间充质细胞特异性ZBTB20基因敲除小鼠模型的建立及表型分析

发布时间：2018-09-17 09:28

【摘要】：锌指蛋白ZBTB20,又被称为树突状细胞来源的POZ锌指蛋白(Dendritic cell-derived POZ zinc finger,DPZF),是BTB锌指蛋白家族成员之一,作为一种转录因子,对于机体生长发育有重要影响。ZBTB20在机体的胰岛、肝脏、海马、心脏等多种组织器官中均有高表达,它不仅调控胰岛?细胞的功能,指导肝脏中甲胎蛋白的表达,而且对痛觉感受有重要调节作用。ZBTB20全身性基因敲除(ZBTB20-/-)小鼠不仅表现为生长发育迟缓、未成年高发性死亡,而且具有海马发育障碍、心脏发育障碍、糖脂代谢异常以及造血功能障碍等重要表型。在人群中ZBTB20基因的突变,会引起Primrose综合症,表现为骨骼发育异常、智力障碍、特殊面容等多系统的临床症状。然而,有关ZBTB20生理功能的了解仍很初步,各个组织器官中ZBTB20的作用还没有完全阐明,关于间充质细胞中ZBTB20的功能还未见报道。间充质干细胞,属于胚胎中胚层的一类多能干细胞,因能分化为间质组织而得名,在特定的体内外环境下,能够诱导分化成为软骨、骨、脂肪、韧带、肌肉、肌腱、神经、内皮等多种组织细胞。ZBTB20在间充质中的表达,预示其强大的调控分化发育的作用。目的:研究间充质细胞中ZBTB20对机体的调控作用,完善ZBTB20的生理功能。方法:本课题通过观察章卫平教授实验室建立的ZBTB20全身性基因敲除(ZBTB20-/-)小鼠模型,研究了ZBTB20对小鼠生长发育的影响,并利用基因打靶技术首次建立了间充质细胞条件特异性敲除小鼠模型(ZBTB20F/F/Prrx1-Cre,MC-ZB20KO),这为我们研究间充质细胞中ZBTB20的功能提供便利条件。ZBTB20在机体的表达一般出现在胚胎13.5-14.5天,而Prrx1-Cre工具鼠的Cre酶在胚胎第9.5天(E9.5)开始表达,为建立间充质细胞条件特异性敲除小鼠模型提供可能。Prrx1-Cre转基因小鼠的出现,为研究四肢相关发育过程中基因的调控提供有效手段,而四肢胚芽历来是研究脊椎动物生长发育的重要模型。结果:通过对该模型进行初步的表型研究,我们发现,MC-ZB20KO小鼠的存活率正常,其生长发育在一定时期内相对正常:4月龄以内小鼠体重、身体长度、脂肪含量、生产率、毛发发育、骨骼发育、垂体发育与对照组相比无明显差异,而血糖代谢有着明显差异。6月龄以后的小鼠,骨骼发育异常现象随年龄增长逐渐明显,四肢的掌、趾关节增长、骨关节增大硬化、脊柱弯曲,行动困难,MC-ZB20KO雌雄小鼠的生殖能力明显下降。结论:我们的研究表明间充质细胞中ZBTB20对小鼠的生长发育有一定程度的调节作用,主要表现在对四肢骨骼发育的调节作用;另外,间充质细胞中ZBTB20利于维持机体血糖平衡。
[Abstract]:Zinc finger protein (ZBTB20,), also known as POZ zinc finger protein (Dendritic cell-derived POZ zinc finger,DPZF) derived from dendritic cells, is a member of BTB zinc finger protein family. As a transcription factor, ZBTB20 plays an important role in the growth and development of human body. Heart and other tissues and organs have high expression, it not only regulate islets? The function of cells, the expression of alpha-fetoprotein in liver, and the important regulation of pain perception. ZBTB20 knockout (ZBTB20-/-) mice not only showed growth retardation, juvenile high incidence of death, but also had hippocampal dysplasia. Cardiac dysplasia, abnormal glucose and lipid metabolism, and hematopoietic dysfunction are important phenotypes. Mutations in the ZBTB20 gene can cause Primrose syndrome in the population, showing multiple systemic clinical symptoms such as abnormal bone development, mental retardation, special facial features, and so on. However, the understanding of physiological function of ZBTB20 is still preliminary, the role of ZBTB20 in various tissues and organs has not been fully clarified, and the function of ZBTB20 in mesenchymal cells has not been reported. Mesenchymal stem cells, a class of pluripotent stem cells belonging to the embryonic mesoderm, derived from their ability to differentiate into mesenchymal tissue, and can be induced to differentiate into cartilage, fat, ligaments, muscles, tendons, nerves in a specific body and outside environment. The expression of ZBTB20 in mesenchymal cells, such as endothelial cells, indicates that ZBTB20 plays a strong role in regulating differentiation and development. Aim: to study the regulation of ZBTB20 in mesenchymal cells and improve the physiological function of ZBTB20. Methods: the effect of ZBTB20 on the growth and development of ZBTB20 systemic gene knockout (ZBTB20-/-) mice was studied by observing the model established by Professor Zhang Weiping's laboratory. The conditional specific knockout mouse model (ZBTB20F/F/Prrx1-Cre,MC-ZB20KO) of mesenchymal cells was established by gene targeting technique for the first time, which provided a convenient condition for the study of the function of ZBTB20 in mesenchymal cells. ZBTB20 expression generally appeared in the embryo 13.5-14.5 days. The expression of Cre in Prrx1-Cre mice began on day 9.5 (E9.5) of embryo, which provided an effective means for the establishment of conditioned knockout mouse model of mesenchymal cells, and the emergence of Prrx1-Cre transgenic mice, which provided an effective means to study the regulation of genes in the process of limb related development. Limb germ has always been an important model to study the growth and development of vertebrates. Results: we found that the survival rate of MC-ZB20KO mice was normal, and the growth and development of MC-ZB20KO mice were relatively normal within 4 months of age, including body weight, body length, fat content, productivity and hair development. There was no significant difference in bone development and pituitary development between the control group and the control group. However, there was a significant difference in blood glucose metabolism in mice over the age of 6 months. The abnormal development of bone and the metacarpal and phalangeal joints of the limbs increased gradually with the increase of age. The reproductive ability of male and female MC-ZB20KO mice decreased significantly. Conclusion: our study shows that ZBTB20 in mesenchymal cells can regulate the growth and development of mice to a certain extent, mainly in the regulation of bone development in limbs, in addition, ZBTB20 in mesenchymal cells is beneficial to maintain the balance of blood glucose.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R363

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