003 Gene Expression Profiles Following Electron Irradiation of Cultured Keloid and Normal Skin Fibroblasts by cDNA Microarray Analysis

Aim: When surgery with postoperative superficial electron irradiation is applied, the recurrence rate of keloid lesion has been found to be <30%. In this study, we assessed the molecular changes underlying the effect of electron irradiation by differential global gene expression analyses of both cultured keloid and normal skin fibroblasts. Materials and Methods: Primary cultured fibroblasts from 4 active keloids and their adjacent normal dermal tissues were irradiated at a calibrated dosage of 15 Gy with 6 MeV electron beam generated by a linear accelerator. Corresponding paired non‐irradiated cells were used as control. RNA isolated from the collected cells was labeled with ³³P, hybridized to the cDNA microarray gene filters and analyzed. Results: After irradiation, the gene expression profiles of keloid and normal skin fibroblasts were closely similar. Electron irradiated keloid fibroblasts showed suppressed levels of collagen typeI (alpha2), collagen typeVI (alpha1), matrix metalloproteinase 2, fibronectin 1, insulin‐like growth factor binding protein 3, alpha‐1‐antichymotorypsin and heparan glucosaminyl 1 as compared with their non–irradiated counterparts. Conclusions: Downregulation of matrix synthesis and upregulation of protease inhibitors and apoptosis promoting genes by electron irradiation may inhibit keloid development. This mode of therapy appears to exert a positive effect toward lowering the recurrence rate of keloid formation.     

Gene expression patterns in isolated keloid fibroblasts

Keloid scars after skin trauma are a significant clinical problem, especially in black populations, in which the incidence of keloids has been estimated at 4-16%. Keloids are abnormal dermal proliferative scars secondary to dysregulated wound healing. Despite several biochemical studies on the role of extracellular matrix proteins and growth factors during keloid formation, we still do not know what molecules and signals induce this change. Fibroblasts are thought to be the major inductive cell for keloid scar formation. The aim of this study was to identify gene expression patterns that characterize keloid fibroblasts; identifying such genetic disequilibrium may shed light on the molecular signaling events responsible for keloid formation. In this study, we performed gene expression analysis of fibroblasts isolated from keloid lesions from three individuals in comparison with the fibroblasts isolated from normal skin using the Affymetrix U133a chip (22,284 genes and expression sequence tags). We found through J5 test score expression analysis that among 22,284 genes, there were 43 genes that were overexpressed and five genes were underexpressed in keloid fibroblasts when compared with dermal fibroblasts from persons without keloids. The overexpression of three genes not previously reported as being up-regulated in keloids (annexin A2, Transgelin, and RPS18) was confirmed by real-time polymerase chain reaction. Certain overexpressed genes were similar to previous biochemical observations on the protein levels of these overexpressed genes during keloid formation. We also report for the first time that a few tumor-related genes are overexpressed in keloid fibroblasts.     

瘢痕疙瘩成纤维细胞的基因组学研究

目的 寻找瘢痕疙瘩致病相关基因，探讨瘢痕疙瘩的发生机理。方法 利用含1100个人类肿瘤相关基因的cDNA芯片(cDNA—microarray)对耳垂和胸部瘢痕疙瘩及正常皮肤成纤维细胞进行检测，初步分析瘢痕疙瘩成纤维细胞与正常皮肤成纤维细胞基因总体表达的差异，并筛选出差异基因。结果 在耳垂及胸部瘢痕疙瘩成纤维细胞中，分别有8种和17种特异性表达基因被检出。在正常皮肤中特异性表达的细胞增殖抑制基因Mda-7，在耳垂及胸部瘢痕疙瘩成纤维细胞中均未被表达。结论 多种基因参与了瘢痕疙瘩的形成过程，瘢痕疙瘩成纤维细胞与正常皮肤成纤维细胞之间存在基因表达的差异，增殖因子受体PAR-1和增殖抑制基因Mda-7可能参与瘢痕疙瘩的形成。     

Involvement of KGF in the Wound Healing Process of Tracheal Cartilage in Rat Longitudinal Tracheotomy Model

