【作者】 唐雪勇；

【导师】 杨志波；

【作者基本信息】 湖南中医药大学 ， 中医外科学， 2012， 博士

【摘要】 背景：银屑病是临床最常见的慢性炎症性皮肤病之一,其发病为多因素参与、多基因改变及多阶段发展的病变过程,随着社会的发展与环境的改变,本病发病率逐年增高,因其发病原因不明,机制不清,治疗较为棘手,易于反复发生。银屑病发病机制的研究一直是皮肤科研究的重点,近年来,随着国内外学术界在小分子RNA领域研究的逐步深入,发现多种miRNA在皮肤生理病理等过程中发挥了极其重要的作用,部分特定的miRNA在某些皮肤病皮损组织中有异常表达,如皮肤肿瘤、银屑病、特应性皮炎等。同时从基因层面探讨银屑病的早期诊断、治疗及预防等课题已成为业界今后研究的主要方向之一。目的：运用基因芯片技术检测银屑病皮损miRNA表达谱,筛选其与正常人皮肤组织差异性表达的miRNA,并对差异miRNA进行靶基因预测及靶基因功能富集分析,构建银屑病差异表达miRNA与靶基因及基因功能调控网络,为银屑病基因层面机制研究提供依据,为银屑病早期诊治提供思路,为下一步开展miRNA靶基因功能及分子标志物研究提供线索。同时研究中药竹黄颗粒剂治疗银屑病患者前后皮损miRNA及血清细胞因子的表达差异,探索中医药治疗银屑病的作用机制,为中医药防治银屑病提供客观依据。方法：招募20名银屑病患者和15名健康志愿者,采集皮肤组织标本并用Trizo法提取RNA,运用Affymetrix miRNA2.0芯片检测正常人皮肤组织与银屑病患者典型皮损组织中miRNA表达谱,运用聚类分析软件(cluster3.0)分析差异表达的miRNA,采用实时荧光定量PCR方法验证部分差异miRNA。通过miRNA靶标基因数据库(miRGen3.0)预测靶基因,基于Gene Ontology数据库,对靶基因进行功能富集分析。20名银屑病志愿者进行2个月竹黄颗粒剂治疗后再次采集原发皮损样本,进行miRNA芯片检测,分析治疗前后miRNA表达差异及意义,同时采用酶联免疫吸附法(ELISA)检测治疗前后血清细胞因子并比较其差异性。结果：银屑病皮损组织中明显上调的miRNA有55个,明显下调的miRNA有12个,5个特异性miRNA (miR-31、miR-155、miR-192、miR-139-5p.miR-497)靶基因预测,得出AIRE、ADRA1D、AREG、IKBKE、POU6F1、BUB1B、 SMC3、SOCS1、DGKH、MAP3K12、OTC、REV3L、SLC26A6等33个关键基因。功能分析得出其调控了促分裂素原活化蛋白激酶信号通路(MAPK signaling pathway)、Wnt信号通路(Wnt signaling pathway)等30项主要的信号通路,主要通过调节炎症发生、细胞凋亡、血管内皮细胞增殖、新血管生成、体液免疫、蛋白激酶C(protein kinase C, PKC)、JNK信号转导与激活、有丝分裂细胞周期等,从而参与银屑病的发病。同时采用竹黄颗粒剂治疗2月后,总有效率为80%,PASI评分较治疗前有显著下降,显示对患者血清中IL-1、IL-2、IL-8、IL-10、IL-18、IFN-γ、TNF-α等细胞因子有明显下调作用,使之趋近于正常对照组。芯片结果显示对miR-192、miR-194、miR-3175、miR-18a、miR-629、miR-21、miR-25、miR-100、miR-199a-5p、miR-99a、miR-409-3p miR-125b、miR-99b等差异miRNA有显著双向调节作用。结论：1.miR-31等67个差异性miRNAs参与了银屑病的发病并扮演着重要角色；2.差异性miRNA所调控的基因可能通过影响MAPKs等信号通路从而参与银屑病的发病,在细胞增殖、炎症形成、血管增生等方面构成影响；3.miR-31等特异性miRNA有望成为银屑病早期诊断的潜在标记物和治疗靶点；4.中药竹黄颗粒剂治疗银屑病疗效显著,对有些差异miRNA有双向调节作用,为中医药防治银屑病提供了客观依据；5.研究结果为下一步开展银屑病相关miRNA靶基因功能及分子标志物研究提供了线索。更多还原

