创伤后瘢痕形成机制及治疗的研究

  本文关键词：创伤后瘢痕形成机制及治疗的研究，由笔耕文化传播整理发布。

        研究背景病理性瘢痕是创伤愈合后最常见的并发症之一,一直是外科修复领域研究的难点与热点。病理性瘢痕不但可以影响患者的容貌,而且产生的挛缩可导致不同程度的功能障碍,为该类患者后期治疗带来沉重经济和心理负担。其形成机制及防治一直是医学界研究的热点。目前其形成机制仍不清楚,尚无疗效确切的治疗方法。多年来,众多学者从不同角度探讨增生性瘢痕的形成、消退机理,期望从中找到治疗瘢痕的突破口。目前较一致的看法是：①增生性瘢痕以细胞过度增殖和细胞外基质过度沉积为特征,其中成纤维细胞是主要的效应细胞；②增生性瘢痕在生物学层面主要表现为胶原代谢紊乱；③TGF-β1/smad信号通路与成纤维细胞的增殖、分化、迁徙、凋亡及胶原代谢等多种生理、病理过程密切相关。成纤维细胞作为创伤愈合过程中的主要效应细胞,常常表现为过度增殖和功能活跃,从而引起一系列级联反应,造成创面修复失控,导致细胞外间质的过多沉积和创伤的过度愈合,形成病理性瘢痕。由此可见,成纤维细胞的功能异常在病理性瘢痕过度增生中扮演着重要的角色。Lekic等认为它是创伤修复的工程师、建筑者和管理员。自Convey等于1959年培养瘢痕成纤维细胞获得成功以来,开始了体外研究瘢痕的新阶段,为从细胞水平上认识瘢痕异常增生的机理提供了有力的证据和资料。近年来,病理性瘢痕的基础研究不断深入,成纤维细胞与瘢痕的关系以及瘢痕增生和侵袭性生长机制已成为医学研究的热点。但是,瘢痕成纤维细胞的原代培养对于初学者仍较为困难,失败率较高。因此,建立高效的培养方法及对瘢痕成纤维细胞进行鉴定及活力测定显得尤为重要。目前普遍的观点认为,增生性瘢痕的形成在生物学层面主要表现为胶原代谢紊乱。较多的研究表明在瘢痕增生过程中,胶原蛋白的大量合成和Ⅰ、Ⅲ型胶原纤维比例发生变化是形成增生性瘢痕的重要机制。胶原代谢紊乱与MMPs、 TIMPs间的动态平衡以及TGF-p/smad信号通路的异常具有密切的关系。但是,目前关于胶原在增生性瘢痕组织中的变化及其调控机制仍不清晰,需进一步澄清。MMPs(?)(?)TIMPs(?)司的动态平衡是影响胶原代谢的重要因素。但是在创伤后增生性瘢痕组织中,胶原酶的变化说法不一。Ghahary等报道在体外培养的人增生性瘢痕成纤维细胞间质胶原酶nRNA表达水平明显低于正常皮肤成纤维细胞,而两者的金属蛋白酶组织抑制因子表达无显著差异。国内赵烨德等研究证实MMP-3mRNA在HS组FB中表达水平低于正常组,而且降解DNP-多肽(胶原酶底物)能力也明显降低；同时将MMP-3基因转入HS成纤维细胞后,提高了MMP-3表达水平,促进了ECM降解,得出了类似的结果。同样,在对皮肤异常瘢痕的研究中发现,异常瘢痕TIMP-1的表达明显高于正常组织。在肝和肾的纤维化病变中也发现TIMP-1表达增加,可通过抑制MMPS的活性阻止过量的胶原降解。另外也存在较多与上述结果相反的报道。探索瘢痕组织内MMPs(?)(?)TIMPs的变化规律及其影响因素,可能为瘢痕形成机制提供线索。SMADs介导的转化生长因子信号通路在增生性瘢痕胶原代谢中发挥着重要的作用。SMADs是信号从受体到核内的细胞内主要转导分子,也是目前瘢痕增生研究最多、最为重要的信号分子。根据不同分型,对成纤维细胞具有双向调控作用。目前大量研究已经证实,其中Smad2/3是具有促进作用的因子,而SAMD7是抑制性的中间作用分子。但是,由于分子内部各种信号通路的相互作用,对于Smad2/3和SAMD7的变化规律及其影响因素仍是研究的重点。虽然大量的研究工作针对HS的发病机制和治疗,并且取得了丰硕的成果,但是对于增生性瘢痕的治疗目前仍缺乏有效的方案。外科手术切除是增生性瘢痕最常用的治疗。尽管各方面的改进,如：“z”或“w”成形术局部减张,切除瘢痕的纤维部分,保留外层皮肤。但是,手术仍可能因愈合失控或再次创伤而导致瘢痕过量形成。本组因功能障碍及影响美观,均采用手术的方法进行治疗。手术标本用作本实验。大量的研究表明,灯盏花素可抑制纤维化病变进展,灯盏花素是否能抑制病理性瘢痕成纤维细胞的增生,尚未见文献报道。也有研究认为细胞凋亡与HS的形成、消退过程密切相关,通过调控成纤维细胞凋亡有望成为有效防治HS的新亮点。本实验通过建立高效的瘢痕成纤维细胞原代培养方法,观察其生长规律,并对其进行生物学鉴定,首次探讨灯盏花素对瘢痕成纤维细胞增殖和凋亡的影响,希望寻找到有效治疗增生性瘢痕的药物。同时对瘢痕组织真皮层内影响胶原纤维代谢相关分子进行研究,挖掘其中重要分子的变化规律；为增生性瘢痕机制的阐明及治疗提供思路。研究目的1、寻求一种高效的瘢痕成纤维细胞体外原代培养方法。2、了解瘢痕成纤维细胞生长过程中的形态学变化和鉴定方法。3、了解病理性瘢痕组织形态学、分子生物学变化规律,更加深入地了解增生性瘢痕的形成机制。4、从细胞学方面初步探讨灯盏花素对瘢痕成纤维细胞生长增殖及凋亡的影响,为寻找有效的天然药物提供理论依据。研究方法1、标本收集、收集标本均来自来自广东省第二人民医院和广州市红十字会医院2011-06～2011-12创伤性瘢痕手术患者标本24例,年龄18～56岁。标本的获取均征得患者的知情同意,并签订知情同意书。标本入选和排除标准：A、经临床医生鉴定为瘢痕组织,并初步进行分类(增生性、瘢痕疙瘩)；B、排除垂体、肾上腺疾病、传染病、皮肤病及免疫性疾病患者,排除局部感染、溃疡患者；C、手术前未接受针对瘢痕的任何治疗。标本的获取均征得病人的同意,签知情同意书。2、标本的处理、分组组织切取后冷冻干燥法保存,均于4小时内进行实验。标本分三部分处理：一部分用于细胞原代培养,另一部分用10%福尔马林甲醛溶液固定,用于形态学鉴定(如免疫组化等)；第三部分置于液氮中冻存备用(提取蛋白和RNA用于后续试验)。选取同期因其他手术切除的正常皮肤组织3例作为正常对照组进行研究。3、组织微粒法进行瘢痕成纤维细胞原代培养,总结和完善瘢痕成纤维培养的方法,并对建立的细胞系进行命名和记录相关资料；观察瘢痕成纤维细胞生长规律和生长特点,MTT法绘制细胞生长曲线图；根据细胞形态以及α-SMA抗体的细胞免疫组化对瘢痕成纤维细胞进行鉴定。4、对增生性瘢痕组织进行HE染色和masson三色染色观察其形态学特征,比较正常组织和瘢痕组织的差异性。5、CollagenⅠ, CollagenⅢ的免疫组织化学染色,分析其在瘢痕组织内分布特点和表达量。6、应用RT-qPCR技术检测病理性瘢痕组织真皮内Collagen Ⅰ, CollagenⅢ, MMP-1, TIMP-1, SMAD3, SMAD7基因表达情况,并与正常组织进行比较,分析其差异性；应用Western Blot技术检测MMP-1、SMAD3、TIMP-1蛋白在瘢痕组织内的表达。