AP3B1基因在雌性生殖和子宫发育中作用的研究及CRISPR-CAS9技术在小鼠基因定点敲除中的应用

发布时间：2018-03-16 22:46

本文选题：AP3B1　切入点：雌性生殖　出处：《山东大学》2014年硕士论文　论文类型：学位论文

【摘要】：受社会快速发展给人们生活环境带来的负面影响,不孕不育的发病率越来越高,据世界卫生组织预测,不孕不育症将成为21世纪的第三大疾病,仅次于肿瘤和心脑血管病。在众多的不孕不育患者中女性子宫发育不良是主要病因之一,且尚无有效的治疗方法。我们的研究发现pearl (pe)雌鼠存在典型的幼稚子宫现象,可以作为研究子宫发育不良的动物模型。pe小鼠是AP3B1基因突变引起的HPSⅡ动物模型。HPS是一种常染色体隐性遗传病,主要的临床症状有肺部纤维化、出血、结肠炎等,目前在HPSⅡ病人和pe小鼠中尚无生殖能力下降的报道。在本部分论文中,我们依次在整体、器官、以及细胞分子水平对pe小鼠的生殖系统进行了全面检测,并对AP3B1基因导致pe雌鼠子宫发育不良的原因进行了深入探索。通过大规模交配实验,我们发现：与对照组相比,pe雌鼠的产仔数显著降低,受孕几率略微降低。进一步通过对各个年龄段pe雌鼠进行器官组织水平分析,我们发现：当发育到一个月时,pe雌鼠开始出现子宫发育不良的明显特征,发育到三个月时pe雌鼠表现出显著的幼稚子宫的特征；而在幼鼠时这一现象并不明显。pe雌鼠的卵巢没有明显的发育异常,但其超排卵数量显著降低,雌激素含量与B6相比没有显著差异。分子蛋白水平分析发现：pe雌鼠的子宫发育的关键基因Hoxa10、Hoxa11、Wnt5a表达明显降低；Western Blot分析证实子宫发育过程重要的通路蛋白β-catenin在细胞质中表达量显著高于B6,在细胞膜上表达量显著低于B6,我们认为受AP3B1基因突变影响,β-catenin的运输过程发生障碍,导致其在细胞质中大量积累。综上所述,pe雌鼠表现出明显的子宫发育不良的特征,是研究幼稚子宫的良好的动物模型,AP3B1基因突变引起子宫发育不良的原因很可能是AP3蛋白复合体的缺失影响了PCP-2和β-catenin的膜质定位,进而导致了子宫发育中重要的调控通路Wnt和Hox异常。对相关途径的深入探究将加深我们对蛋白转运的认识,同时可以为临床不孕不育研究提供新的思路。对基因组中特定位点进行修饰的实验手段称为基因组编辑。它在研究基因的功能和基因修复以及细胞替代治疗上有广泛的应用前景。CRISPR-CAS9技术作为一门新兴的基因修饰方法,凭借其众多的优势正逐步成为传统基因编辑方法强有力的替代者。2013年CRISPR-CAS9技术在小鼠基因定点敲除中得到了前所未有的普及和发展,本部分论文的目的在于搭建小鼠受精卵显微注射平台,并在此基础上初步探索CRISPR-CAS9技术在小鼠基因定点敲除中的应用。小鼠受精卵显微注射平台主要由四部分组成,分别为受精卵的收集,受精卵的处理,受精卵的显微注射和二细胞期胚胎移植。我们通过大量的重复实验针对上述四个步骤摸索了一套合适的实验条件,包括受精卵消化用的透明质酸酶浓度最佳为0.3mg/ml,受精卵收集的时间设在下午13：00最合适,胚胎移植的假孕雌鼠品系最好用CD1。最终我们胚胎移植的多只CD1雌鼠成功生育,说明显微注射平台初步建设完成。为了利用CRISPR-CAS9技术在我们建设的显微注射平台上进行小鼠基因敲除工作,我们选择了附睾特异基因Teddml和serpina1f,由于时间关系,我们完成了上述基因的靶点设计工作,后续的gRNA的构建和Cas9RNA的制备工作正在进行中。相信在未来CRISPR/Cas技术会在基因修饰领域得到全面的发展,并有效地推动基因功能的研究进程。
[Abstract]:By the rapid development of society and brings negative influences to the living environment of people, the incidence of infertility is more and more high, according to WHO, infertility will become the third largest disease in twenty-first Century, after cancer and cardiovascular disease. In many infertile women with infertile patients with uterine dysplasia is one of the main reason, and there is no effective treatment method. We found that Pearl (PE) female rats are a typical phenomenon of infantile uterus, animal model of.Pe mice can be used to study the uterine dysplasia is caused by mutations in the AP3B1 gene HPS II animal model.HPS is an autosomal recessive genetic disease, the main clinical symptoms of pulmonary fibrosis, bleeding, colitis. At present in the HPS II patients and PE mice have decreased reproductive capacity is reported.
In this part, we turn on the whole, organ, cell and molecular level and reproductive system of PE mice conducted a comprehensive inspection, and the AP3B1 gene causes uterine dysplasia PE female rats were deeply explored. Through large-scale mating experiments, we found that: compared with the control group, number of litter PE female rats the lower the chances of pregnancy decreased slightly. Through further analysis, the level of organs and tissues of all ages PE female rats we found that when the development of a month, PE female rats began to appear obvious features of hypoplasia of uterus, development to three months PE female rats showed significant features of infantile uterus in; this phenomenon is not obvious when the rats.Pe female rats