【Abstract】 Background:Psoriasis is one of the most common clinical chronic inflammation skin diseases. Many factors influence its incidence process, involving polygenetic change, complicated progress of pathological changes. With the development of the society and the environment changes, the rate of the disease increased year by year, due to its unclear etiology and mechanism, its treatment is difficult and easy to repeat. Therefore, research for psoriasis mechanism has caused the wild attention in the field of dermatology. In recent years, with the exploring to psoriasis progressing from domestic and foreign academic circles, it has been found that many minimal RNA (miRNA) play an important role in its pathogenesis and some of them were found abnormal expression in some skin lesions, such as skin cancer, psoriasis, atopic dermatitis. At the same time, from the perspectives of gene, studying on the psoriasis diagnosis, treatment, and prevention early has become one of the main research fields in the future all over the world.Purpose:Using gene chip technology testing psoriasis lesions miRNA expression patterns, the different miRNA expression from both abnormal and normal skin tissue are screened, and the differences in miRNA target genes prediction and target genes function enrichment are analysed, so as to construct a gene function regulation web for psoriasis differentially expressed miRNA and target genes and, providing the basis for the research of diagnosis and treatment of the psoriasis at the gene level, giving the clues for the next step in miRNA target genes function and molecular markers study. At the same time, the studies on influence of the traditional Chinese medicine ZhuHuang granules on the differences in psoriasis lesions and serum miRNA patients have been carried out before and after the treatment, so that the mechanism of Chinese medicine on the psoriasis treatment was explored, and more objective basis were provided for the Chinese medicine preventing and treating psoriasisMethods:20patients with psoriasis and15healthy volunteers were recruited, the skin and tissue samples from Trizo law to extract RNA were collected. Using Affymetrix miRNA2.0chip test the miRNA expression profile of normal skin organization and patients with psoriasis lesions in the typical organization. With clustering analysis software (cluster3.0) to analyze the differences of expression miRNA, and by use real time fluorescence quantitative polymerase chain reaction (PCR) to verify part miRNA differences. Through the miRNA target genes database (miRGen3.0) forecast target genes, Gene Ontology based on database of target genes function enrichment was analysed.20psoriasis volunteers lesions samples were collected again2months after the treatment of ZhuHuang granules for miRNA chip testing. Analyse the differences and significance on the miRNA expression before and after treatment. At the same time, using the method of ELISA to test differences on the serum cells factor before and after treatment.Results:There are55miRNAs obviously up-regulated in psoriasis lesions and12miRNAs of them obviously down-regulated. The result of target genes forecast for5specific miRNA (miR-31, miR-155, miR-192, miR-139-5p, miR-497) draw about33key genes,such as AIRE, ADRA1D, AREG, POU6F1, BUB1B IKBKE, SMC3, SOCS1, DGKH, MAP3K12, OTC, REV3L, SLC26A6etc. From the function analysis, its control the original mitogen activated protein kinase signal pathway (MAPK signaling pathway), Wnt signal pathway (Wnt signaling pathway)30other major signaling pathways, mainly by adjusting the inflammation occurs, the cell apoptosis, endothelial cell proliferation and angiogenesis, new humoral immune, protein kinase C (protein kinase C, PKC), JNK signal transduction and activation, mitotic cells cycle, so as to participate in the pathogenesis of psoriasis. At the same time the ZhuHuang granules miRNA expression of the intervention, and the results show the miR-192, miR-194, miR-3175, miR-18a, miR-629, miR-21, miR-25, miR-100, miR-199a-5p, miR-99a, miR-409-3p miR-125-b, miR-99-b miRNA have significant differences such as two-way of regulating functions.Conclusion:1.67differenct miRNAs such as miR-31involved in the incidence of psoriasis and played a very important role;2. The regulation of genes miRNA differences may be affected by the MAPK signals of pathways that participate in the pathogenesis of psoriasis and prognosis, in cell proliferation, inflammation formation, the growth of blood vessels to constitute the influence, etc;3. Five specific miRNAs such as miR-31is expected to become potential markers of psoriasis early diagnosis and therapeutic targets;4. The effect of treating psoriasis with Chinese medicine Zhu-Huang granules is distinct. The granule have the two-way adjustment function to different miRNAs such as miR-192. The research provide the objective basis for treatment of psoriasis with traditional Chinese medicine;5. The results provide clues for the next step for psoriasis miRNA target genes relating to function and molecular markers.更多还原