【作者】 陈兵；

【导师】 孙怀昌；

【作者基本信息】 扬州大学 ， 预防兽医学， 2012， 博士

【摘要】 乙烷亚硝基脲(ethylnitrosourea, ENU)是一种能够引起基因高效突变的烷化剂,通过使用ENU诱导小鼠突变的手段,经过筛选可获得大量具有突变表型的G1代小鼠,用于基因功能的研究及人类疾病动物模型的建立。通过ENU诱导小鼠突变,我们获得一种瞳孔散大小鼠,研究发现该突变表型是由Nrg1基因突变引起,药理及免疫组化实验表明该突变导致小鼠瞳孔括约肌上M受体减少。该小鼠被命名为NrglmlYzcm (neuregulin1; mutation l,Yangzhou University Comparative Medicine Center, hereafter Dp1)。遗传实验表明突变杂合子小鼠瞳孔散大表型外显率极低,而纯合子小鼠外显率为100%。本研究工作分为以下五个部分：1ENU诱变获得一种瞳孔散大小鼠通过ENU诱导小鼠突变手段获得一种瞳孔部分散大杂合子小鼠,当用光线照射小鼠眼球时,该小鼠瞳孔对光线无明显反射现象(无明显收缩)。将瞳孔散大杂合子小鼠与正常B6小鼠配种后共观察并记录115只后代小鼠,其中3只小鼠出现单侧且部分瞳孔散大表型；将瞳孔散大杂合子互交,观察并记录34只后代,共有5只后代小鼠表现为瞳孔散大表型,其中1只小鼠表现为单侧且部分瞳孔散大表型,4只小鼠表现为双侧且严重瞳孔散大表型；4只双侧且严重瞳孔散大表型小鼠互交后代全部表现为瞳孔散大表型。2瞳孔散大突变基因的定位在初步染色体定位过程中,采用B6背景瞳孔散大突变小鼠与C3H或D2小鼠配种获得的F1代小鼠,并将F1代小鼠与B6背景野生型小鼠回交获得的N2代突变小鼠配种方案繁殖用于定位小鼠。定位结果发现在25个N2瞳孔散大小鼠样品中突变基因与微卫星D8mit171(距着丝粒11.43cM)和D8mit4(距着丝粒18.89cM)均未发生交换,从而确定该突变基因位于小鼠第8号染色体D8mit171和D8mit4之间(或附近)。另外突变基因与其他染色体上所选微卫星均无明显连锁。由于以上配种方案获得具有突变表型的N2代小鼠的外显率极低。在精确定位过程中改用将瞳孔散大突变小鼠与D2或C3H小鼠配种得到F1代,再将Fl代小鼠互交获得F2代突变小鼠方案进行定位。结果共获得118只F2代突变小鼠,最终将瞳孔散大突变基因定位于遗传标记rs32829041(单核苷酸多态性标记)与D8Mit4之间,两标记间距离约1.52Mb。在此区间目前已报道Dusp26, Rnfl22, BC019943, Mak16, Fut10,7420700N18Rik, Snord13,Mir1186, Nrg1共9种基因,但无相关基因突变引起瞳孔散大异常表型的相关报道。3瞳孔散大突变基因的克隆与序列分析对上述9种基因分别进行测序分析。序列比对结果显示Dusp26、Mir1186、 Rnf122、Snordl3、BC019943、Mak16、Fut10、7420700N18Rik序列与数据库或本中心B6小鼠相应基因序列一致,基本排除这些基因的突变引起Dp1小鼠瞳孔散大表型的可能。在对Nrg1基因测序分析后发现在Nrg1外显子E59(该外显子编码NRG1蛋白EGFβ结构域)后第5个碱基处发生G到A的转换。由于瞳孔散大小鼠G到A的碱基突变位于mRNA剪接供体位点,推测该突变可能影响剪接体对该位点剪接能力,从而引起Dp1小鼠瞳孔散大表型。为了证实以上设想,设计分别扩增Ig及CRD型Nrg1局部序列的引物(扩增区域包含突变位点)。通过RT-PCR方法,分别从瞳孔散大及野生型B6小鼠脑组织总RNA中扩增相应片段,RT-PCR产物经1.5%的琼脂糖凝胶电泳。电泳结果显示对瞳孔散大及野生型B6小鼠Ig及CRD型Nrg1基因分别扩增出3个条带(band a、band b、band c)。其中band b对应于β1-type EGF Nrgl,该类型Nrgl在大脑中高度表达。定量分析结果表明与野生型小鼠相比突变纯合子小鼠Nrg1的CRD及Ig型Nrg1中band b基因表达量仅分别为野生型小鼠的42.6%和32.8%。对野生型B6和瞳孔散大小鼠Ig型及CRD型条带a直接测序表明：野生型B6和瞳孔散大小鼠Ig型及CRD型4种a条带测序信号图都出现明显的重叠信号。对野生型B6小鼠Ig型及CRD型b条带直接测序表明：所测序列测序信号无明显重叠,序列分别对应于CRD-β1和Ig-β1Nrg1基因序列,而对瞳孔散大小鼠Ig型及CRD型条带b测序都出现明显的重叠信号。对野生型B6和瞳孔散大小鼠Ig型及CRD型c条带测序结果表明：1)野生型B6和瞳孔散大小鼠Ig型c带序列一致,野生型B6和瞳孔散大小鼠CRD型c带序列一致；2)与对应野生型B6小鼠b条带相比c条带缺少外显子E59和E24对应序列共83个碱基,该转录产物目前尚未有文献报道。将以上RT-PCR产物直接测序出现重叠信号的条带进行T-A克隆后进一步测序分析,结果显示：所有所测序的野生型B6克隆的mRNA模板在剪接过程中,剪接体在外显子E59后发生剪接；而由于G到A碱基突变,削弱剪接体对该剪接位点的剪接能力,从而绝大多数测序的严重瞳孔散大小鼠克隆的mRNA模板在剪接过程中,剪接体未对紧接外显子E59后的剪接位点进行剪接,部分克隆序列显示剪接体对其他潜在的剪接位点进行了剪接。所以在测序的克隆中存在部分克隆不包含外显子E59、部分克隆存在正常情况下尚未检测到得“异常”剪接所产生的外显子、剪接体跳过紧接外显子E59后剪接位点而对其后潜在的剪接位点进行剪接等结果。蛋白预测表明突变引起部分EGFβ-type Nrg1蛋白的减少,并被部分EGF a-type Nrg1蛋白代替。4Nrg1基因突变对瞳孔括约肌毒蕈碱受体表达的影响单独使用毛果芸香碱眼药水滴眼后,瞳孔散大纯合子及瞳孔部分散大杂合子小鼠瞳孔大小无明显变化。单独使用新斯的明或与毛果芸香碱联合对瞳孔散大纯合子滴眼后,瞳孔散大纯合子小鼠瞳孔大小无明显变化。毛果芸香碱、新斯的明腹腔注射后,瞳孔散大纯合子小鼠瞳孔大小无明显变化,但注射后小鼠出现较严重的流涎及轻度流泪反应。阿托品腹腔注射瞳孔部分散大杂合子小鼠后,小鼠瞳孔出现完全散大表型。以上结果显示,瞳孔散大小鼠括约肌上M受体可能减少,且杂合子小鼠M受体比纯合子数量多,其M受体经过阿托品的封闭后,使得肌肉不能收缩。肌肉收缩程度在一定范围内与神经递质(配体)与受体数量有关。过量的神经递质,但有限的受体同样不能使瞳孔散大杂合子小鼠括约肌完全收缩。胆碱型受体可分为烟碱型受体(Nicotinic Acetylcholine receptor,N受体)和毒蕈碱型受体(Muscarinic Acetylcholine receptor,M受体)两大类。M受体分为M1-M5五个亚型,括约肌上受体属于M受体且其中以M3受体为主,M3基因敲除小鼠表现为部分瞳孔散大表型。文献报道Nrg1对神经-骨骼肌接头处骨骼肌表面N受体具有聚集作用。为进一步验证瞳孔散大小鼠瞳孔括约肌上M受体减少的可能,使用免疫组化技术对括约肌上M3受体进行分析,结果显示与野生型小鼠相比Dp1/Dp1小鼠瞳孔括约肌上M3受体分布明显减少。5瞳孔散大小鼠模型的遗传特性在使用酶切进行基因分型基础上,将Nrg1突变杂合子(Dp1/+)互交,Nrg1突变纯合子(Dp1/Dp1)与突变杂合子(Dp1/+)配种,将Nrg1突变纯合子(Dp1/Dp1)与野生型小鼠(+/+)配种,将Nrg1突变纯合子(Dp1/Dp1)互交,分别记录后代正常与瞳孔散大表型小鼠的数目(包括散大严重程度)及对应的基因型。结果表明所有突变纯合子小鼠表现为瞳孔散大表型,而杂合子小鼠瞳孔散大表型外显率极低(5/111)。另外Dpl/+×Dp1/+配种后代小鼠基因型Dp1/Dp1:Dp1/+:+/+为16：63：29,与孟德尔遗传理论比1：2：1不符(0.01<P<0.05),提示存在部分纯合子小鼠死亡。更多还原

