Analysis of differences of gene expressions in keloid and normal skin with the aid of cDNA microarray

Background: Microarray analysis is a popular tool to investigate the function of genes that are responsible for the phenotype of the disease. Keloid is a intricate lesion which is probably modulated by interplay of many genes. We ventured to study the differences of gene expressions between keloids and normal skins with the aid of cDNA microarray in order to explore the molecular mechanism underlying keloid formation. Methods: The PCR products of 8400 human genes were spotted on a chip in array. The DNAs were then fixed on the glass plate by a series of treatments. Total RNAs was isolated from freshly excised human keloids and normal skin, and then was purified to mRNA by Oligotex. Both the mRNA from keloids and normal skin was reversely transcribed to cDNAs with the incorporations of fluorescent dUTP, for preparing the hybridization probes. The mixed probes were then hybridized to the cDNA microarray. After highly stringent washing, the cDNA microarray was scanned for the fluorescent signals to display the differences between two kinds of tissues. Results: Among 8400 human genes, there were 402 genes (4.79%) with different expression levels between the keloids and normal skins in all cases, 250were up-regulated (2.98%) and 152 down-regulated (1.81%). Analyses of collagen, fibronectin, proteoglycan,growth factors and apoptosis related molecule gene expression confirmed that our molecular data obtained by cDNA microarray were consistent with published biochemical and clinical observations of keloids. Conclusions: DNA microarray technology is an effective technique in screening for differences in gene expression between keloid and normal skin. Many genes are involved in the formation of keloids. Further analysis of the obtained genes will help understand the molecular mechanism of keloid formation.     

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.     

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。     

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。     

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目的 利用基因芯片技术分析原发性下肢深静脉瓣膜功能不全(PDVI)所致大隐静脉曲张的基因表达谱和差异基因。方法 对10例PDVI所致的曲张大隐静脉和10例正常对照组织进行mRNA抽提，分别给予荧光素Cy5和Cy3标记以制备cDNA探针，并与含有1220个人类全长互补DNA的基因芯片进行杂交和分析。结果 在PVDI所致的曲张大隐静脉组织中共检测到21个差异基因，其中9个为上调基因，12个为下调基因。差异表达基因包括参与信号传导、细胞周期以及细胞生长、分化、代谢、增殖和凋亡的相关基因。结论 运用基因芯片技术筛选出的差异基因与PVDI所致大隐静脉曲张发病相关。     

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。     

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。     

TGF-βRⅠ、Smad2、Smad 3及Smad7在瘢痕疙瘩中的表达

目的 观察比较瘢痕疙瘩、正常瘢痕及正常皮肤中转化生长因子β1型受体（TGFβRⅠ）、Smad2、3、7的表达情况，探讨以上信号传导分子在瘢痕疙瘩形成过程中的作用。方法 运用免疫组化技术检测上述4种蛋白在3种不同组织44例标本中的表达，寻找其规律。结果 与正常瘢痕及正常皮肤相比，瘢痕疙瘩中TGF-βRⅠ表达增强，Smad7表达减弱（P〈0．05），Smad2及Smad3表达增强不显著，但在核内积聚较为明显。结论 瘢痕疙瘩中TGF-βRⅠ表达增强，Smad2、3核内持久积聚及抑制性因子Smad7表达减少，可能是瘢痕疙瘩形成的重要原因。     

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。     

增生性瘢痕和瘢痕疙瘩组织中TGF—β1及Ⅰ,Ⅲ型胶原基因 …

目的 检测增生性瘢痕（Ｈ）和瘢痕疙瘩（Ｋ）组织中ＴＧＦ－β１及Ⅰ、Ⅲ型前胶原ｍＲＮＡ的表达,了解其相互关系及意义。方法 斑点杂交分析检测Ｈ和Ｋ组织中Ⅰ、Ⅲ前胶原及ＴＧＦ－β１ｍＲＢＡ稳态水平的改变;原位杂交检测ＴＧＦ－β１ｍＲＮＡ在组织中的空间分布。结果 ①Ｋ和Ｈ组织中ＴＧＦ－β１ｍＲＮＡ稳态水平明显高于Ｎ组和Ｓ组;②Ｋ选择性Ｉ型前胶原ｍＲＮＡ表达增强,而Ｈ组织中Ⅰ、Ⅲ前胶原ｍＲＮＡ表达均增强。     

