Cyp4v3基因敲除小鼠模型的表型分析

doi:10.19723/j.issn.1671-167X.2021.06.016

摘要/Abstract

摘要：

目的:构建Cyp4v3^-^/^-小鼠模型以模拟人类结晶样视网膜变性(Bietti crystalline dystrophy,BCD)患者的临床症状,为进一步探索BCD的致病机制和基因治疗方案奠定基础。方法:利用clustered regularly interspaced short palindromic repeats (CRISPR) /Cas9技术,设计sgRNA,注射入C57BL/6J小鼠受精卵中构建携带定点突变的小鼠模型。提取小鼠DNA确定其基因型,分别在其3、6、12月龄时以野生型(wild type, WT)的C57BL/6J小鼠为对照组,进行眼底彩照检查观察其眼底结晶沉积情况;用视网膜电生理(electroretinogram,ERG)检查视网膜功能;用冰冻切片免疫荧光染色观察视网膜组织结构;视网膜色素上皮(retinal pigment epithelium,RPE)铺片鬼笔环肽染色观察RPE形态结构。结果:Cyp4v3^-^/^-小鼠随着年龄增长,可模拟BCD患者的一些临床症状。在疾病早期未发现眼底有结晶样沉积,ERG检测其视网膜功能未发现明显下降,神经视网膜及RPE的形态结构及数量均未发生明显变化。随着Cyp4v3^-^/^-小鼠年龄增长,眼底彩照在6月龄时发现有结晶样沉积,12月龄时沉积消失但色素沉积,RPE萎缩;ERG检查在6月龄时发现有暗适应波幅下降,12月龄时暗适应和明适应波幅均有明显下降;免疫荧光染色显示Cyp4v3^-^/^-小鼠神经视网膜层形态结构受疾病影响不严重;RPE铺片鬼笔环肽染色显示,12月龄时Cyp4v3^-^/^-小鼠RPE细胞六边形形态改变,排列松散,与WT小鼠相比同等大小视野范围内RPE细胞数量明显减少且差异有统计学意义(P=0.011)。结论:Cyp4v3^-^/^-小鼠疾病表型与年龄相关,与人类BCD患者临床症状有相似之处,为进一步研究BCD发病机制和基因治疗策略提供了好的模型;本研究发现BCD病理改变首先发生在RPE,但是具体机制还需进一步研究。

关键词: 结晶样视网膜变性, 小鼠模型, 视网膜电生理

Abstract:

Objective: Bietti crystalline dystrophy (BCD) is a rare degenerative eye disease caused by mutations in the CYP4V2 gene, and Cyp4v3 is the murine ortholog to CYP4V2. To better understand the molecular pathogenesis of this disease and to explore the potential treatment we have established a Cyp4v3 knock-out mouse model. Methods: Cyp4v3 ^-^/^- mice were generated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in embryonic stem cells of C57BL/6J mice. Ocular morphologic characteristics were evaluated via fundus imaging, histologic analysis of rods and cones via immunofluorescence, and phalloidin stain to observe retinal pigment epithelium (RPE) in whole-mounts, electroretinogram (ERG) was also conducted to examine the retinal function. Results: The characteristic features of BCD recurred in the Cyp4v3 ^-^/^- mice, including retinal crystalline deposits, atrophy and degeneration of RPE cells, and ERG amplitude decline of dark and light adapted a- and b- wave; however, the immunofluorescence stain of rod and cone cells did not show obvious differences when compared with the wild type (WT) mice. In the early stage of the disease, no crystal-like deposits were found in the fundus, ERG detection of the retinal function did not find a significant decline, and the morphological structure and quantity of the neural retina and RPE did not change significantly. Crystalline deposits occurred and converged when the Cyp4v3 ^-^/^- mice at the end of 6 months, and the deposits disappeared when the Cyp4v3 ^-^/^- mice at the end of 12 months. The ERG amplitude started to decline when the Cyp4v3 ^-^/^- mice at the end of 6 months and deteriorated at the end of 12 months. The RPE cells of the 12-month old Cyp4v3 ^-^/^- mice showed irregular shape by phalloidin staining of F-actin. The Cyp4v3 ^-^/^- mice behaved normally and were viable and fertile when maintained under specific pathogen-free (SPF) housing conditions. Conclusion: Just like BCD patients, the disease progress of Cyp4v3 ^-^/^- mouse is correlated with the age, which provides a good model for pathogenesis and gene therapy study in the future. The atrophy and degeneration of RPE take the lead in progressing of the disease, but the mechanism is not clear yet.

