乙烷基亚硝基脲诱变眼睑开放小鼠模型角膜组织学及突变基因研究

发布时间：2018-01-26 02:51

本文关键词： 乙烷基亚硝基脲眼睑开放 Map3k1基因人类疾病动物模型　出处：《扬州大学》2017年硕士论文　论文类型：学位论文

【摘要】：在哺乳动物正常发育过程中,眼睑发育经历融合及重新开放,牵涉细胞增殖、分化和迁移,并受到多种因子的调控,眼睑闭合缺陷将导致包括角膜混浊在内的多种眼病。由于人眼睑发育在胚胎期完成,很难确定先天性眼病是否与眼睑闭合缺陷有关。本文以一例由乙烷基亚硝基脲(ENU)诱变获得的出生时眼睑开放,之后出现角膜混浊表型小鼠模型为研究对象,对该小鼠角膜进行H.E染色及免疫组织化学分析;对引起眼睑开放的突变基因进行定位与鉴定并初步研究引起眼睑开放的机制。现汇报如下:1.眼睑开放小鼠模型角膜组织学研究石蜡切片取8-10周龄初生时表现为眼睑开放,之后出现角膜混浊表型及同窝正常小鼠眼球。H.E染色显示正常小鼠角膜上皮为非角质化上皮,角膜基质无血管;突变小鼠角膜上皮出现颗粒层和角化层,角膜基质形成新生血管。免疫组化分析发现,在突变小鼠中,细胞角蛋白12(CK12),细胞角蛋白14(CK14)和细胞角蛋白10(CK10)均表达异常。2.眼睑开放小鼠突变基因的精确定位及鉴定将B6背景眼睑开放杂合子小鼠与D2小鼠繁殖获得F1代小鼠,F1代小鼠回交B6获得[(B6×D2)×B6]N2代眼睑开放杂合子小鼠。在先前定位基础上进一步选择微卫星和SNP标记对引起小鼠眼睑开放的突变基因进行精确定位,最终将突变基因定位于D13 M i t2 91(62.07cM)与rs3697199(112598216bp)之间。通过对突变基因定位区域内逐个基因功能的分析发现其候选基因Map3k1。对Map3k1编码区进行PCR或RT-PCR扩增,切胶回收后,委托生物公司测序,并与野生型小鼠进行序列对比,结果发现突变小鼠Map3k1基因编码区941nt处发生T-A颠换,引起其编码蛋白MEKK1第314位氨基酸由亮氨酸变为谷氨酰胺(L314Q),该突变位于该蛋白N端调控区SWIM结构域。3.眼睑闭合缺陷机制的初步研究通过配种检栓获得胚胎期14.5d(E14.5)小鼠,采用免疫组织化学对胚胎眼睑部位c-Jun与P-c-Jun进行检测。结果发现:与正常小鼠相比,突变小鼠眼睑c-Jun与P-c-Jun的表达水平均明显下降。结论:本文通过对一例眼睑开放表型小鼠的突变基因进行精确定位与鉴定,为研究人类眼睑闭合缺陷机制提供参考并为该小鼠作为人类疾病的动物模型的开发与应用奠定基础。
[Abstract]:During the normal development of mammals, eyelid development experiences fusion and reopening, which involves cell proliferation, differentiation and migration, and is regulated by many factors. Eyelid closure defects can lead to a variety of eye diseases, including corneal opacity. Human eyelid development is completed at the embryonic stage. It is difficult to determine whether congenital ophthalmopathy is related to eyelid closure defect. In this paper we present a case of opening of the eyelid at birth induced by ethylnitrosourea (ENUU). The corneal opacity phenotypic mouse model was used as the research object. The cornea of the mouse was stained with e and analyzed by immunohistochemistry. The mutated genes causing eyelid opening were identified and the mechanism of eyelid opening was preliminarily studied. 1. Histological study on cornea of mouse model of eyelid opening: paraffin sections were taken from 8-10 weeks of age to show eyelid opening at birth. Then the corneal opacity phenotype and the eyeball. H. E staining showed that the corneal epithelium of the normal mice was non-keratinized epithelium and the corneal stroma had no blood vessels. Keratinocytes and keratinocytes appeared in the corneal epithelium of mutant mice and neovascularization in corneal stroma. Immunohistochemistry analysis showed that cytokeratin 12 (CK12) was found in mutant mice. Cytokeratin 14 (CK14) and cytokeratin 10 (CK10). The mutation gene of eyelid opening mice was accurately located and identified. F1 generation mice were obtained by breeding B6 background eyelid open heterozygotes and D2 mice. F _ 1 generation mouse backcross B6 obtained. [B6 脳 D2 脳 B6 脳 B6] N2 generation open heterozygote mice. On the basis of previous localization, microsatellite and SNP markers were further selected to locate the mutated genes that caused eyelid opening in mice. Finally, the mutant gene was mapped to D13Mi t291n62.07cM) and rs3697199112598216bp). The candidate gene Map3k1.The Map3k1 coding region was amplified by PCR or RT-PCR. After the recovery of the gel, the biological company was commissioned to sequence and compared with the wild type mice. The results showed that T-A transversion occurred at 941 NT of the Map3k1 gene coding region of the mutant mice. The amino acid of the protein MEKK1 was changed from leucine to glutamine (L314Q). The mutation was located in the SWIM domain of the N-terminal regulatory region of the protein .3.The mechanism of eyelid closure defect was studied. Immunohistochemistry was used to detect c-Jun and P-c-Jun in the eyelid of embryo. The expression levels of c-Jun and P-c-Jun in eyelid of mutant mice were significantly decreased. Conclusion: the mutation gene of a mouse eyelid open phenotype was accurately located and identified. It provides a reference for studying the mechanism of human eyelid closure defect and lays a foundation for the development and application of this mouse as an animal model of human disease.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R777.1;R-332

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