7、使用MTT法检测不同浓度组瘢痕成纤维细胞增殖情况,绘制抑制率曲线图,并进行统计学分析各组差异性；使用流式细胞仪Annexin V/PI双染色法检测不同浓度灯盏花素对增生性瘢痕成纤维细胞凋亡的影响；半定量RT-PCR检测细胞Collagen Ⅰ和Collagen ⅢmRNA表达。研究结果1、瘢痕成纤维细胞形态学观察和鉴定(1)病理性瘢痕组织微粒在接种后大约2-7天,少量细胞从组织周围放射状或者旋涡状爬出(图1-1a、b)。原代成纤维细胞呈梭形或不规则三角形,胞质向外伸出多个长短不同的突起,多呈放射状、编织状或漩涡状排列,有时细胞排列紊乱,有明显的交叉重叠现象并呈团块状。细胞免疫组化α-SMA染色显示细胞核呈类圆形或长椭圆。(2)病理性瘢痕成纤维细胞传代培养时,成纤维细胞刚接种尚未贴壁时呈球形(图1-1c),贴壁后细胞逐渐伸展,呈梭形、长条形、多角形或不规则形(图1-1d)。2-3d即呈汇合状态(图1-1e)。细胞密度低时细胞之间排列疏松,有较大细胞间隙(图1-1e)。细胞密度高时,细胞相互平行排列或呈放射状和漩涡状排列(图1-1f)。随着细胞代龄的增高,细胞逐步出现老化现象,镜下观察可见细胞体积变大、形状扁平,过度伸展,伸展末端细长有分支,生长缓慢。(3)生长曲线显示：瘢痕成纤维细胞接种后经过短暂的潜伏期,开始增长的趋势,于6-8天生长进入对数期,10天以后进入停滞期(图1-3)。(4) α-SMA细胞免疫组化染色,出现较多阳性染色细胞的结果。结合其分离时的组织来源,以及其长梭形、栅栏状、漩涡状生长的特点,证实其为成纤维细胞。2、瘢痕组织HE染色观察结果：(1)正常皮肤成纤维细胞和微血管数目较少(图2-3a)。(2)瘢痕组织内可见成纤维细胞和微血管增多,微血管呈缩窄倾向(图2-3e)。真皮内见编织状排列的大片致密嗜伊红染同质性粗大胶原纤维,排列紊乱,无一定的方向,胶原纤维相互交叉呈漩涡状或结节状分布,结节中的胶原纤维较细,各结节之间由粗大的胶原纤维束分隔,粗大的胶原纤维束间可见分布有数量不等的成纤维细胞和小血管。(3)瘢痕组织(图2-3b、d)表皮厚度较正常表皮(图2-3a)明显增厚。3、Masson三色染色观察发现：瘢痕组织真皮层大量胶原纤维,粗细不一,显示真皮乳头层及附近血管增生,胶原纤维排列紊乱(图2-4a)；真皮网状层胶原纤维排列致密,近表皮处紊乱,近皮下组织处排列规则,较多血管分布,部分胶原纤维交叉形成胶原结节,以近乳头层处明显(图2-4b)。4、免疫组化结果(图)显示：瘢痕组织Ⅰ型胶原抗体在真皮层的细胞间质中,主要分布于真皮网状层,较为粗大,交织成结节状,周围散在分布成纤维细胞和血管壁。Ⅲ型胶原散在分布于真皮层的细胞间质中,以真皮乳头层分布较多,胶原束较细,排列疏松,走行方向不一,互相交织。血管壁上均有两种纤维的分布。5、RT-qPCR结果显示：瘢痕真皮组织和正常皮肤真皮组织内均可检测到collagen Ⅰ、collagen Ⅲ、MMP-1、TIMP-1、SMAD3、SMAD7的基因表达。与正常组织相比,瘢痕组织内collagen Ⅰ (t=5.85, P<0.01)、collagenⅢ(t=4.515, P<0.05)、MMP-1(t=-13.22,P<0.01)、TIMP-1(t=2.52,P<0.05),smad7(t=-2.503,P<0.05)基因表达差异具有显著性；由图可见collagen Ⅰ、collagenⅢ、TIMP-1表达较正常组织升高,而MMP-1表达较正常组织降低；Smad3(t=-0.339,P>0.05)基因表达未见明显差异。(图2-7,表2-1)6、Western Blot显示：这些样品中瘢痕真皮组织和正常皮肤真皮组织内均可检测到MMP-1、TIMP-1、SMAD3蛋白表达,但是表达量均低于正常组织。(图2-9)7、灯盏花素作用前后成纤维细胞形态未见明显变化；灯盏花素对体外培养FB生长增殖具有明显的抑制作用((F=3.309,P<0.01)(表3-1)。与对照组相比,(80、100、150)μmol·mL-1浓度(实验)组明显抑制瘢痕成纤维细胞生长(P<0.05)。结合抑制率曲线可知,灯盏花素对瘢痕成纤维细胞的抑制作用呈剂量依赖性(图1)。IC50=123.5μmol·mL-1。8、流式细胞仪Annexin V/PI双染色法检测FB凋亡结果显示：IC50处瘢痕成纤维细胞凋亡率为14.2050±0.95553,未加药组瘢痕成纤维细胞凋亡率为4.0220±0.31412,两者相比差异具有统计学意义(t=-22.653,P<0.05)。：RT-PCR结果显示随着灯盏花素浓度的升高,对瘢痕成纤维细胞collagen Ⅰ和collagen Ⅲ mRNA产生明显的影响,差异具有统计学意义。(如图3-4,表3-1)结论1、本研究改良了瘢痕成纤维细胞培养方法,减少了培养失败率,进一步补充了瘢痕成纤维细胞培养相关理论,为细胞培养提供一种高效方法。2、瘢痕成纤维细胞和正常成纤维细胞形态上无明显的区别,可利用特异性标志α-SMA对其进行鉴别。3、增生性瘢痕组织中collagen Ⅰ和collagen Ⅲ明显的增加,MMPs表达减少,以及TIMPs表达增高,与创面愈合纤维瘢痕形成关系密切。4、瘢痕增生过程中是多种信号通道、多因素相互作用的复杂过程。5、灯盏花素可通过抑制瘢痕成纤维细胞的增殖,并且诱导其发生凋亡,在瘢痕的治疗方面存在较大的应用前景,为寻找有效治疗增生性瘢痕的药物提供了新的思路。

    Background:Pathological scar, one of complications after skin trauma and post-operative, has a bad effect on the patient’s appearance and psychological. Its mechanism and prevention has been the focus of the medical profession.There is no clear conclusion on its mechanism, prevention and treatment.For many years, many scholars approach the problem from different perspectives on hyperplastic scar formation and regression,expect to find a breakthrough in the treatment of scar. A more consistent view are that:①hypertrophic scar is characterized by excessive cell proliferation and extracellular matrix, in which fibroblasts are the major effector cells;②The hypertrophic