【Abstract】 N-ethy1-N-nitrosourea (ENU) is a powerful point mutagen that can generate random mutations in the mouse genome. Following an ENU-mutagenesis screen for dominant and recessive mutations, a large number of mouse mutants with a variety of phenotypes were recovered for the study of gene function and the generation of human disease models.NrglmlYzcm (neuregulin1; mutation1, Yangzhou University Comparative Medicine Center, hereafter Dp1) was identified as a new ENU-induced mutant with a dilated pupil phenotype. Here, we report that the abnormal phenotype is due to a mutation in the Nrgl gene, which causes a reduction in muscarinic receptors in the sphincter papillae. Interestingly, the Dp1dilated pupil phenotype is inherited with very low penetrance in heterozygous mice and with complete penetrance in homozygous mice.1A Dilated pupil phenotype obtained by ENU mutagenesisDp1is an ENU-induced mutation conveying either a unilateral or bilaterally dilated pupil phenotype that can range from partial to severe. When illuminated, the eyes of affected Dp1mice have no pupillary response to light, and the sphincter pupillae fail to act.The founder Dp1male mouse, which was the progeny of an ENU-treated B6male mouse and an untreated B6female mouse,had a unilateral dilated pupil phenotype. After mating the mutant with B6mice, a very low percentage of the progeny (3/115) were recorded to have the unilateral dilated pupil phenotype. Interestingly, crosses among the three heterozygous progeny resulted in five out of34abnormal progeny, of which four had bilaterally dilated pupils. All progeny generated by crossing the above four bilaterally dilated pupil individuals with each other had a dilated pupil phenotype.2Mapping of the mutation gene that causes dilated pupil phenotype of Dp1mouseFor initial mapping, we tested genomic DNA from25N2samples with microsatellite markers across the whole genome. We observed no exchange of the markers D8Mit171and D8Mit4with the dilated pupil phenotype and found no significant linkages with other chromosomal loci.In order to further refine the map position, we crossbred F1mice and reduced the critical interval to a1.52-Mb region between the single nucleotide polymorphism (SNP) rs32829041and D8Mit4using118F2, dilated pupil offspring. The region contained7protein coding genes (Dusp26, Rnf122, BC019943, Mak16, Fut10,7420700N18Rik. and Nrgl),1miRNA gene (Mir1186) and1snoRNA gene (Snord13).3Identification of the mutation gene that