Key words: Bietti crystalline dystrophy, Mouse model, Electroretinogram

中图分类号:

R774.1

贾睿璇,姜尚伟,赵琳,杨丽萍. Cyp4v3基因敲除小鼠模型的表型分析[J]. 北京大学学报（医学版）, 2021, 53(6): 1099-1106.

JIA Rui-xuan,JIANG Shang-wei,ZHAO Lin,YANG Li-ping. Generation and characterization of Cyp4v3 gene knockout mice[J]. Journal of Peking University (Health Sciences), 2021, 53(6): 1099-1106.

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参考文献 31

[1]	Vargas M, Mitchell A, Yang P, et al. Bietti crystalline dystrophy[M]. Seattle: University of Washington,Seattle, 2012: 12.
[2]	Bietti G. Ueber familiaeres Vorkommen von ‘Retinitis punctata albescens’ (verbunden mit ‘Dystrophia marginalis cristallinea corneae’), Glitzern des Glaskoerpers und anderen degenerativen Augenveraenderungen[J]. Klin Monatsbl Augenheilkd, 1937, 99:737-745.
[3]	Xiao X, Mai G, Li S, et al. Identification of CYP4V2 mutation in 21 families and overview of mutation spectrum in Bietti crystalline corneoretinal dystrophy[J]. Biochem Biophys Res Commun, 2011, 409(2):181-186. doi: 10.1016/j.bbrc.2011.04.112
[4]	Meng XH, Guo H, Xu HW, et al. Identification of novel CYP4V2 gene mutations in 92 Chinese families with Bietti’s crystalline corneoretinal dystrophy[J]. Mol Vis, 2014, 20:1806-1814.
[5]	Fong AMY, Koh A, Lee K, et al. Bietti’s crystalline dystrophy in Asians: Clinical, angiographic and electrophysiological charac-teristics[J]. Int Ophthalmol, 2009, 29(6):459-470. doi: 10.1007/s10792-008-9266-7
[6]	Kaiser-Kupfer MI, Chan CC, Markello TC, et al. Clinical biochemical and pathologic correlations in Bietti’s crystalline dystrophy[J]. Am J Ophthalmol, 1994, 118(5):569-582. pmid: 7977570
[7]	Yuzawa M, Mae Y, Matsui M. Bietti’s crystalline retinopathy[J]. Ophthalmic Genetics, 1986, 7(1):9-20.
[8]	Wilson DJ, Weleber RG, Klein ML, et al. Bietti’s crystalline dystrophy: A clinicopathologic correlative study[J]. Arch Ophthalmol, 1989, 107(2):213-221. doi: 10.1001/archopht.1989.01070010219026
[9]	Bernauer W, Daicker B. Bietti’s corneal-retinal dystrophy: A 16-year progression[J]. Retina, 1992, 12(1):18-20. doi: 10.1097/00006982-199212010-00004
[10]	Rossi S, Testa F, Li A, et al. Clinical and genetic features in Italian Bietti crystalline dystrophy patients[J]. Br J Ophthalmol, 2013, 97(2):174-179. doi: 10.1136/bjophthalmol-2012-302469
[11]	Toto L, Carpineto P, Parodi MB, et al. Spectral domain optical coherence tomography and in vivo confocal microscopy imaging of a case of Bietti’s crystalline dystrophy[J]. Clin Exp Optom, 2013, 96(1):39-45. doi: 10.1111/j.1444-0938.2012.00784.x
[12]	Saatci AO, Doruk HC, Yaman A, et al. Spectral domain optical coherence tomographic findings of Bietti crystalline dystrophy[J]. J Ophthalmol, 2014, 2014:739271.
[13]	Özkiriş A, Evereklioğlu C, et al. A comparison of electroretinographic values of patients with Bietti’s crystalline dystrophy with normal individuals[J]. Erciyes Tip Dergisi, 2004, 26(3):113-118.
[14]	Lai TY, Ng TK, Tam PO, et al. Genotype phenotype analysis of Bietti’s crystalline dystrophy in patients with CYP4V2 mutations[J]. Invest Ophthalmol Vis Sci, 2007, 48(11):5212-5220. doi: 10.1167/iovs.07-0660
[15]	Mansour AM, Uwaydat SH, Chan CC. Long-term follow-up in Bietti crystalline dystrophy[J]. Eur J Ophthalmol, 2007, 17(4):680-682. pmid: 17671952