scar is mainly the result of collagen metabolism disorders at the biological level;③TGF-β1/smad signaling pathways are closely related with physiological and pathological procession of fibroblasts, such as proliferation, differentiation, migration, apoptosis and collagen metabolism and so on.Fibroblasts,as the major effector cells in the wound healing process, often shows excessive proliferation and active function, causing a cascade of reactions, out of control in wound repair, excessive deposition of extracellular matrix, the formation of pathological scar. Thus, the abnormal function of fibroblasts plays an important role in pathological scar hyperplasia. Lekic etal. describes fibroblasts as engineers, builders and administrators of wound repair.Since Convey etal. cultured hypertrophic scar fibroblasts in1959,it started a new stage of understanding of the mechanism of scar in vitro and the cellular level. In recent years, with the deepening basic research of pathological scar,relationship of fibroblasts and scar and mechanism of scar formation and invasive growth have become a hot topic of medical research. However, the primary culture of scar fibroblasts is still a more difficult for beginners for a high failure rate.Therefore, the establishment of efficient cultivation methods is particularly necessary for scar fibroblasts.The prevailing view is that The hypertrophic scar is mainly the result of collagen metabolism disorders at the biological level. The excessive synthesis of collagen and collagen proportion change during the scar formation process is the important reason of hypertrophic scar.Collagen metabolism disorder has a close relationship with the dynamic balance of TIMPs and MMPs and TGF-beta signaling pathway. However,the changes of collagen and its regulation in hypertrophic scar is still unknown.The dynamic balance between MMPs and TIMPs is an important factor affecting the metabolism of collagen.But there are different opinions on changes of collagenase.Ghahary et al reported that mRNA expression of MMP-1in human scar fibroblast derived fibroblast collagenase was significantly lower than normal skin fibroblasts, while there was no significant difference in the mRNA expression of TIMP-1.Zhao etal. confirmed that MMP-3mRNA expression level in the HS group was lower than the normal group,which reached the same results.Likely, in the study of abnormal skin scars,the expression of TIMP-1was significantly higher than that of normal tissue. The same thing occurred in the liver and kidney fibrosis.In addition,many opposite conclusions were reported.it is possible to provide clues for the mechanism of scar formation by exploring the changes of MMPs and TIMPs in the scar tissue and its related factors.TGF-β1/smad signaling pathways plays an important role in the collagen metabolism of hypertrophic scar.SMADs is the main transduction molecules from the receptor