【作者】 高峰；

【导师】 霍然；

【作者基本信息】 山东大学 ， 整形外科， 2009， 硕士

【摘要】 目的瘢痕疙瘩是皮肤损伤后纤维组织过度修复的产物,具有一定的良性肿瘤的特征,过度增生并超过初始创伤的边界,瘢痕疙瘩的发病机制复杂,尽管皮肤的创伤和感染是公认的发病原因,具体的发病机制仍然未知。正常的创面修复需要胶原纤维增生和基质降解之间的巧妙平衡,但是瘢痕疙瘩的胶原合成量明显高于增生性瘢痕,而且瘢痕疙瘩存在着胶原纤维的降解不足。肥大细胞类胰蛋白酶(mast cell tryptase,MCT)是一种中性丝氨酸蛋白酶,以活性形式储存在肥大细胞分泌颗粒中,在肥大细胞脱颗粒过程中被释放到细胞的周围并在局部产生相应的生物学作用。MCT是肥大细胞最主要的蛋白酶,约占其细胞蛋白总量的25%,基因定位于人16号染色体的短臂末端,其活化形式为一个约140KD大小的四聚体。研究发现MCT能够促进表皮成纤维细胞增殖和α1型前胶原合成,提示可能与瘢痕疙瘩的形成有一定关系。我们将应用免疫组化的方法确定MCT在瘢痕疙瘩中是否有表达及表达的位置,在应用实时荧光PCR方法确定MCT基因在瘢痕疙瘩、增生性瘢痕及正常皮肤中是否存在差别。根据以上的结果我们就能够结合MCT的生物学特性,探讨MCT在瘢痕疙瘩的形成过程中是否有一定的作用及其可能的作用机制。资料与方法一.临床资料瘢痕疙瘩,20例,来自于20个患者,年龄15到32岁,瘢痕形成时间1到4年;增生性瘢痕,20例,来自于20个患者,年龄10到45岁,瘢痕形成时间6个月到2年;正常皮肤,20例,来自于20个患者,年龄8到30岁。标本全部由山东大学附属山东省立医院烧伤整形科提供,根据临床表现和病理结果[42-46]分类。瘢痕及皮肤均无感染、无皮肤病变,手术切除前未接受激素、放射等治疗。标本切取后立即放入液氮中保存备用。二.方法1.荧光免疫组化当瘢痕组织中有MCT表达时,MCT抗体(一抗)将于MCT结合。再用荧光标记的二抗与一抗结合,就能够在荧光显微镜下观察到二抗的位置,也就是MCT的位置。2.实时荧光相对定量pcr(1)提取瘢痕组织中的RNA。(2)反转录并扩增MCT的基因。(3)然后应用实时荧光半定量pcr方法观察MCT在瘢痕疙瘩、增生性瘢痕及正常皮肤中是否存在差异。相对定量分析MCT在这三者之间的表达倍数关系。三.统计学方法本实验应用相对定量的C_T值比较法,7500PCR仪系统软件将根据扩增过程中荧光信号的变化情况来设定相关参数并计算样本中基因的相对含量,再应用SPSS10.0的F检验对相对含量值进行数据统计分析。结果一.荧光免疫组化荧光显微镜下,在瘢痕疙瘩中可见较强的而且多的荧光信号,主要集中在胶原纤维束之间,以真皮浅层较多。在增生性瘢痕和皮肤中只有较少或者较弱的荧光信号。二.实时荧光相对定量pcr统计学证实瘢痕疙瘩、增生性瘢痕及皮肽三者之间在MCT基因表达上存在统计学差异(P＜0.01)。在瘢痕疙瘩中的MCT基因量约为增生性瘢痕的2.5倍、皮肤的5.4倍,而增生性瘢痕中MCT基因量约为皮肤的2.13倍。结论本实验发现MCT基因在皮肤、增生性瘢痕和瘢痕疙瘩之间存在递增的梯度变比趋势及表达倍数关系。这与我们的预期结果相一致,也写以前研究发现的MCT在纤维化组织中的高表达结果相一致免疫荧光组化又发现MCT主要定位于瘢痕疙瘩的真皮浅层胶原纤维束之间,而Ghildyal N等发现MCT释放后主要作用于其周围局部组织,以前的研究发现MCT能够促进降解纤连蛋白等细胞外基质,还能够促进成纤维细胞的增生。因此我们认为,MCT可能通过在真皮浅层降解细胞外基质屏障和促进某些成纤维细胞的增生两种方式在瘢痕疙瘩的形成中起到了一定的作用。更多还原