ovary had no obvious abnormalities, but the superovulation quantity decreased, estrogen content had no significant difference compared with B6. Molecular analysis of protein level Found: key genes Hoxa10, PE female mice uterus Hoxa11, Wnt5a expression decreased significantly; Western Blot analysis confirmed the expression of -catenin protein beta pathway important developmental processes in the cytoplasm of uterine volume was significantly higher than that of B6, the expression of B6 was significantly lower than in the cell membrane, we believe the impact of AP3B1 gene mutation, the transport process of beta the occurrence of -catenin disorder, lead to the accumulation in the cytoplasm.
In conclusion, PE female rats showed obvious features of hypoplasia of uterus, is a good animal model for the study of infantile uterus, AP3B1 gene mutation causes uterine dysplasia is likely to lack AP3 protein complexes influence membrane localization of PCP-2 and beta -catenin, which led to the development of uterus in important regulatory pathway of Wnt and the Hox anomaly. On way of in-depth study will deepen our understanding of the protein transport, and can provide new ideas for clinical infertility research.
The specific points in the genome of experimental method called modified genome editing. It in the study of gene function and gene repair and cell replacement therapy has broad application prospect of.CRISPR-CAS9 technology as a new gene modified method, with its many advantages are gradually become a traditional gene editing method strong substitute.2013 CRISPR-CAS9 gene knockout mouse technology in point has been the development and popularization of hitherto unknown, the purpose of this paper is to build a mouse zygotes microinjection platform, and on the basis of preliminary exploration of CRISPR-CAS9 technology in gene knockout mice designated in the application.
Mouse zygotes microinjection system consists of four parts, respectively, the fertilized egg collection, processing of fertilized eggs, microinjection of fertilized eggs and two cell stage embryos. We through a lot of experiments according to the above four steps to explore a set of suitable experimental conditions, including the fertilized egg is digested with transparent hyaluronidase optimum concentration is 0.3mg/ml, the fertilized egg collection time at 13:00 in the afternoon the most appropriate, pregnant female mouse strain of embryo transplantation is best to use CD1. in the end we embryos only CD1 female rat reproductive success, that microinjection platform preliminary construction is completed.
In order to carry out the work in the knockout mice were injected on the construction of our platform by using CRISPR-CAS9 technology, we chose the epididymis specific gene Teddml and serpina1f, because of the time, we completed the target gene design work, Cas9RNA construction and subsequent gRNA preparation work is underway. I believe in the future of CRISPR/Cas technology in the field of genetically modified to obtain the comprehensive development, and effectively promote the process of gene function.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R711.74

【共引文献】