to the cell nucleus.Different Smad have two-way regulation on fibroblast. Numerous studies have confirmed that Smad2/3is the promoting factor, while SAMD7being the inhibitory role.However, variation and its influencing factors of Smad2/3and SAMD7is also the focus of the study because of the interaction of various signaling pathways.Lots of work have been done for the pathogenesis and treatment of the HS and fruitful results have been achieved, but there still no effective or satisfactory treatment for HS so far. Surgical resection is the most common treatment for hypertrophic scar. Despite the improvements were done, Surgical resection may still be re-trauma healing and lead to excessive scar formation. This group have adopted surgical treatment due to the dysfunction and ugly appearance.The surgical specimens were used for this experiment. It was reported that breviscapine can inhibit fibrosis progression,but it has not been reported whether it can inhibit proliferation of pathological scar fibroblast.Other studies suggest that apoptosis is closely related to the formation of HS.And the regulation of fibroblast apoptosis is expected to become a new bright spot of the effective prevention and treatment of HS.In this study,we observe the growth variation and biological identification in the establishment of efficient primary culture of scar fibroblasts, and firstly explore the impact of Breviscapine on proliferation and apoptosis of scar fibroblast, hope to find effective drugs for the treatment of hypertrophic scars. The metabolism of collagen fibers in the dermis layer of scar tissue related molecules, and tap one of the important molecular variation; to provide ideas for the clarification and treatment of mechanism of hypertrophic scar. Objective:1. Searching for an efficient method of scar fibroblasts culture in vitro.2.Detecting the morphology, molecular biology of pathological scar,to understand formation mechanism of hyperplastic scar in depth.3.To study the effects of Breviscapine on proliferation and apoptosis of cultured scar fibroblasts. It may be developed as a new drug for the treatment of pathological.Methods:1. Specimen collectionThe24Traumatic scar specimens, age18to56years, was from the Second People’s Hospital of Guangdong Province and Guangzhou Red Cross Hospital from2011.06to2011.12. Access to specimens with the patient’s informed consent, and signed informed consent. Inclusion and exclusion criteria of specimen:1. The clinician identification of scar tissue, and a preliminary classification (hypertrophic, atrophic, keloid);2. Without the pituitary, adrenal diseases, infectious diseases, skin diseases and autoimmune diseases, local infection, ulcer;3. Receiving nothing treatment for the scar. Obtaining of specimens had the patient’s informed consent, and signed the Informed Consent Form.2. Specimen handling, groupingSpecimen was saved with freeze-drying