[16]	Li A, Jiao X, Munier FL, et al. Bietti crystalline corneoretinal dystrophy is caused by mutations in the novel gene CYP4V2[J]. Am J Hum Genet, 2004, 74(5):817-826. doi: 10.1086/383228
[17]	Shan M, Dong B, Zhao X, et al. Novel mutations in the CYP4V2 gene associated with Bietti crystalline corneoretinal dystrophy[J]. Mol Vis, 2005, 11:738-743.
[18]	Yin H, Jin C, Fang X, et al. Molecular analysis and phenotypic study in 14 Chinese families with Bietti crystalline dystrophy[J]. PLoS One, 2014, 9(4):e94960. doi: 10.1371/journal.pone.0094960
[19]	Yin X, Yang L, Chen N, et al. Identification of CYP4V2 mutation in 36 Chinese families with Bietti crystalline corneoretinal dystrophy[J]. Exp Eye Res, 2016, 146:154-162. doi: 10.1016/j.exer.2016.03.007
[20]	Darki F, Fekri S, Farhangmehr S, et al. CYP4V2 mutation screening in an Iranian Bietti crystalline dystrophy pedigree and evidence for clustering of CYP4V2 mutations[J]. J Curr Ophthalmol, 2019, 31(2):172-179. doi: 10.1016/j.joco.2019.01.007
[21]	Nakano M, Kelly EJ, Rettie AE. Expression and characterization of CYP4V2 as a fatty acid omega-hydroxylase[J]. Drug Metab Dispos, 2009, 37(11):2119-2122. doi: 10.1124/dmd.109.028530
[22]	Lai TY, Chu KO, Chan KP, et al. Alterations in serum fatty acid concentrations and desaturase activities in Bietti crystalline dystrophy unaffected by CYP4V2 genotypes[J]. Invest Ophthalmol Vis Sci, 2010, 51(2):1092-1097. doi: 10.1167/iovs.09-3665
[23]	Kumar S. Comparative modeling and molecular docking of orphan human CYP4V2 protein with fatty acid substrates: Insights into substrate specificity[J]. Bioinformation, 2011, 7(7):360-365. doi: 10.6026/bioinformation
[24]	Lockhart CM, Smith TB, Yang P, et al. Longitudinal characterisation of function and structure of Bietti crystalline dystrophy: Report on a novel homozygous mutation in CYP4V2[J]. Br J Ophthalmol, 2018, 102(2):187-194. doi: 10.1136/bjophthalmol-2016-309696
[25]	Lockhart CM, Nakano M, Rettie AE, et al. Generation and characterization of a murine model of Bietti crystalline dystrophy[J]. Invest Ophthalmol Vis Sci, 2014, 55(9):5572-5581. doi: 10.1167/iovs.13-13717
[26]	Hirashima T, Miyata M, Ishihara K, et al. Choroidal vasculature in Bietti crystalline dystrophy with CYP4V2 mutations and in retinitis pigmentosa with EYS mutations[J]. Invest Ophthalmol Vis Sci, 2017, 58(10):3871-3878. doi: 10.1167/iovs.17-21515
[27]	Xiong W, Wu DM, Xue Y, et al. AAV cis-regulatory sequences are correlated with ocular toxicity[J]. Proc Natl Acad Sci USA, 2019, 116(12):5785-5794. doi: 10.1073/pnas.1821000116
[28]	Strauss O. The retinal pigment epithelium in visual function[J]. Physiol Rev, 2005, 85(3):845-881. pmid: 15987797
[29]	Rando RR. The Biochemistry of the visual cycle[J]. Chem Rev, 2001, 101(7):1881-1896. pmid: 11710234
[30]	Nakano M, Kelly EJ, Wiek C, et al. CYP4V2 in Bietti’s crystalline dystrophy: Ocular localization, metabolism of omega-3-polyunsaturated fatty acids, and functional deficit of the p.H331P variant[J]. Mol Pharmacol, 2012, 82(4):679-686. doi: 10.1124/mol.112.080085
[31]	Hata M, Ikeda HO, Iwai S, et al. Reduction of lipid accumulation rescues Bietti’s crystalline dystrophy phenotypes[J]. Proc Natl Acad Sci USA, 2018, 115(15):3936-3941. doi: 10.1073/pnas.1717338115

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