角膜病是人类第4大致盲眼病.通过乙烷基亚硝基脲(N-ethyl-N-nitrosourea,ENU)诱导小鼠突变建立遗传性角膜病小鼠模型,对角膜病小鼠角膜组织进行病理分析并确定致病基因的染色体位置.HE染色发现:角膜病小鼠角膜基质层出现新生血管及较多的角膜细胞,角膜与虹膜发生粘连.采用免疫组织化学技术检测角膜上皮Keratin 12(K12)、Keratin 14(K14)、Keratin 10(K10)及PAX6的表达情况,结果发现:角膜病小鼠K12信号局部减弱或为阴性,K10局部免疫组织化学染色结果为阳性,K14可表达于整个角膜上皮层且局部信号增强,PAX6阳性细胞明显减少或消失.连锁分析将致病基因定位于小鼠第10号染色体D10Mit162与D10Mit233之间.该研究有望为人类角膜病研究提供一种新的小鼠模型.

In this study, we describe an N-ethyl-N-nitrosourea-induced mouse model with a corneal opacity phenotype that was associated with "eye open at birth" (EOB). Histological and immunohistochemistry staining analysis showed abnormal differentiation of the corneal epithelial cells in the mutant mice. The EOB phenotype was dominantly inherited on a C57BL/6 (B6) background. This allele carries a T941A substitution in exon 4 that leads to an L314Q amino acid change in the open reading frame of MAP3K1 (MEEK1). We named this novel Map3k1 allele Map3k1L314Q. Phalloidin staining of F-actin was reduced in the mutant epithelial leading edge cells, which is indicative of abnormality in epithelial cell migration. Interestingly enough, not only p-c-Jun and p-JNK but also c-Jun levels were decreased in the mutant epithelial leading edge cells. This study identifies a novel mouse Map3k1 allele causing EOB phenotype and the EOB phenotype in Map3k1L314Q mouse may be associated with the reduced level of p-JNK and c-Jun.

Ip3r1 encodes an inositol 1,4,5-trisphosphate-responsive calcium channel. Mutations in the IP3R1 gene in humans may cause Gillespie syndrome (GS) typically presents as fixed dilated pupils in affected infants, which was referred to as iris hypoplasia. However, there is no report of mice with Ip3r1 heterozygous mutations showing dilated pupils. Here, we report a new Ip3r1 allele with short-term dilated pupil phenotype derived from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. This allele carries a G5927A transition mutation in Ip3r1 gene (NM_010585), which is predicted to result in a C1976Y amino acid change in the open reading frame of IP3R1 (NP_034715). We named this novel Ip3r1 allele Ip3r1(C1976Y). Histology and pharmacological tests show that the dilated pupil phenotype is a mydriasis caused by the functional defect in the iris constrictor muscles in Ip3r1(C1976Y). The dilated pupil phenotype in Ip3r1(C1976Y) was referred to as mydriasis and excluding iris hypoplasia. IHC analysis revealed increased expression of BIP protein, the master regulator of unfolded protein response (UPR) signaling, in Ip3r1(C1976Y) mice that did not recover. This study is the first report of an Ip3r1 mutation being associated with the mydriasis phenotype. Ip3r1(C1976Y) mice represent a self-healing model that may be used to study the therapeutic approach for Ip3r1-related diseases.

Objective To describe the mortality trend of major malignant tumors in Shandong province,from 1970 to 2013.Methods Data related to cancer mortality were obtained from the Shandong Death Registration System and three nationwide retrospective cause-of-death surveys.Trends of overall mortality and major causes of death were described using the indicators as:mortality rates and age-standardized mortality rates,through comparing the three large-scale mortality surveys in Shandong province.Difference decomposing method was applied to estimate the contribution of demographic and non-demographic factors for the change of mortality.Results From 1970 to 2013,the crude mortality rate of malignant tumors in Shandong was increasing.The age standard mortality rate was increasing and then decreasing.The composition of cancer deaths in the all-cause-deaths was seen increasing and then decreasing as well.Both demographic and non-demographic factors contributed to the increase of crude cancer mortality rate.With the gradual increase of the proportion of population,its role exceeded the non-demographic factors.The age-standardized mortality rate of malignant tumors in 2011-2013 was lower than that in 2004-2005.Lung cancer mortality rose from the fifth to the first place,with an increase of 6.81 times from 1970-1974 to 2011-2013.Ranking of gastric cancer mortality dropped from first to the third place,with esophageal cancer dropped from second to the fourth.After adjusted by China's standard population in 1964,the mortality rate of lung cancer was still rapidly increasing,but the age-standardized mortality rates of esophageal cancer was gradually decreasing.The crude and age-standardized mortality rates of cervical cancer showed a rapid downward trend,reduced 87.00％ and 93.00％ respectively from 1970-1974 to 2011-2013.Conclusions Malignant tumors were still major threats to the residents of Shandong province.The changing trend of different malignant tumors presented an inconsistent nature which called for different intervention strategies be carried out,accordingly.