Aim:  To investigate the expression of human HtrA1 in keloid lesions, and to clarify a possible role of human HtrA1 in keloid pathogenesis.
Methods:  Total RNA was isolated from six keloids and two normal skins by single‐step method. The expression level of human HtrA1 was examined by using Northern blot analysis. Keloid and normal skin tissue samples were fixed in paraformaldehyde, and paraffin sections were obtained. Immunohistochemical analysis was performed with anti‐human HtrA1 polyclonal antibody.
Results:  The mRNA level of human HtrA1 was markedly elevated in keloid samples, compared with normal skin. Using immunohistochemical analysis, fibroblast‐like cells abundantly found in the margin of keloid lesions, were stained with anti‐human HtrA1 antibody. No human HtrA1 staining of fibroblasts in normal skin was observed. Interestingly, no significant staining was detected in hypertrophyic scar lesions, which is a similar dermal disease to keloid.
Conclusion:  Human HtrA1 expression was found to be up‐regulated in keloid lesions, especially in their margin, compared to normal skins and hypertrophic scars. Our data suggest that human HtrA1 could play a critical role in an expression of keloid specific phenotype and in the development of keloid lesions.     

瘢痕疙瘩组织p53基因突变的实验研究

目的：分析瘢痕疙瘩中p53基因第4—8外显子的突变及其意义。方法：取手术切除的瘢痕疙瘩和正常瘢痕各12例，并分别取同一患者正常皮肤对照，采用聚合酶链反应-单链构象多态性分析方法(PCR-SSCP)和基因测序，检测各组织p53基因的突变情况。结果：12例瘢痕疙瘩标本中有9例p53基因外显子4、5、6、7出现点突变和移码突变，正常瘢痕标本、正常皮肤标本均未检出突变。结论：p53基因突变是瘢痕疙瘩形成和发展的重要因素之一。     

增殖瘢痕成纤维细胞接触后增殖及生物合成特性对异常瘢痕形成的机理

目的 为明确不同异常瘢痕成纤维细胞在体外完全接触后其增殖活性及生物全成功能的特性。方法 以瘢痕疙瘩、增生性瘢痕和正常皮肤（各6例）为材料，通过细胞培养、免疫组织化学及分子生物学等方法，对不同成纤维细胞在细胞接触及未接触时通过检测增殖细胞核内抗原、P16、Ⅰ、Ⅲ型胶原蛋白及前胶原基因表达对成纤维细胞的增殖、抑制及生物合成进行了研究。结果 瘢痕疙瘩成纤维细胞接触表现为细胞交叉重叠及较高的增殖活性及旺盛的生物合成功能，提示其失去了接触性抑制及密度抑制。皮肤成纤维细胞接触后则增殖及生物合成功能明显下降。增生性瘢痕成纤维细胞接触后表现为旺盛的生物合成功能，但其增殖活性处于瘢痕疙瘩和正常皮肤成纤维细胞之间。结论 不同瘢痕成纤维细胞接触后增殖及生物合成的特性可能是形成不同瘢痕的机理之一。     

表达谱芯片筛选粥样硬化性腹主动脉瘤的差异表达基因

目的运用基因芯片研究粥样硬化性腹主动脉瘤中基因表达谱的变化,筛选差异表达基因。方法选取粥样硬化性腹主动脉瘤(AAA)标本5例,以5例正常腹主动脉作对照。抽提总RNA,纯化mRNA后逆转录制备杂交探针,采用含有4096种人类基因全长cDNA的芯片进行差异表达谱分析。结果在粥样硬化性AAA的基因表达谱中差异表达基因共有186条,其中上调的基因102条,下调的基因84条;共存性基因有37条(上调基因26条,下调基因11条),其中有细胞凋亡相关基因、原癌基因和抑癌基因、细胞信号和传递蛋白等多种基因。反转录聚合酶链反应(RTPCR)及蛋白印迹验证了Caspase9及周期素依赖蛋白激酶(CDK)4在腹主动脉瘤中的差异表达。结论表达谱芯片筛选差异表达基因,为AAA发病机制的研究提供新思路;凋亡/增殖相关基因的表达失衡可能是粥样硬化性AAA的重要发病机制。     

Gene expression of early hypertrophic scar tissue screened by means of cDNA microarrays