causes dilated pupil phenotype of Dp1mouseSequence analysis of the exons and flanking intronic sequences using DNA or mRNA of these genes revealed no apparent nucleotide changes in Dp1mice except for Neuregulin-1(Nrg1). In the Nrg1gene, we discovered a G to A transition mutation, which flanked exon E59, encoding for the EGFβ domain, in the5’splice donor site.In order to assess the effect of the G to A substitution on EGFβ-type Nrg1mRNA splicing, we amplified the sequences from RNA harvested from the brains of mice homozygous for the Nrgl mutation and wild-type B6mice using primers specific for CRD-Nrg1and Ig-Nrg1, respectively. For both CRD-Nrgl and Ig-Nrgl RT-PCR products, electrophoresis results revealed three bands from both Dp] and wild-type B6mice. The middle band (band b), corresponding to NRG Is with a β1-type EGF sequence (see below), was predominant in the brain of wild-type B6mice. Quantitative analysis by densitometry shows that, in homozygous mice, the relative yield of band b in CRD-Nrgl and Ig-Nrgl is only42.6%and32.8%, respectively, of that in wild-type B6mice.To determine the sequences amplified by RT-PCR analysis, individual bands generated from both wild-type and Dp1mice were purified and used directly as templates for nucleotide sequencing. Sequence analysis of the middle band of CRD-Nrgl and Ig-Nrgl from wild-type mice revealed wild-type sequences corresponding to CRD-β1and Ig-β1Nrg1. However, sequence analysis of the middle band of CRD-Nrgl and Ig-Nrgl in Dpl/Dpl mice revealed a mixed sequence. Sequence analysis of the lowest band (band c) from both wild-type and Dpl/Dpl mice revealed alternative splicing transcripts that lacked exons E59and E24when compared to the corresponding band b sequence from wildtype mice. To our knowledge, these are new transcripts that have not previously been reported. Sequence analysis of the top band (band a) of CRDNrg1and Ig-Nrgl from wild-type and Dpl/Dpl mice revealed a mixed sequence.We cloned the bands with mixed sequences into a T vector and then sequenced the plasmids. Sequencing of these bands revealed differences in the transcripts between Dpl/Dpl and wild-type mice. The majority of the transcripts found in Dpl/Dpl bypass the mutated splice donor site by splicing over exon E59, activating a cryptic splice site, or transcribing through exon E59into the adjacent sequence. These types of protein isoforms were expected, and the results show that, in the mutant, there is a decrease in, but not an elimination of, EGFβ-type Nrgl isoforms. This decrease is partially compensated for by increased expression of the alpha forms, inactive isoforms (without EGFβ,and EGFa domains), and truncated proteins (without the EGFc, EGFβ,and EGFa domains).4Mechanism analysis of dileatd pupil phenotype caused by Nrgl mutation in miceTo analyze the defect that caused the dilated pupil phenotype and cure the abnormal phenotype, we used