HECT类泛素连接酶Smurf1 C426A基因敲入小鼠模型构建及表型初步分析

发布时间：2018-01-11 02:03

本文关键词：HECT类泛素连接酶Smurf1 C426A基因敲入小鼠模型构建及表型初步分析　出处：《安徽医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：泛素-蛋白酶体系统调控着细胞内绝大多数蛋白的降解。在泛素化过程中,泛素分子经过泛素活化酶(E1)、泛素结合酶(E2)和泛素连接酶(E3)的逐级呈递,最后结合到底物蛋白的赖氨酸残基上,并进一步形成泛素链,其中E3具有底物识别的特异性。研究发现NEDD4超家族HECT类泛素连接酶Smurf1广泛参与到免疫调节、骨动态平衡、肿瘤发生发展及神经系统发育等重要的生理、病理过程。Smurf1可以通过降解经典BMP通路的Smads如Smad1、Smad5、Smad7和BMP受体等负调控BMP通路,进而上调了成骨特异基因的转录。随后又陆续鉴定出Smurf1的其它底物分子如Rho A、Runx2、Myd88以及激活形式的MEKK2等。2005年对Smurf1基因敲除小鼠的研究发现,敲除Smurf1后小鼠骨量会随着年龄增长而增长,骨形成特异增加,Smurf1激活因子CKIP-1的小鼠敲除的研究则发现其骨密度随年龄增长有升高的现象。本课题组前期研究表明,Smurf1能够催化自身发生类泛素化Neddylation修饰,该修饰能够增强Smurf1对E2的募集作用,提高了其泛素连接酶活性,从而促进底物降解。催化反应的活性位点位于Smurf1蛋白的N-lobe区域的426位半胱氨酸。以上结论均为细胞和分子水平的研究得出,我们进而希望能够在生理条件下证实这一结论,并且探究Smurf1的Neddylation修饰对生物整体的影响。由于小鼠、发育过程、途径都与人接近,小鼠可作为一种研究模型。我们利用基因工程技术构建了Smurf1 C426A转基因小鼠模型,并对其表型进行了分析研究。首先获得了小鼠模型后进行扩繁,并利在小鼠MEF细胞内进行体内类泛素化修饰实验,确定该模型构建成功;分离小鼠顶骨前成骨细胞进行诱导培养,探究小鼠成骨细胞能成骨能力,在动物的整体表型上,我们对小鼠股骨进行HE脱钙切片染色,对二维切片获得的骨组织静态参数,对小鼠骨量进行了估测,发现转基因小鼠在骨量上有上升的趋势。综上,本研究在国际上率先建立了Smurf1 C426A基因敲入的小鼠模型,获得了关于Smurf1 C426A基因生理功能的遗传学证据,发现了类泛素化修饰Neddylation与骨形成调控的联系。这些结果将为HECT类泛素连接酶Smurf1 E3功能研究打下基础,并且加深了对HECT类泛素连接酶Smurf1 E3功能调控的认识。
[Abstract]:The ubiquitin proteasome system regulates the degradation of most proteins in the cell. During the process of ubiquification, the ubiquitin molecule passes through the ubiquitin activating enzyme E1). The Ubiquitin binding enzyme E2) and the ubiquitin ligase E3) were presented step by step. Finally, the lysine residues of the protein were bound to the end, and the ubiquitin chain was further formed. E3 has the specificity of substrate recognition. It was found that NEDD4 superfamily HECT ubiquitin ligase Smurf1 is widely involved in immunomodulation and bone homeostasis. Smurf1 can degrade Smads such as Smad1 and Smad5 via classical BMP pathway. Smad7 and BMP receptors negatively regulated the BMP pathway and up-regulated the transcription of osteoblast-specific genes, and then identified other Smurf1 substrates such as Rho Agnon Runx2. Myd88 and activated form of MEKK2 et al. In 2005, a study of Smurf1 gene knockout mice found that the bone mass increased with age after Smurf1 knockout. Bone formation specifically increased the knockout of murf1 activator CKIP-1 in mice and found that bone mineral density increased with age. Smurf1 can catalyze the autogenesis of ubiquitin-like Neddylation modification which can enhance the recruitment of Smurf1 to E2 and the activity of ubiquitin ligase. The active site of catalytic reaction is located at the 426-cysteine in N-lobe region of Smurf1 protein. We then hope to be able to confirm this conclusion under physiological conditions and to explore the effects of Smurf1 Neddylation modification on the overall biology, due to the developmental process in mice. The pathway is similar to that of human, and mice can be used as a research model. We constructed Smurf1 C426A transgenic mice model by genetic engineering. The phenotypes were analyzed and studied. Firstly, the mouse model was obtained, and then expanded and modified in MEF cells. The results showed that the model was successfully constructed. Mouse preparietal osteoblasts were isolated and cultured to explore the osteogenic ability of mouse osteoblasts. In the overall phenotype of animals we stained the femur of mice with HE decalcification sections. The static parameters of bone tissue obtained from two-dimensional sections were estimated, and the bone mass of transgenic mice was found to be increasing. In this study, we first established the mouse model of Smurf1 C426A gene knockin in the world, and obtained the genetic evidence about the physiological function of Smurf1 C426A gene. The relationship between Ubiquitin modified Neddylation and the regulation of bone formation was found. These results will lay a foundation for the study of the function of HECT ubiquitin ligase Smurf1 E3. Furthermore, the function regulation of HECT ubiquitin ligase Smurf1 E 3 was deepened.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78

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