BACKGROUND: Hypertrophic scar is an excessive healing response that often follows thermal injury. The most outstanding morphologic change is the overdeposition of collagen, which is caused by imbalance between synthesis and metabolism of collagen. Previous studies also found that transforming growth factor-beta was the key factor controlling scar formation. However, neither anti-transforming growth factor-beta nor other methods could completely control scar formation and contraction. This fact suggests a multifactorial cause. Fortunately, cDNA microarray throws light on the general alteration at the gene level, and thus could allow us to find some new clues for understanding scar formation and contraction. METHODS: In this article, we report the results obtained from the scanning of gene expression of hypertrophic scar by means of cDNA microarray. Five cases of early human postburn hypertrophic scars were selected. Total tissue RNA was extracted from each hypertrophic scar sample and the corresponding uninjured region skin tissue; mRNA was further purified by Oligotex and then was reversely transcribed to cDNAs with the incorporation of fluorescent dUTP to prepare the hybridization probes. The mixed probes were hybridized to the cDNA microarray containing 4,096 genes on a type of chemical material-coated glass slide. After high-stringent washing, the hybridized slides were scanned for fluorescent signal detection. Then, the expression and distribution of cytoskeletal genes such as alpha-smooth muscle actin (alpha-SMA) gene; fibroblast tropomyosin TM30(pl) gene; vimentin gene; profilin gene; and BM40 gene of hypertrophic scar at 3, 6, 9, and 12 months age were further quantitatively studied by in situ hybridization or immunohistochemistry. RESULTS: Our data indicated that there were 94 genes overexpressed and 3 genes down-regulated in early postburn hypertrophic scar. These altered genes were related to proto-oncogenes, apoptosis, immune regulatory genes, cytoskeletal elements, metabolism, and so forth. We also found that the detected cytoskeletal gene expression was much more intense at all time points than the control group. Consistent with clinical observation, cytoskeletal genes reached a peak at an early stage and gradually decreased. CONCLUSION: Our study implied that multiple genes are involved in scar formation and contraction. Interferon is an autosecreted cytokine that might be responsible for self-control of overgrowth of cells in wounds. The early period of hypertrophic scar formation might be a good time for preventing overgrowth and contraction of hypertrophic scar by gene therapy.     

人类乳腺癌基因表达分析

目的研究乳腺癌发生和发展相关基因群的表达及其初步功能。方法用4096种人类基因多聚酶链反应(PcR)产物制成BioDoor4096型表达谱芯片，分离纯化正常乳腺组织和乳腺癌组织mRNA，制备表达谱探针，用ScanArray3000荧光扫描仪扫描芯片荧光信号图像，利用计算机分析正常乳腺组织和乳腺癌组织之间差异表达的基因。结果在4096种基因中，正常乳腺组织和乳腺癌组织之间差异表达的基因有619条(15．11％)。生物信息学分析显示，这些差异表达的基因可能与乳腺癌的发生和发展密切相关。结论乳腺癌的发生、发展中存在多基因表达调控的改变，对于相关基因群的研究有助于认识肿瘤发病机制，并为乳腺癌个体化治疗提供依据。     

Tenascin-C在瘢痕疙瘩和增生性瘢痕中的基因表达研究

目的 探讨Tenascin-C基因在瘢痕疙瘩和增生性瘢痕中的表达。方法 取正常成人皮肤组织RNA，构建正义、反义Tenascin-C(Tn-C)mRNA探针，运用原位杂交技术，观测10例瘢痕疙瘩、10例增生性瘢痕和5例正常成人皮肤组织中Tn-C mRNA的表达。结果 Tn-C mRNA在正常皮肤表皮中无表达，真皮中表达稀少，局限于乳头真皮层的成纤维细胞和皮肤附属器；10例瘢痕疙瘩表皮均有表达，真皮分布较广，如成纤维细胞、血管内皮和皮肤附属器；Tn-C mRNA在3例增生性瘢痕表皮表达，7例无表达，真皮中表达与瘢痕疙瘩相同但较弱，比正常皮肤增多，但差异无显著性。结论 Tenascin-C mRNA在瘢痕疙瘩表皮和真皮中有高表达。     

基因芯片在筛选胆管癌相关基因差异性表达的应用研究

目的　应用cDNA芯片技术进行肝外胆管癌相关基因表达谱差异分析 ,筛选胆管癌相关基因。方法　按一步法分别抽提 6例胆管癌和正常人胆管黏膜总RNA、纯化 ,逆转录合成掺入荧光分子的cDNA链探针 ,与 10 68条人PCR微矩阵芯片杂交 ,扫描芯片荧光信号图像 ,计算机分析比较二种组织基因表达谱差异。结果　胆管癌与正常胆管黏膜的基因表达谱分析 ,发现有 194条基因表达差异 ,6组标本 47条与肿瘤相关的基因一致向上或向下表达 ,2 3条表达上调 ,2 4条表达下调。结论　基因芯片能快速筛选胆管癌相关基因 ,分析这些差异表达的基因能够阐明胆管癌复杂的生物学特性与基因表达之间的内在联系 ,并识别肿瘤的标记物。     