细胞色素氧化酶10在非梗阻性无精子症患者睾丸组织中的表达

目的:评价细胞色素氧化酶10(COX10)基因在非梗阻性人无精子症及正常睾丸组织中的表达及意义。方法:应用包含有人COX10及RBM、EIF1AY等基因的微矩阵芯片对人正常睾丸及无精子症睾丸组织中差异表达基因谱进行了研究:通过RT-PCR方法获得两种组织mRNA,再分别用Cy5-dUTP及Cy3-dUTP标记制备cDNA探针。两种探针混合后与包含有人4096条cDNA序列的cDNA微矩阵芯片杂交,经扫描、计算机处理分析比较杂交结果;随后利用原位杂交技术对COX10mRNA在10例正常生育及39例非梗阻性无精子症睾丸组织中的表达进行了研究。结果:获得128个可能与无精子症相关的差异表达基因,其中56个基因表达上调,72个基因表达下调,其中COX10下调明显。与cDNA微矩阵杂交结果相同,原位杂交证实COX10mRNA在正常睾丸组织生精细胞中表达强于非梗阻性无精子症睾丸组织。结论:COX10可能在无精子症的发生与进展过程中起一定的作用;cDNA微矩阵技术可以应用于筛选非梗阻性无精子症相关基因的研究中。     

缺血预处理对大鼠移植小肠基因表达谱的影响

目的:研究缺血预处理(IPC)后大鼠移植小肠基因表达谱的变化，探讨IPC保护移植物的机制。方法:大鼠随机分为假手术组（S组）、小肠移植组（SBT组）和缺血预处理后小肠移植组（ISBT组）。移植肠冷保存/再灌注1 h后，抽提各组小肠总RNA并纯化mRNA;逆转录合成cDNA荧光探针，与cDNA芯片杂交。洗片后扫描芯片荧光信号图像，将SBT组和S组杂交结果与ISBT组和S组杂交结果行生物信息学分析。结果:在4 096条基因中，正常小肠与ISBT组差异表达基因共有297条，已报道有GeneBank（基因库）登录号的基因84条。其中表达下调的基因共18条，表达上调基因共66条，这些差异表达基因与IPC保护效应有关。结论:IPC通过调节细胞黏附相关基因、细胞能量和物质代谢相关基因及细胞信号转导相关基因的表达，发挥移植物细胞保护效应。     

大鼠移植小肠冷保存/再灌注后基因表达谱的变化

目的　研究冷保存 /再灌注后大鼠移植小肠基因表达谱的变化 ,以探讨移植物损伤的机制。方法　建立改良式大鼠异位节段小肠移植和正常对照模型。抽提对照组正常小肠和移植肠冷保存 /再灌注 1h后总RNA并纯化mRNA将两组等量抽提纯化的小肠mRNA逆转录合成cDNA荧光探针 ,与cDNA芯片杂交。严格洗片后扫描芯片荧光信号图像 ,分析比较两组中差异表达基因。结果　在 40 96条基因中 ,两组间存在差异表达的基因共 82条 ,新发现基因 18条 ,表达序列标识 (EST ) 3 3条 ,已报道基因 3 1条。该 3 1条差异表达基因与移植物冷保存 /再灌注损伤存在相关性。结论　移植小肠冷保存 /再灌注损伤与中性多形核白细胞和微血管内皮细胞异常黏附、移植物细胞能量和葡萄糖、蛋白质代谢障碍等因素有关     

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.     

Expression of transforming growth factor beta 1, 2, and 3 proteins in keloids.

Keloids represent a pathological response to cutaneous injury, creating disfiguring scars with no known satisfactory treatment. They are characterized by an excessive accumulation of extracellular matrix, especially collagen. Transforming growth factor beta (TGF-beta) has been implicated in the pathogenesis of keloids. The three TGF-beta isoforms identified in mammals (TGF-beta1, -beta2, and -beta3), are thought to have different biological activities in wound healing. TGF-beta1 and TGF-beta2 are believed to promote fibrosis and scar formation, whereas TGF-beta3 has been shown to be either scar inducing or reducing, depending on the study. The aim of this study was to characterize expression of TGF-beta isoforms in keloids at the protein level using Western blot analysis. The authors found that TGF-beta1 and -beta2 proteins were at higher levels in keloid fibroblast cultures compared with normal human dermal fibroblast cultures. In contrast, the expression of TGF-beta3 protein was comparable in both the normal (N = 3) and keloid (N = 3) cell lines. These findings, demonstrating increased TGF-beta1 and -beta2 protein expression in keloids relative to normal human dermal fibroblasts further support the roles of TGF-beta1 and -beta2 as fibrosis-inducing cytokines.     

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。