【作者】 周西瑾；

【导师】 吴秀山； 王跃群；

【作者基本信息】 湖南师范大学 ， 遗传学， 2008， 硕士

【摘要】 心血管疾病是危害人类健康的严重疾病。它是造成死亡的主要原因之一。为攻克这一顽症,首先必须从分子水平上系统地研究控制心脏发育的基因及其相互调控关系。本实验室在研究基因FoxP4时,通过酵母双杂交筛选获得了一个相互作用蛋白FBL5, FBL5属于F-box家族蛋白成员,这类蛋白质在泛素-蛋白酶体途径中起非常重要的作用,近来的研究资料表明泛素-蛋白酶体途径与心血管疾病有关。因此对FBL5基因的深入研究对于探讨心血管疾病的基因调控机制,以及心血管疾病的诊断、治疗具有重大的理论和实践意义。为了研究FBL5的表达与功能,从人类脑cDNA文库中克隆了FBL5基因的开放阅读框全长。亚细胞定位分析表明,FBL5定位于细胞质。荧光素酶报告基因分析表明,FBL5是个转录抑制子,并抑制SRF的调控活性。利用原核表达系统表达并获得GST-FBL5融合蛋白,免疫新西兰大白兔获得兔抗人FBL5蛋白的多克隆抗体。利用生物信息学从NCBI上搜索到FBL5的斑马鱼同源基因,克隆了其中特异性较强的一段,制备带有地高辛标记的探针,进行斑马鱼各个时期的整体胚胎原位杂交。实验结果显示FBL5在斑马鱼1细胞期到48小时的胚胎中均有表达,且在大脑、心脏和骨骼中表达强烈。利用morpholino oligo显微注射法,沉默FBL5在斑马鱼中的表达,结果表明在1-2细胞期注射,并培养到48小时,得到的胚胎畸形率最高,畸形特征表现为胚胎体轴弯曲,心脏发育畸形,心率减慢。将FBL5-MO与FoxP4-MO分别注射到斑马鱼中,用FBL5的探针进行整体原位杂交实验,发现注射了FBL5-MO与FoxP4-MO的胚胎,FBL5表达量都下降。初步研究表明,FBL5在心脏发育过程中起调控作用,与FoxP4共同调控心脏的发育过程。更多还原

【Abstract】 Cardiovascular diseases have become one of the most serious diseases that threaten human beings. It is a critical prerequisite to understand the normal progress of cardiovascular development and the mechanisms underlying the causes of this kind of diseases.In previous work, we performed yeast two-hybrid screen and obtained FBL5 as a binding partner of FoxP4. FBL5 belongs to F-box protein family.This protein family constitute one of the four subunits of ubiquitin protein ligase complex called SCFs (SKP-cullin-F-box), which governs ubiquitin-proteasome pathway. Recent study showed that ubiquitin-proteasome pathway is related to cardiovascular diseases.In order to promote our understanding the expression and function of FBL5, the full-length cDNA was isolated from a human brain cDNA library. Using immunofluorescence staining, FBL5 alone distributes in the cytoplasm and still distributes in the cytoplasm when FoxP4 was coexpressed in COS-7 cells.Luciferase assay showed that FBL5 is a transcriptional inhibitor,and can inhibit the regulation activity of SRFUsing bioinformatics we found the zebrafish homologous gene of FBL5. A fragment of FBL5 was amplified by the PCR and inserted into pGEM-T. Antisense digoxigenin-labeled riboprobe was prepared using this construct. Whole-mount in situ hybridization revealed FBL5 expression in the heart, the brain and the bones. Injection of zebrafish embryos with a morpholino directed against FBL5 resulted in embryos with a notochord and an unlooped heart tube. The in situ hybridization assay used embryos injected with FBL5-MO or FoxP4-MO at 48hpf, showed that FBL5 expression decreased in FBL5-MO and FoxP4-MO samples. Conclusion, FBL5 have important function in the zebrafish heart development.更多还原