Ip3r1 encodes an inositol 1,4,5-triphosphate-responsive calcium channel. Mutations in the Ip3r1 gene in humans may cause Gillespie syndrome (GS) typically presents as fixed dilated pupils in affected infants, which was referred to as iris hypoplasia. However, there is no report of mice with Ip3r1 heterozygous mutations showing dilated pupils. Here, we report a new Ip3r1 allele (dilated pupil 2; Dp2 ) with short-term dilated pupil phenotype derived from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. This allele carries a G5927A transition mutation, which is predicted to result in a C1976Y amino acid change in the open reading frame. Histology and pharmacological tests show that the dilated pupil phenotype is a mydriasis caused by the functional defect in the iris constrictor muscles in Dp2 . The dilated pupil phenotype in Dp2 was referred to as mydriasis and excluding iris hypoplasia. IHC analysis revealed increased expression of BIP protein, the master regulator of unfolded protein response (UPR) signaling, in Dp2 mice that did not recover. Apart from the dilated pupil phenotype (mydriasis), there are no other abnormal phenotypes including Ip3r1 -related ataxia that may be found. This study is the first report of an Ip3r1 mutation being associated with the mydriasis phenotype. Dp2 mice represent a valuable self-healing model that may be used to study the therapeutic approach for Ip3r1 -related diseases or diseases caused by similar pathomechanisms.

Objective To analyze the epidemiological and temporal-spatial distribution characteristics of hemorrhagic fever with renal syndrome (HFRS) in Shandong province during 2010-2016 and provide references for developing prevention and control measures.Methods Based on the data of Infectious Disease Reporting Information System in China,the incidence and temporal-spatial distribution of HFRS in Shandong from 2010 to 2016 were analyzed by spatial autocorrelation and space-time scan statistics.Results A total of 9 114 HFRS cases were reported in Shandong during this period.The cases were mainly distributed in age group 30-70 years,and the male to female ratio of the cases was 2.63 ∶ 1.Most cases were farmers.The higher incidence rate was reported in southeastern Shandong,while the lower incidence rate was reported in northwestern Shandong.Among the epidemic periods,the highest incidence rate was 1.87/100 000 in 2013.The results of spatial autocorrelation and space-time scanning indicated that the high-high clusters of HFRS were concentrated in southeastern Shandong and then spread to central Shandong.The cluster mainly occurred from the end of 2011 to the first half of 2015.Both the incidence rate and the cluster decreased in 2016.Conclusions The epidemic and cluster of HFRS still existed in Shandong from 2010 to 2016.The key areas for the prevention and control of HFRS were in southeastern and central Shandong.

目的分析阿尔茨海默病（AD）模型树鼩大脑影像学特征。方法在脑立体定位仪上侧脑室注射Aβ1-40建立AD动物模型。经视觉-空间行为学检测确定模型成功后,用MRI作脑冠状面T2加权成像（T2WI）和弥散张量成像（DTI）分析。结果模型组模型组参考记忆错误（3周,4周）和工作记忆错误（2周,3周,4周）显著多于对照组（P〈0.05）。模型组完成任务的时间（2周,3周）显著多于对照组（P〈0.05）。3周起模型组树鼩单侧或双侧海马减小,相应侧脑室或双侧脑室增大。12周时模型组树鼩双侧颞角宽度显著大于对照组和治疗组（P〈0.01）。弥散张量成像扫描显示,模型组树鼩海马双侧表观弥散系数（ACD）大于对照组（P〈0.01）。模型组胼胝体纤维束缺失严重。结论侧脑室注射Aβ1-40可引起树鼩学习记忆障碍。MRI能显示AD树鼩脑部的特征性改变,颞角宽度、海马ADC值、胼胝体纤维受损对痴呆的诊断有参考价值。