【Abstract】 Objectiveto investigate the expression and distribution of mast cell tryptase（MCT） in keloids,and to find out the difference of MCT among keloids,hypertrophic scar and normal skins.IntroductionKeloids develop as a result of a proliferation of dermal tissue following skin injury. They differ from hypertrophic scars clinically and histologically.Clinically,keloids are a deep red or purple color with raised indurated tissue that extends beyond the original wound borders.Hypertrophic scars have a less impressive white or pink color, with firm tissue limited to original wound border.Histologically,keloids are composed of disorganized thick hyalinized collagen with a prominent mucoid matrix, whereas hypertrophic scars are characterized by fewer,more organized collagen fibers with a scanty mucoid matrix.Mast cell tryptase（MCT） is a tetrameric neutral serine protease with a molecular weight of 134 kDa and is the most abundant mediator stored in mast cell granules. The genes encoding mast cell tryptase are located on the short arm of chromosome 16. The enzyme is made up of four non-covalently bound subunits,and each subunit has one active enzyme site.MCT has ability to stimulate proliferation of dermal fibroblasts and to stimulate type al pro-collagen synthesis,which may be the great related to the formation of scars.In our research,we detected the expressing differences of MCT among the keloids,hypertrophic scars and skins by means of fluorescence immunohistochemistry and relative quantification real time pcr. Materials20 keloids from 17 patients aged from 15 to 32,20 hypertrophic scars from 18 patients aged from 10 to 45,and 20 healthy skins as control from 20 patients aged from 8 to 30.All the samples were supplied by Shandong Province Hospital Burn and Platic Department.The scars had developped for 6 months to 5 years and were classified clinically and histologically.All the samples undercovered no theropies before and were put into liquid nitrogen quickly after they were incised.MethodsFluorescence Immunohistochemistry After the frozen samples were sliced to 4-μm sections and fixed,50μl 1:100 Anti-Humin Tryptase mAb Biotin was incubated at 4℃for a whole night,the following day,50μl 1:100 Immunopure Goat Anti-Mouse IgG was incubated at 37℃for lh in darkroom.And then the slices were viewed with a Leica DMIREZ IM50 fluorescence microscope powered by a 100-W mercury lamp and equipped with a Hamamatsu ORCA-ER camera.RNA isolation About 50 mg sample was homogenized in 1 mL RNAiso Reagent and RNA was separated according to the manufacturer’s instruction.The amount of the isolated RNA were measured spectrophotometrically,and the purity was checked by the OD260/OD280 values.Electrophoresis was done to make sure the isolated RNA had not degradeted.RNA was dissolved in 20μL of RNase-free H₂O.Reverse transcription The reaction system was composed of 2μl 5×PtimeScript Buffer,0.5μl PtimeScript RT Enzyme Mix I,0.5μlOligodTPrimer（50μM）,0.5μl Random 6mers（100μM）,500ng Total RNA,and proper RNase Free dH₂O to make sure the total amount is 10μl.All the reagents were added on the ice.Then the system reacted on a Biometra T1 Rt-pcr Instrument（Biometra）with a thermocycle of 37℃15min,85℃5sec and cDNA was stored at -20℃until use.Semi-quantative realtime-pcrβ-Tryptase appears to be the main form of tryptase [10]and we had proliferated its gene TPSB2.Dilute the cDNA by 20 times,and A 20-μL reaction mixture including 10μlSYBR Premix Ex Taq（2×）,0.4μlROX Reference DyeⅡ,5μl primer（0.8μm）,4μl diluted cDNA and 0.6μl dH₂O was added to the wells of the MicroAmp Optical 96-Well Reaction Plate（Applied Biosystems,US）,shortly centrifuged（3,000 x g） and then reacted in7500 Real-Time PCR System（Applied Biosystems,US）.Products were controlled by SYBR Green I dissociation curves and 2.5%agarose gel electrophoresis to ensure only one single targetspecific product was amplified.ACTB was chosen as a reference house-keeping gene.7500 PCR system software was used to estimate scar mRNA expression as well as the expressing differences of each sample relative to controls and based on an efficiency corrected mathematical model and a pair-wise fixed reallocation randomization test.Statistical analysisExperiments were carried out in triplicates.Data analysis was performed using SPSS10.0 software.Unless otherwise indicated,significance was determined at p＜0.01.ResultsFluorescence Immunohistochemistry Strong fluorescent signals were detected in the collagen fiber bundles of the keloids,espicially in the upper dermis.（figure6） Whereas less or weaker signals were detected in hypertrophic scars and skins.RNA isolation The OD260/OD280 values of the isolated RNA ranged from 1.8 to 2.0,means the high purity of the isolated RNA.Electrophoresis showed two bands of 18s and 28s,and 28s had a stronger fluor intensity,which means the RNA had not degradeted.Real time-pcr The proliferative curve run regularly.Only one hump appeared in the dissociation curve and 2.5%agarose gel electrophoresis showed only one single band of 155bp.（figure8） Both the keloid and hypertrophic scars expressed more tryptase genes than the controls（p＜0.01）,and the keloids expressed more tryptase than the hypertrophic scars（p＜0.01）.Averagely,there were 5.4- and 2.13-fold increase of tryptase gene in keloids and hypertrophic scars compared to the skins,and 2.5-fold increase in keloids compared to the hypertrophic scars. DiscussionKeloids exhibit 20 times the rate of collagen synthesis of normal skin and three times the rate of hypertrophic scars.In our research,strong fluorescent signals of tryptase were detected between the collagen fiber bundles of the keloids,and rt-pcr showed there were 5- and 1.7-fold increase of tryptase gene in keloids and hypertrophic scars compared to the skins,and 3-fold increase in keloids compared to the hypertrophic scars,considering that tryptase can be mitogenic for fibroblasts from various sources and stimulate collagen synthesis,we consider tryptase as a promoting factor of formation of keloids by stimulating collagen synthesis.更多还原