小发夹RNA对板层状鱼鳞病TGM1突变基因表达的调控研究

项目名称： 小发夹RNA对板层状鱼鳞病TGM1突变基因表达的调控研究

项目编号： No.81201223

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 医学四处

项目作者： 何玉清

作者单位： 广东医学院

项目金额： 23万元

中文摘要： 转谷氨酰胺酶 1（TGM1）基因突变是板层状鱼鳞病（LI）的主要致病基因。研究表明小发夹RNA（shRNA）介导的RNAi能高特异性持久地沉默特定基因，降低突变。本课题拟构建针对TGM1突变基因的shRNA慢病毒载体，转入裸鼠表皮内后建立表达TGM1突变基因的动物模型。在此基础上设计荧光标记探针，注入裸鼠表皮内后与TGM1基因进行体内荧光杂交。通过探测裸鼠活体细胞内荧光信号强度来判断TGM1突变基因mRNA的表达情况；并用酶敏感可激发探针检测裸鼠体内微量的TGM1活性，探讨shRNA对TGM1突变基因mRNA表达的调控作用，期望使突变的等位基因恢复互补状态、功能性多肽表达并发挥疗效，表皮屏障功能得以重建。本研究首次提出用shRNA介导的RNAi 高特异性地沉默板层状鱼鳞病TGM1 突变基因，研究结果将为鱼鳞病的基因治疗建立经济有效的途径，并对其他遗传性皮肤病的基因治疗提供科学的理论基础。

中文关键词： 板层状鱼鳞病；转谷氨酰胺酶 1；基因突变；表达调控；

英文摘要： Ichthyosis. Studies indecated that RNA interference technology based on short hairpin RNA(shRNA) has a very strong ability to make the target gene keeping silence for a long time and decreaseing the gene mutation. In this project we designed a virus expression carrier, witch can interfere with TGM 1gene mutation. We can get the mouse model with TGM1 mutant gene after injection the virus expression carriers into the mouse's epidermal cells. Based on the above studies, we then designed a fluorenscence labeled probe which can hybridised with TGM 1 mRNA in epidermal cells of TGM1 mutant gene mouse model. Through this method we can clearly observed the expresstion of TGM1 mutant gene in alive mouse model, and we can estimate the shRNA intere effect at anytime only by read fluorenscence signal. Further more, we designed a enzyme sensitivity and excitated probe to detect the activity fo TGM1 in alive mouse model, advanced to investigate the regulation funtion of shRNA interference. The results may show a promise, potencial and very useful approach of gene therapy in lamellar Ichthyosis, and provide a basic knowledge for treatment other inherited skin disorders.

英文关键词： Lamellar Ichthyosis；Transglutaminase 1；gene mutation；expression regulation；