Objective To analyze the neuroimaging changes of tree shrew models of Alzheimer’ s disease.Methods Nineteen healthy adult female tree shrews were randomly divided into control (5 animals) and model group (14 animals). The model of Alzheimer’s disease was induced by intracerebroventricular injection of Aβ1-40 using a stereotaxic devise and proved successfully by visuospatial congnitive task.The in vivo microstructural changes in the brain of tree shrew AD models and control group (0, 1, 2, 3, 4 weeks) were observed on 1.5T MRI (T2WI), and on 7.0T MRI (12 week)(T2WI, DTI).&nbsp;Results Reference memory errors were increased in the model group at 3 or 4 weeks (P<0.05), and so working memory errors (P<0.05) and period of time to perform (P<0.05, P<0.05, P<0.01) from 2 to 4 weeks.Thus the model was proved to be established successfully.T2WI test and DTI test were carried out.Hippocampus atrophy of the model group at 3 and 4 weeks was observed compared with that at 0 or 1 week or 2 weeks on a 1.5T Philips Gyroscan.Compared with the control group, the temporal horn width in the model group was significantly increased (P<0.01) at 12 weeks on a 7.0T Bruker Biospec Scanner.DTI test at 12 weeks showed that ADC of bilateral hippocampus was up-regulated in the model group ( P<0.01 ) .In the color coded orientation view, loss of the corpus callosum fibers was obvious in the model group. Conclusions Intracerebroventricular injection of Aβ1-40 can lead to learing and memory impairment in tree shrews.There are abnomal MRI signal changes in the brain, and the temporal horn width, hypocampal apparent diffusion coefficient ( ADC) value and corpus callosum damage may provide reference value for the diagnosis of Alzheimer’ s disease.

Hirschsprung disease (HSCR), or colonic aganglionosis, is a congenital disorder characterized by the absence of intramural ganglia along variable lengths of the colon, resulting in intestinal obstruction. It is the most common cause of congenital intestinal obstruction, with an incidence of 1 in 5,000 live births. N-ethyl-N-nitrosourea (ENU)-induced mutagenesis is a powerful tool for the study of gene function and the generation of human disease models. In the current study, a novel mutant mouse with aganglionic megacolon and coat color spotting was generated by ENU-induced mutagenesis. Histological and acetylcholinesterase (AChE) whole-mount staining analysis showed a lack of ganglion cells in the colon in mutant mice. The mutation was mapped to chromosome 14 between markers rs30928624 and D14Mit205 (Chr 14 positions 103723921 bp and 105054651 bp). The Ednrb (Chr 14 position 103814625-103844173 bp) was identified as a potential candidate gene in this location. Mutation analysis revealed a T>C missense mutation at nucleotide 857 of the cDNA encoding endothelin receptor B (EDNRB) in which a proline was substituted for the highly conserved Lys-286 residue (L286P) in the fifth transmembrane (TM V) domain of this G protein coupled receptor. The mutant mouse was named Ednrb(m1yzcm) (Ednrb; mutation 1, Yangzhou University Comparative Medicine Center). The results of the present study implicate the structural importance of the TM V domain in Ednrb function, and the Ednrb(m1yzcm) mouse represents a valuable model for the study of HSCR in humans.

实验动物学是实验动物专业的重要课程,笔者结合本院实验动物学的教学实际,对课程内容和设置作了介绍,通过提升教师的业务水平,实施分层次授课,结合传统的教学方法,针对不同的教学内容,灵活采用合适的教学方法,取得了良好的教学效果.

在实验动物学实验教学改革的探索和实践中,采用提升教师业务水平、改进教学方法、增强学生的实验动物伦理观念、强化基本操作,优化实验内容,搭建开放实验室平台、改变考核方法等措施;取得了显著效果,增强了学生的操作技能,培养了学生的创新意识和创新能力.