核心蛋白聚糖和光蛋白聚糖在体外培养的瘢痕疙瘩成纤维细胞中的表达

目的:研究体外培养的瘢痕疙瘩成纤维细胞中两种小分子蛋白聚糖(核心蛋白聚糖和光蛋白聚糖)的表达水平,探讨这两种蛋白聚糖与瘢痕疙瘩发生机制的关系。方法:采用实时PCR(Real-timePCR)和WesternBlot方法,研究体外培养的瘢痕疙瘩和正常皮肤成纤维细胞中核心蛋白聚糖和光蛋白聚糖的mRNA和蛋白表达水平,并进行比较分析。结果:不同患者来源的瘢痕疙瘩成纤维细胞中核心蛋白聚糖的mRNA表达水平差异较大,最高表达水平是最低表达水平的5.6倍。瘢痕疙瘩和正常皮肤的培养成纤维细胞中,核心蛋白聚糖和光蛋白聚糖在基因和蛋白表达水平上无显著性差异。结论:核心蛋白聚糖和光蛋白聚糖在体外培养的单层瘢痕疙瘩和正常皮肤成纤维细胞中的表达无特异性改变,它们在瘢痕疙瘩形成过程中所起的作用尚有待进一步研究。     

瘢痕疙瘩中Smads表达的研究

目的 探讨不同类型Smads在瘢痕疙瘩、正常瘢痕和正常皮肤中的差异表达及其意义.方法 采用RT-PCR和Western Blot法分别对10例瘢痕疙瘩、10例正常瘢痕及10例正常皮肤组织,以及体外培养瘢痕疙瘩、正常瘢痕及正常皮肤成纤维细胞中的Smads mRNA及蛋白的表达水平进行检测.用t检验比较其表达差异,P<0.05为差异具有统计学意义.结果 在瘢痕疙瘩组织及瘢痕疙瘩成纤维细胞中,Smad7的mRNA及蛋白水平表达明显低于正常瘢痕(P<0.05)和正常皮肤(P<0.05),而Smad2、3的mRNA及蛋白水平表达以及磷酸化的Smad2、3的蛋白水平表达并无明显改变(P>0.05).结论 在瘢痕疙瘩中,存在有Smad7的表达缺陷,这可能是增高的转化生长因子-β1(TGF-β1)/Smads信号传导不能被自身负反馈循环终止的重要原因.     

瘢痕疙瘩Fas基因外显子6,8,9的突变分析

目的：研究瘢痕疙瘩Fas基因中外显子6，8，9的突变情况，了解该基因突变与瘢痕疙瘩形成之间的关系。方法：实验分正常皮肤、增生性瘢痕和瘢痕疙瘩组织三组，采用PCR-SSCP(聚合酶链反应-单链构象多态性)方法筛选突变，然后进行序列分析以确定突变。结果：经银染SSCP分析，在23例瘢痕疙瘩标本中，出现异常电泳带的有18例，经测序，外显子6，8，9突变分别有8例、12例和7例，6和8同时存在突变的有2例，6和9同时存在突变的有3例，8和9同时存在突变的有2例，三个外显子同时有突变的有l例，而且发现了新的突变，在正常皮肤组织和增生性瘢痕组织中均未检出突变。结论：瘢痕疙瘩组织中Fas基因的突变可能与该病的发生有密切关系；PCR-SSCP是检测基因点突变的一种快速、敏感、有效的方法。     

利用抑制消减杂交筛选瘢痕疙瘩差异表达基因

目的 利用抑制消减杂交（suppression subtractive hybridization，SSH）技术，比较瘢痕疙瘩与正常皮肤组织之间基因表达差异，筛选瘢痕疙瘩特有的差异表达基因片段，为揭示瘢痕疙瘩的病因提供实验依据。方法提取瘢痕疙瘩与正常皮肤组织细胞mRNA，逆转录成cDNA。以瘢痕疙瘩cDNA为检测子，正常皮肤组织细胞cDNA为驱动子，选择四碱基内切酶RsaI将cDNA酶切；连接特殊设计的接头进行消减杂交和PCR反应，得到消减混合物，与T载体连接，转化DH5a感受态细菌，建立差异消减文库。Southern杂交筛选鉴定差异基因，进行测序和同源分析。结果经SSH筛选、Southern杂交鉴定得到13个瘢痕疙瘩差异表达基因，其中已知功能的基因11个，如TA结合因子1、E4基因转录因子1、ephrinB2型受体基因、血小板生长因子受体基因等；有与肿瘤发生有关的基因，如G抗原7基因等；未知功能基因2个。结论筛选得到的瘢痕疙瘩差异表达基因，对了解瘢痕疙瘩发生发展的病因学基础和指导临床治疗具有重要意义。