method,and was used for primary culture within4hours. The specimens were divided into three parts:1. Cells primary culture;2. Fixed in10%formaldehyde solution for morphological identification (eg, immunohistochemistry, etc.);3. Frozen in liquid nitrogen alternate (extraction of protein and RNA). three normal skin used as normal controls3.Primary culture of scar fibroblasts:Observing the scar fibroblasts growth regulation and the growth characteristics; Drawing cell growth curves by MTT assay; identifition of scar fibroblasts. 4.Observing the differences morphological characteristics between hypertrophic scar and normal tissue by HE staining and masson trichrome staining.5.Distribution characteristics and expression of Collagen Ⅰ and Collagen Ⅲ was analyzed in the scar tissue by immunohistochemical staining.6.Gene expression of Collagen Ⅰ, Collagen Ⅲ, MMP-1, TIMP-1, SMAD3, SMAD7within the dermis is detected by RT-qPCR and the difference is compared between HS and the normal tissue. Protein expression of MMP-1, of SMAD3and TIMP-1by Western Blot technology.7.Fibroblasts were cultured as an experimental model and the effect of breviscapine were studied. Cell proliferation was determined by MTT assay, and Apoptosis of cultured fibroblasts is induced by breviscapine at the IC50with Annexin Ⅴ/PI staining and flow-cytometry.Results:1. Morphology and identification of scar fibroblast(1) A small number of cells climb out from HS organization in the shape of radial or spiral between two and seven days.(Fig1-1a, b) Primary fibroblasts were in the shape of spindle-shaped or irregular triangle. Nuclei were round or oblong ina-SMA of Immunohistochemistry staining.(2) It is spherical when the fibroblast has not been adherent in HS fibroblast subculture (Fig1-1c), and then extended, showing spindle-shaped, long rectangular, polygonal or rules-shaped (Fig1-1d). Cell was in the state of the convergence at2～3d (Fig1-1e). It is arranged loosely and in larger cell gap in Low cell density (Fig1-1e) and arranged in parallel or radial and swirling arrangement in high cell density,(Fig1-1f).(3) Growth curve:After a short incubation period, Scar fibroblasts showed a gradually increasing trend growth in6to8days (Fig1-3) and into the stagnation of the number after10days.(4) The results of α-SMA of Immunohistochemical staining is shown in fig1-5. We can identify fibroblasts by positive staining cells and its morphology.2. HE staining and Masson staining:1. Fewer fibroblast and microvascular in normal skin (Fig2-3a).2. Increased fibroblast and microvascular can be seen in Scar tissue (Fig2-3e). Thick collagen in dermis is disorganized, the collagen fiber was cross-cutting,swirling, nodular, among which there are collagen fibers,vessels and fibroblast(Fig2-3b, d).3. Marked thickening of the epidermal Scar tissue is thicker than normal epidermis (Fig2-3a).3. Result of IHC:Collagen Ⅰ is mainly distributed