实验动物是生命科学相关学科基础理论研究以及生药制造，化药筛选、检定等必不可少的试验材料和发展基础。实验动物的生物学特性及其感染情况，不仅直接影响实验动物本身的发展，而且还关系到生命科学基础理论与应用结果的判定，这些都属于实验动物医学研究范畴，因此开设实验动物医学课程，对推动实验动物科学健康发展具有积极意义。

To obtain a suitable method for an evaluation of the cognitive abilities of tree shrews( Tupaia belangeri),the wrong choices and repeated choices of male adult tree shrews for baits were observed through the food searching test based on holeboard. The result showed that the time that the animals found out all the baits gradually shortened. The time to complete the task entered a relatively stable period( P 0. 05)since the 2nd d( visual spatial task) and the 4th d( space task). The number of wrong choices of tree shrews reached relative stability( P 0. 05) between 3 d and 5 d with or without visual cues,it showed that the spatial reference memory of tree shrews was relatively stable. In the visual space experiments,the repeated choices of tree shrews reached relative stability( P 0. 05) between 2 d and 4 d,and then were relatively significantly decreased at 5 d. The repeated choices of tree shrews reached relative stability( P 0. 05) between 4 d and 5 d in the space experiments without visual cues,it was suggested that spatial working memory of tree shrews had greater relative volatile frequency.

目的 制备bcr启动子驱动-增强绿色荧光蛋白(bcr-EGFP)转基因小鼠模型,在活体水平验证bcr启动子的启动特异性.方法 以慢性髓性细胞白血病(CML)细胞株K562细胞基因组为模板,PCR扩增1.1 kb bcr启动子,在扩增产物的上、下游引物加入了Ase Ⅰ、EcoR Ⅰ酶切位点,AseⅠ、EcoRⅠ双酶切pEGFP-N1真核表达质粒载体,去除载体自身的CMV启动子,并将1.1 kbbcr启动子定向克隆到EGFP绿色荧光蛋白报告基因上游,构建pbcr-EGFP真核表达载体,并将此重组质粒瞬时转染K562细胞和NIH3T3细胞,进行表达验证.显微注射法制备bcr-EGFP转基因小鼠,采用PCR方法进行初步整合检测.结果 显微注射583枚受精卵,移植到30只受体鼠输卵管中,受体鼠妊娠26只,共生产首建鼠90只,经过PCR检测,4#(♀)、36#(♂)和81#(♂)等3只F0代为转基因整合阳性鼠.结论 成功制备了bcr-EGFP转基因小鼠,为进一步研究bcr启动子在CML转基因小鼠模型制备的安全性和可靠性提供线索和帮助.

Objective To investigate the expression levels of BDNF, trkB and ChAT mRNA and proteins in the brain of adult tree shrews ( Tupaia belangeri ) .Methods Quantitative real-time PCR was employed to detect the expression levels of BDNF, trkB and ChAT mRNA in the hippocampus, basal ganglia and frontal cortex of adult tree shrews.The expression levels of BDNF, trkB and ChAT proteins andβ-actin was used as internal standard.Results The expression level of BDNF mRNA was highest in the hippocampus of adult tree shrew, and there were significant differences between that in the hippocampus, and basal ganglia and frontal cortex (P<0.01).The expression level of trkB mRNA was higher in the frontal cortex than in the basal ganglia and hippocampus, showing a significant difference between them ( P<0.05).The expression level of BDNF protein was significantly higher in the basal ganglia than in the hippocampus or frontal cortex (P<0.01).There were no significant difference (P>0.05) in the expressions of trkB protein among the hippocampus, basal ganglia and frontal cortex of the adult tree shrews.There were no significant differences in expressions of ChAT mRNA and protein among the hippocampus, basal ganglia and frontal cortex in adult tree shrews ( P>0.05 ) . Conclusions The expression levels of ChAT mRNA were consistent with that of ChAT protein in the hippocampus, basal ganglia and frontal cortex of adult tree shrews, while the expression levels of BDNF and trkB mRNA were not consistent with their proteins, which might indicate that the transcriptional regulation pattern might be more complex.Tree shrew is a valuable animal model in the study of mechanism of BDNF/trkB gene expression.

