核仁素对热变性成纤维细胞自噬的影响及其机制研究

项目名称： 核仁素对热变性成纤维细胞自噬的影响及其机制研究

项目编号： No.81471868

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： 梁鹏飞

作者单位： 中南大学

项目金额： 72万元

中文摘要： 变性真皮的转归机制仍是烧伤创面修复研究的热点。核仁素具有多生物学功能，申请者在前一个课题中发现核仁素在皮肤成纤维细胞热损伤转归中起着显著促修复作用，但其促修复机制尚不得而知。最近申请者发现热变性皮肤成纤维细胞转归中伴有自噬增多，与核仁素的表达存在一定相关性，而核仁素低表达可抑制自噬的发生，提示核仁素可能是重要的自噬调控分子，其可能通过调控自噬发生从而发挥促修复作用。本项目拟在上述研究的基础上，从整体及细胞水平探讨热变性皮肤成纤维细胞修复中自噬发生情况及其生物学意义；采用基因转染和RNA干扰技术探讨核仁素对皮肤成纤维细胞自噬的影响；采用RNA凝胶滞留实验、紫外交联、蛋白质-RNA免疫共沉淀、mRNA稳定性、报告基因分析及基因突变等方法深入探讨核仁素对自噬相关基因的调控机制。开展本项目有助于阐释核仁素对皮肤成纤维细胞热损伤转归中自噬的影响及其机制，为深度烧伤创面的治疗提供新的思路和实验线索。

中文关键词： 细胞自噬；核仁素；成纤维细胞；热变性；mRNA稳定性

英文摘要： Nucleolin is an abundant, multifunctional protein that exhibits an unusual range of activities, including DNA binding and RNA binding. Our previous studies found nucleolin enhanced the recovery of heat-denatured human dermal fibroblasts via increasing cell proliferation and migration. However, whether nucleolin enhances the autophagy during the recovery of heat-denatured human dermal fibroblasts or not is still unclear. Recently,our studies showed that autophagy increases during the recovery of heat-denatured dermis and fibroblasts and nucleolin ablation decreased the autophagy significantly. These results suggest nucleolin probably plays a vital role in autophagy. However, little is known about the molecular mechanism by which nucleolin regulates autophagy. Since nucleolin contains four RNA binding domains and can selectively bind to (T/G)CCCG(A/G),as nucleolin recognition element (NRE), we hypothesized that nucleolin might regulate the expression of autophagy-related genes through post-transcriptional mechanisms, i.e., stabilization of mRNA. We analyzed the mRNA sequence of autophagy-related genes by bioinformatics and found the mRNA molecules of Bnip3、ATG5、ATG3、Beclin 1、FOXO1、STMN1、RAB5A genes containing a NRE and some of genes expression were regulated by nucleolin. In this project, we will study the change and role of autophagy during the recovery of heat-denatured dermis and fibroblasts;the role of nucleolin in autophagy by using full-length nucleolin cDNA and nucleolin RNA interfere and approach the molecular mechanism by which nucleolin regulates cellular autophagy by using Bioinformatics, RNA Transcription, RNA Gel Mobility Shift Assays, UV Cross-linking, Coimmunoprecipitation of protein and RNA, Measurement of mRNA Stability, and luciferase reporter assay system. Carrying out this project can help us to explain the effect of nucleolin on autophagy and its molecular mechanism during the recovery of heat-denatured fibroblasts and to provide new ideas and experimental clues for the recovery of heat-denatured dermis.

英文关键词： autophagy;Nucleolin;fibroblasts;heat-denatured;mRNA stability