drugs to clinically constrict or enlarge the pupils. A1%pilocarpine solution (a nonselective muscarinic cholinergic receptor agonist) was first applied as drops in the eyes of mice with severe and partially dilated pupil phenotypes. but it did not alter pupil size in mice with either phenotype (full or partial dilation of the pupil). In contrast, administration of a1%atropine solution, a muscarinic cholinergic receptor antagonist which can compete for AchR with Ach (we considered the drug effect to be a blocking of the AchR), resulted in the further loosening of the sphincter pupillae, which led to complete mydriasis in partially dilated pupil mice. Taken together, these results suggest that the number of muscarinic receptors might be decreased in the iris constrictor muscles of mutant mice, while the number of muscarinic receptors in partially dilated pupil mice is higher than in mice with a severely dilated pupil phenotype. The contractile extent of the sphincter corresponds to the amount of Ach and the number of AchR. Due to the decrease in AchR in partially dilated pupil mice, an excess of Ach with a limited number of AchR cannot lead to complete muscle contraction. In addition to pilocarpine, we used drops of a1%neostigmine solution (a cholinesterase inhibitor) or both solutions together in dilated pupil mice, but both drugs failed to constrict the mutant pupils (full or partial dilation of the pupil). After intraperitoneal administration of pilocarpine or neostigmine in Dp1mice, severe salivation and mild lachrimation were observed, suggesting that AchRs in the glands of Dp1mice were not (or only slightly) affected.M receptors comprise five distinct subtypes (M1-M5). M3is known to play a dominant role in eliciting smooth muscles contraction, and M3-/-mice showing a partially dilated pupil phenotype have been reported. To further validate the reduction of M receptors in the sphincter papillae of Dp1mice, we performed immunohistochemistry tests using an anti-M3antibody and found a significant reduction of M3receptors in the sphincter pupillae of Dp1/Dp1mice.5Genetic model testing of dilated pupil phenotypeTo determine the inheritance fashion of the dilated pupil phenotype, genetic model testing based on genotyping for a Tai I restriction site polymorphism was carried out by intercrossing unaffected Dpl/+mice, crossing Dp1/Dp1mice with unaffected Dp1/+mice, crossing Dp1/Dp1mice with wild-type mice, and intercrossing Dp1/Dp1mice, respectively. The results show that Dp1is a mutation with dilated pupil phenotype that is inherited with a very low penetrance (5/111) when heterozygous and with complete penetrance when homozygous.Additionally, in Dp1/+×Dp1/+offspring, the ratio of genotype of Dp1/+×Dp1/+offspring differed significantly from the expected1:2:1(homozygous mice: heterozygous mice:wild type) ratio (0.01<P<0.05, chi-square test) indicating viability effect.更多还原