线粒体融合基因2在原发性高血压患者血管平滑肌细胞中低表达的分子遗传机制研究

The mobidity of essential hypertension (EH) is estimated about 20% in Chinese, which is a heavy burden for patients and the government. Millions of people died of EH and its complications every year. The basic pathophysiology character of EH is hyperplasia of smooth vascular muscle cells (VSMCs). Mitofusion 2 (Mfn2) is a novel gene with the function of negatively regulating the hyperplasia of VSMCs in EH patients.It was reported that this gene was down-regulated in VSMCs of EH. However, the potential mechanism is remaining undiscovered. In the previous study, we found some new single nucleotide polymorphsims in EH patients, particularly, a new variation in the 5'-uncoding region of Mfn2, which was the binding site of activator protein-1 (AP-1). Our preliminary study confirmed that the transcript activity of the variated promoter down-regulated about 35%. In the present study,firstly, combined with SNP database, we will analyse the 5'-uncoding region of Mfn2 gene by bioinformatics methods, by combing with QuikChange Multi Site-Directed Mutagenesis System,design the primers and clone the variated promotor. After the transcript activity of the promoter were tested by Dual luciferase reporter gene system, promoter will be screened for a key one.Then, transgenic mouse analysis is used for indentifiction of the function of the key promoter. Secondly, CHiP and EMSA will be used to identify the transcript factors and their binding sites, in vivo and vitro. Then,RNA interference and over expressed of transcription factors will be used to tested the function of transcription factors by the measurement of Mfn2 expression in VSMCs. At last, bioinformatics will be performed to study the structure-activity relationship between promotor structure and its function. Since this study may uncover the mechanism of Mfn2 down regulation in VSMCs, it will be valuable in the prevention,morbidity and motality of EH, as well as in the development of antihyperplasia drugs. Therefore, it has a prospective future on the aspects of basic study and clinical practice as well.

原发性高血压(EH)的发病率极高(占人群的20%)，危害巨大。线粒体融合基因2(Mfn2)是一个新发现的负调控血管平滑肌细胞（VSMCs）增殖的基因，在EH患者VSMCs中表达极低,但其分子机制还不明确。我们前期发现一些新的Mfn2启动子元件变异,可导致转录活性下降35%。本课题拟以此为切入点，对Mfn2的转录调控进行深入研究。首先综合前期研究结果及dbSNPs资源对Mfn2启动子进行结构和功能分析，结合定点突变技术，体外克隆启动子。通过双荧光素酶报告基因系统检测各启动子的转录活性；应用转基因鼠验证其功能，筛选出关键启动子及其变异。然后应用CHiP和EMSA对关键启动子及与其互作的转录因子进行研究。再利用RNA干扰和过表达技术验证转录因子的功能。最后，对关键启动子的构效机制进行研究。这对于阐明EH患者VSMCs中Mfn2的低表达分子遗传机制，降低该病的致残致死率及药物开发，意义深远。

本课题针对原发性高血压（EH）的发病率极高，结合线粒体融合基因 2（Mfn2）是一个新发现负调控血管平滑肌细胞（VSMCs）增殖的基因，在 EH 患者 VSMCs 中表达极低的特点，我们从分子遗传学角度对 Mfn2 的转录调控机制进行深入研究。首先利用dbSNPs 资源对 Mfn2 基因的启动子进行结构和功能的生物信息学研究，结果发现了5个启动子区域。其中2000bp以内存在着3个启动子和39种转录因子。然后按照JNC VII标准筛选原发性高血压患者和正常对照组共800例，对Mfn2上游2000bp的调控区进行DNA提取、引物设计和和PCR扩增，然后进行DNA测序，最后对测序结果进行序列分析。通过与GENE bank的标准序列和正常对照的序列进行比对分析，我们发现了5个新的单核苷酸多态性点，且这5个位点与原发性高血压的发病率存在着明显的相关。为了进一步筛选 Mfn2 基因启动子区内可能影响该基因转录的 SNPs。我们通过双荧光素酶报告基因系统检测了各启动子的转录活性，筛选出了核心启动子，大概在538bp以内。最后综合前期研究和本研究的病例-对照研究结果以及 NCBI dbSNP 数据库资料，现已基本明确原发性高血压患者Mfn2 基因上游2000bp以内的启动子区有5个转录因子结合元件的变异，这些位点的变异可能会影响了转录因子与DNA元件的结合，从而影响了转录因子的功能。