建立BALB/c小鼠肝螺杆菌(Helicobacter hepaticus,Hh)感染模型,旨在研究Hh在小鼠感染不同时期的病理特征.以Hh灌饲SPF级BALB/c雄性小鼠,于最后1次接种后第2、4、6、8周收集小鼠粪便提取DNA,PCR扩增Hh 16S rRNA基因检测小鼠感染率;结果表明随时间延长,小鼠的Hh感染率逐步增加,获得了稳定的BALB c小鼠Hh感染试验模型.接种后2、3、4和5个月,分别取小鼠肝脏、胃、盲肠和结肠,进行Hh16S rRNA基因扩增和病理组织学检查.结果显示,小鼠的肝脏、胃、盲肠和结肠组织均检测到了Hh的定植;小鼠肝细胞发生脂肪变性、肝小叶局灶性坏死、坏死灶周围有不同程度的炎性细胞浸润等病变,随时间延长,病变严重程度增加;少量小鼠胃黏膜下层有炎性细胞浸润、胃黏膜上皮坏死脱落;盲肠表现不同程度的炎性细胞浸润.该研究结果表明,Hh可引起小鼠肝炎、胃炎和盲肠炎.

为了探索树鼩食物识别的机制,试验采用4种处理方法:(A)空白对照,(B)黄粉虫气味,(C)密封的黄粉虫,(D)黄粉虫,每个个体每种处理均进行6次试验.结果表明:不同处理条件下的探究频次、啃咬频次、行为持续时间差异显著(P＜0.05).树鼩在处理C(视觉刺激)及处理D(既有化学刺激又有视觉刺激)条件下的行为持续时间、探究频次和攻击频次显著高于处理B(化学刺激)(P＜0.05).在无视觉刺激的条件下,树鼩在处理B的行为持续时间、探究频次、啃咬频次显著高于处理A(P＜0.05).在视觉刺激相同的条件下,树鼩在处理D的行为持续时间显著长于处理C(P ＜0.05).树鼩主要利用视觉捕食,化学识别为辅.

目的 通过乙烷基亚硝基脲(ENU)诱导小鼠突变手段获得眼部异常小鼠模型.方法 选用8～ 10周龄C57BL/6J (B6)雄鼠40只,腹腔内注射ENU.将处理雄鼠与同品系母鼠交配,在其后代小鼠中筛选眼部异常个体,并对突变个体进行遗传实验.结果 通过ENU诱变手段获得一例可稳定遗传的小眼畸形小鼠,该小鼠小眼畸形表型受小鼠遗传背景影响,在B6背景中外显率为90％,BDF1背景中外显率为0,BDBN2背景为27.8％.结论 ENU诱变获得了类似人类小眼畸形疾病的小鼠模型,为人类相应眼病研究提供一种新模型.

Objective To define the loci of the mutant gene in the loop-tail mouse.Methods To study the heredity pattern, loop-tail mice were mated with normal C57BL/6J and C3H mice.Their offsprings with loop-tail or normal phenotype were registered respectively.Microsatellite marker D1Mit113 and D1Mit149 were used to locate the mutant gene.Based on fine mapping, the candidate gene Vangl2 was found.Vangl2 gene from the loop-tail mice was amplified by PCR followed by sequencing.Incision enzyme FspBI ( BfaI ) identified the genotype of offspring from loop-tail mice intercrossing.Results Heredity test indicated that the loop-tail phenotype was controlled by a single dominant gene not with 100%penetrance but was affected by genetic background.A C-to-T transversion was at the 1345bp in Vangl2 gene of the loop-tail mice.Conclusions The C-to-T transversion introduces a pre-termination codon of amino acids and causes the phenotype of loop-tail phenotype.None homozygous mice were found in the offsprings, suggesting that the homozygous mice are lethal.

为获得树鼩行为学特征的基础数据,通过视线阻隔处理,观察雄性成年树鼩的行为变化.结果表明树鼩的活跃度和取食逐步提高,阻隔时间与运动树鼩数及取食数有回归关系,回归方程为:运动树鼩数=50.954+时间×0.607(R2=0.870,n=80).取食树鼩数=e(4 356-0.721/时间)(R2 =0.996,n=80).树鼩对4种水果(F=294.234;d=3;P =0.000)、动物性食物(F =279.692;d =3;P =0.000)及混合食谱(F=239.692;d =3;P =0.000)的取食量有显著差异(P＜0.01),树鼩喜好甜而多汁的石榴和面包虫.本研究建立了一种行为学观察的基础方法和数据,为树鼩行为学研究提供依据.