本文關(guān)鍵詞： 乙烷基亞硝基脲 眼瞼開放 Map3k1基因 人類疾病動物模型　出處：《揚(yáng)州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：在哺乳動物正常發(fā)育過程中,眼瞼發(fā)育經(jīng)歷融合及重新開放,牽涉細(xì)胞增殖、分化和遷移,并受到多種因子的調(diào)控,眼瞼閉合缺陷將導(dǎo)致包括角膜混濁在內(nèi)的多種眼病。由于人眼瞼發(fā)育在胚胎期完成,很難確定先天性眼病是否與眼瞼閉合缺陷有關(guān)。本文以一例由乙烷基亞硝基脲(ENU)誘變獲得的出生時眼瞼開放,之后出現(xiàn)角膜混濁表型小鼠模型為研究對象,對該小鼠角膜進(jìn)行H.E染色及免疫組織化學(xué)分析;對引起眼瞼開放的突變基因進(jìn)行定位與鑒定并初步研究引起眼瞼開放的機(jī)制。現(xiàn)匯報如下:1.眼瞼開放小鼠模型角膜組織學(xué)研究石蠟切片取8-10周齡初生時表現(xiàn)為眼瞼開放,之后出現(xiàn)角膜混濁表型及同窩正常小鼠眼球。H.E染色顯示正常小鼠角膜上皮為非角質(zhì)化上皮,角膜基質(zhì)無血管;突變小鼠角膜上皮出現(xiàn)顆粒層和角化層,角膜基質(zhì)形成新生血管。免疫組化分析發(fā)現(xiàn),在突變小鼠中,細(xì)胞角蛋白12(CK12),細(xì)胞角蛋白14(CK14)和細(xì)胞角蛋白10(CK10)均表達(dá)異常。2.眼瞼開放小鼠突變基因的精確定位及鑒定將B6背景眼瞼開放雜合子小鼠與D2小鼠繁殖獲得F1代小鼠,F1代小鼠回交B6獲得[(B6×D2)×B6]N2代眼瞼開放雜合子小鼠。在先前定位基礎(chǔ)上進(jìn)一步選擇微衛(wèi)星和SNP標(biāo)記對引起小鼠眼瞼開放的突變基因進(jìn)行精確定位,最終將突變基因定位于D13 M i t2 91(62.07cM)與rs3697199(112598216bp)之間。通過對突變基因定位區(qū)域內(nèi)逐個基因功能的分析發(fā)現(xiàn)其候選基因Map3k1。對Map3k1編碼區(qū)進(jìn)行PCR或RT-PCR擴(kuò)增,切膠回收后,委托生物公司測序,并與野生型小鼠進(jìn)行序列對比,結(jié)果發(fā)現(xiàn)突變小鼠Map3k1基因編碼區(qū)941nt處發(fā)生T-A顛換,引起其編碼蛋白MEKK1第314位氨基酸由亮氨酸變?yōu)楣劝滨０?L314Q),該突變位于該蛋白N端調(diào)控區(qū)SWIM結(jié)構(gòu)域。3.眼瞼閉合缺陷機(jī)制的初步研究通過配種檢栓獲得胚胎期14.5d(E14.5)小鼠,采用免疫組織化學(xué)對胚胎眼瞼部位c-Jun與P-c-Jun進(jìn)行檢測。結(jié)果發(fā)現(xiàn):與正常小鼠相比,突變小鼠眼瞼c-Jun與P-c-Jun的表達(dá)水平均明顯下降。結(jié)論:本文通過對一例眼瞼開放表型小鼠的突變基因進(jìn)行精確定位與鑒定,為研究人類眼瞼閉合缺陷機(jī)制提供參考并為該小鼠作為人類疾病的動物模型的開發(fā)與應(yīng)用奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R777.1;R-332

【參考文獻(xiàn)】

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本文編號：1464452