscope the scars show more thicken epidermis and a large number of fibroblasts and collagen arranged in disorder, or in nodules. The proliferation of capillaries and inflammatory cell infiltration were noticed in the dermis. SEI of DCN injection group was1.716±0.290, significantly lower than PBS group (2.785±0.435)and control group (2.981±0.475). The PBS group and the control group had no significant difference (P>0.05).
Immunohistochemical staining showed that collagenⅠprotein expression signals were brown staining and mainly located in the extracellular matrix and the fibroblast cytoplasm. It was noticed that the extracellular matrix of PBS group and control group were filled with dark brown, thick collagen fibrous bundle, or irregular fiber network. The collagen I staining of DCN group were slightly deeper brown. TGF-β1 expression signals were also brown staining, mainly located in the cytoplasm of fibroblasts, extracellular matrix lightly stained. In the fibroblasts cytoplasm of PBS group and the control group, dense brown particles, or clumps of deep brown staining could be seen. In DCN group the number of positive cells were reduced, most of which were lightly and uneven brown staining. The relative protein express level of typeⅠcollagen and TGF-β1 were quantifed with mean absorbance value caculated in computer image analysis system. The results showed that the mean absorbance of typeⅠcollagen and TGF-β1of DCN group were significantly lower than PBS group (P=0.000) and the control group (P=0.000). There was no significant statistical difference between the PBS group and the control group (P>0.05).
The pro-collagen I and TGF-β1 mRNA relative expression level were indicated with 2-△Ct value, assessed by real-time PCR. The results showed that the pro-collagen I and TGF-β1 mRNA relative expression level of DCN group were statistically lower than PBS group and control group (P<0.01). There were no significant difference between PBS group and control group (P>0.05).
Conclusion This part of the experiment confirmed that exogenous decorin, injected into the rabbit ear hypertrophic scar at the early stage of scar formation, inhibit the scar proliferation and remarkably decrease the degree of fibrosis in the scar.
Part II The effect of decorin on the rabbit ear scar model in the different wound healing stage
Objective:To investigate the effect of decorin on the wound healing and scar formation of the rabbit dermal ear wound model, the wounds were treated with decorin in the different early and late wound healing time respectively. Methods: 180 full-thickness dermal wounds,8mm in diameter, were made over the ventral side of ears in 15 adult New Zealand white rabbits, each side of rabbit ear had 6 wounds on, a total of 180 wounds in the study. Then the rabbits were divided into 3 groups: early treatment group (E-DCN group) were treated with lOμg/ml decorin on postwounding days 1,3,5; late treatment group (L-DCN group) were treated with 10μg/ml decorin on postwounding days 9,11,13; the wounds of control group were exposed to natural healing. On days 7 and 30 after wounding samples were harvested. Half of the samples stained with immunohistochemistry monocolonal antibodies and sirrus red to quantify Scar Elevation Index (SEI) and the protein expression level of type I collagen and TGF-β1 The other half of samples were stored for subsequent RNA extraction and real-time polymerase chain reaction and examined the mRNA expression level of pro-collagen I and TGF-β1.
Results:The average wound healing time of early treatment group was 16.188±1.856 days. Compared with late treatment group (15.338±1.615 d) and the untreated control group (15.388±1.559 d), the difference was statistically significant (P<0.05). There was no significant difference between late treatment group and control group (P> 0.05). On ays 20 after wounding the scar formation rate of three groups were not significantly different.The SEI of early treatment group was 80±6.847%, that of late treatment group was 85±5.590%, and the control group was 82.5±6.847%.
On postwounding days 30, SEI of late treatment group was 1.951±0.312, significantly lower than those of early treatment group (2.770±0.519) and control group (2.858±0.358). There was no significant difference between early treatment group and control group (P> 0.05). Immunohistochemical staining of the granulation tissue of 7 days after wounding showed that in the early treatment group, the mean absorbance value of type I collagen and TGF-β1 were lower than late treatment group (P<0.01, P<0.05) and the control group (P<0.01, P<0.05), the difference was significant statistically. No significant difference between late treatment group and control group (P> 0.05). Immunohistochemical staining of the scar of 30 days after wounding indicated that the mean absorbance value of type I collagen and TGF-β1 of late treatment group lower than early treatment group and control group, the difference was statistically significant (P<0.05). No significant difference between early treatment group and control group (P> 0.05).
2-△Ct value indicates the relative mRNA expression level of procollagenⅠand TGF-β1. The results displayed that in the early treatment group procollagenⅠ(1.196±0.310) and TGF-β1 mRNA level were significantly lower than in late treatment group (procollagenⅠ:1.619±0.380) and the control group (procollagen I:1.747±0.486) (P<0.01). No significant difference between late treatment group and control group (P>0.05). On days 30 after wounding, the results showed that procollagen I (1.619±0.380) and TGF-β1 mRNA level of the late treatment group were significantly lower than early treatment group (procollagenⅠ3.926±0.666) and the control group(procollagen I 4.012±0.797) (P<0.05). No significant difference between early treatment group and control group (P> 0.05).
Compared with days 7 after wounding, the procollagen I mRNA level of each group increased significantly (P<0.01) on postwounding days 30. For early treatment group TGF-β1 mRNA level on postwounding days 7 increased from 0.025±0.087 up to 0.080±0.029 on postwounding days 30, late treatment group decreased from 0.067±0.022 to 0.038±0.012, the difference were significant statistically. Although TGF-β1 mRNA expression level of control group increased from 0.061±0.020 to 0.074±0.032, no significant difference between days 7 and 30 after wounding
Conclusion:This part of the experiment confirmed that exogenous decorin, injected into the rabbit ear hypertrophic scar at the early stage of wounding healing period, prolonged the wounding healing time, without the effect of inhibiting the scar proliferation. When decorin was choosed at the late stage of wound healing time, the wounds healed without delay and the formation of hypertrophic scar was inhibited successfully.
TGF-β1 has been identified as a critical factor in hypertrophic scar formation. Our findings suggest that to decrease hypertrophic scar formation, treatment with TGF-β1 inhibitor, decorin, needs to occur when TGF-β1 mRNA expression level are near their peak numbers, and collagen synthesis is very active. Otherwise, inhibition the activity of TGF-β1 earlier may affect the wound healing process and fail to suppress the role of scar formation. This may be clinically significant, because strategies to decrease scarring can potentially impair healing. If wound healing has progressed sufficiently, various strategies to reduce collagen synthesis may be more successful.
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