项目名称: NCSTN基因-Notch信号通路在反常性痤疮发病过程中的作用及其分子机制
项目编号: No.81472872
项目类型: 面上项目
立项/批准年度: 2015
项目学科: 医药、卫生
项目作者: 李诚让
作者单位: 中国医学科学院
项目金额: 70万元
中文摘要: 反常性痤疮(AI)是一种慢性、炎症性皮肤病,γ-分泌酶是其致病基因,后者由早老素、早老素增强子-2、NCSTN和Aph-1四个亚基组成。目前,在报道的23个基因突变中,19个出现在NCSTN亚基上,可见NCSTN的改变与AI发病密切相关。本课题以培养的角质形成细胞HaCaT为基础,采用RNA干扰的方法,建立NCSTN基因低表达的角质形成细胞模型,开展如下研究:(1)通过MTT、流式细胞仪和TUNEL法检测细胞生长和凋亡;(2)利用Notch信号通路PCR芯片检测细胞中Notch信号通路重要分子的改变;(3)生物信息学筛选影响细胞增殖、角化和炎症有关的分子;(4)检测AI患者的基因突变,选取具有NCSTN基因突变患者,免疫组化验或原位杂交验证候选靶基因,激光共聚焦显微镜观察上述分子在细胞中的不同分布;(5)干预候选靶基因表达,研究AI毛发角栓和炎症的发生机制。
中文关键词: 反常性痤疮;致病基因;发病机制;RNA干扰;HaCaT细胞
英文摘要: Acne inversa (AI) is a chronic and inflammatory skin disease. The causative gene is γ-secretase, which has four subunits including is PSEN1, PSENEN, NCSTN and Aph-1. Up to now, 23 mutations of γ-secretase were reported; of which 19 appear on NCSTN subunit, indicating closely relation of NCSTN with AI disease. We cultured keratinocytes HaCaT cell, created NCSTN gene low expression cell model using RNA interference method, and carried out research as follows: (1) To assay for cell growth and apoptosis by MTT, flow cytometry and TUNEL; (2) To detect changes in the cells Notch signaling pathway using Notch Signaling Pathway PCR Array; (3) To screening molecules associated cell proliferation, keratinocytes and inflammatory by bioinformatics;(4) To detect the AI patients' mutations, and selecte patients with NCSTN mutation to validate candidate target genes by immunohistochemistry or in situ hybridization, and observe the distribution in the cell by laser scanning confocal microscopy; (5) To study AI hair angle plug and inflammation mechanisms by intervention candidate target gene expression.
英文关键词: Acne inversa;Disease genes;Pathogenesis;RNA interference;HaCaT cell