in the reticular dermis. The distribution of Collagen Ⅲ is in the papillary dermis. Both of two fibers are found in the vessel wall.4. RT-qPCR results:collagen Ⅰ, collagen Ⅲ, MMP-1and TIMP-1, SMAD3, SMAD7gene expression in dermal tissue of the scar and normal skin can be detected. Compared with normal tissue, gene expression of collagenⅠ (t=5.85, P<0.01)、 collagenⅢ (t=4.515, P<0.05), MMP-1(t=-13.22, P<0.01), TIMP-1(t=2.52, P<0.05)、smad7(t=-2.503, P<0.05) were significant different in scar. We can see collagenⅠ、collagenⅢ、TIMP-1is increased and MMP-1is decreased. There was no significant difference in gene expression of Smad3(t=-0.339, P>0.05), and Smad7(p=0.0629>0.05).(Fig2-7and Tab2-1).5. Western blot:MMP-1and TIMP-1, of SMAD3can be detected in dermal of the scar tissue samples and normal skin.But with a lower Protein expression than normal tissue.(Fig2-9).6. In vitro, the proliferation of fibroblasts is significantly inhibited by breviscapine in the manner of dose-dependent(F=3.309,P<0.01). Compared with the control group, proliferation of fibroblasts is significantly inhibited in the concentration of80,100,150μmol·ml-1(P<0.05). Apoptosis rate of fibroblasts induced by breviscapine at the IC50is significantly increased.7. The results of Flow cytometric Annexin V/PI double dyeing show apoptosis rate of experimental group is14.2050±0.95553in IC50, Apoptosis rate of control group is4.0220±0.31412, a statistically significant difference in both (t=22.653, P<0.05). The results of RT-PCR show that with increasing of concentration, the scar fibroblasts collagen Ⅰ and collagen Ⅲ mRNA are influenced obviously, there is a statistically significant difference,(fig3-4, tab3-1)Conclusion:1. Establishing scar fibroblast cell lines and naming them, and complementing primary culture theory of scar fibroblast.2. A better understanding of different fibroblasts between HS and normal skin is observed by observing the morphological changes during the growth prosess.3. Collagen Ⅰ and collagen Ⅲ in the hypertrophic scar tissue increased significantly. The formation of HS is closely related with the reduced expression of MMPs, and increased expression of TIMPs.4. The formation process of scar is a complex process of multiple signal paths, multi-factor interactions.5. Breviscapine can significantly inhibit the proliferation and induce the apoptosis of cultured scar fibroblasts. It may be developed as a new drug for the treatment of pathological scar.

        创伤后瘢痕形成机制及治疗的研究

摘要3-10ABSTRACT10-16第一部分 增生性瘢痕成纤维细胞的原代培养与鉴定19-33    1 材料和方法19-24    2 结果24-28    3 讨论28-30    4 结论30    参考文献30-33第二部分 病理性瘢痕胶原代谢及其影响因素的研究33-54    1 材料和方法33-40    2 结果40-49    3 讨论49-52    4 结论52    参考文献52-54第三部分 灯盏花素对创伤性瘢痕成纤维细胞增殖、凋亡及mRNA表达的影响54-64    1 材料和方法54-57    2. 结果57-60    3 讨论60-62    4 结论62    参考文献62-64全文总结64-65综述65-76    参考文献72-76中英文缩略词对照表76-77攻读学位期间主要的研究成果77-78致谢78-80附件80

  本文关键词：创伤后瘢痕形成机制及治疗的研究，由笔耕文化传播整理发布。