Construction of mouse ESC line stably expressing Pdx1 by Tet-On system

AIM To construct the mouse embryonic stem cell （ESC） line with stable pancreatic and duodenal homeobox 1 （Pdx1） expression by Tet-On system, which may lay a foundation for further research on the differentiation of Pdx1⁺ definitive endoderm cells into pancreatic cells. METHODS The Pdx1-overexpressing lentiviral vector with green fluorescent protein marker and puromycin resistance was constructed by Tet-On system and was used to infect the mouse ESC. The cells were divided into 3 groups： blank control group （ESC group）, empty lentivirus control group （PDX1^- ESC group） and Pdx1 lentivirus transfection group （PDX1⁺ ESC group）. Flow cytometry was used to detect the transfected cells after screening by doxycycline （DOX）. The function of Tet-On system and the expression of Pdx1 gene were detected. The transfected cells in PDX1^- ESC group and PDX1⁺ ESC group were sorted by flow cytometry, and constructed ESC line with stable expression of Pdx1 and negative control ESC line were verified. RESULTS （1） The positive rates of transfected cells in PDX1^- ESC group and PDX1⁺ ESC group were 90.72% and 94.01% after screening by DOX, respectively. The positive rates of transfected cells in PDX1^- ESC group and PDX1⁺ ESC group was 97.84% and 98.13% after sorting by flow cytometry, respectively. （2） With DOX, green fluorescence was observed in PDX1^- ESC group and PDX1⁺ ESC group. The mRNA and protein expression of Pdx1 was significantly increased in PDX1⁺ ESC group （P<0.05）. Without DOX, no green fluorescence was observed in the cells of the 3 groups, and no significant difference in the mRNA and protein expression of Pdx1 was observed （P>0.05）. （3） After 3 months of cryopreservation, the cell lines still survived in resuscitation culture and were regulated by DOX. CONCLUSION Using Tet-On system, the mouse ESC line with inducible Pdx1 expression were successfully established and could be used as an effective cell model to research the differentiation of Pdx1⁺ definitive endoderm cells into pancreatic cells.     

济宁市农村土地流转现状及对策

在深入调查研究的基础上,分析了济宁市农村土地流转现状及其存在的问题,并提出相关建议,以期促进农村土地有序流转,合理利用配置土地资源,解决经济发展与土地资源紧缺的矛盾。     

犬基底细胞癌的病理组织学观察

基底细胞癌(basal cell carcinoma,BCC)是一种常见的低度皮肤恶性肿瘤,又名基底细胞上皮癌、基底细胞癌和侵蚀性溃疡。本文主要探讨皮肤基底细胞癌的临床病理诊断和鉴别诊断要点,为提高基底细胞癌的诊治水平提供依据。通过病理组织学观察,皮肤破溃、出血,肿瘤细胞成团块状增生,由结缔组织分割为不规则小叶状。细胞呈梭形、多边形及近圆形;细胞核圆形,核膜清晰,有一明显核仁,有一定异形性,偶见分裂相,作为确诊基底细胞肉瘤的主要依据,并对疾病的处理和治疗方法进行了归纳和总结。     

鸭源致病性大肠杆菌的血清型鉴定及其相关毒力基因分析

自规模化养鸭场患典型大肠杆菌败血症雏鸭分离的282株致病性大肠杆菌（E.coli）中鉴定出210株（包含37种血清型）,其中O93、O78、O92、O76占43.8%（92/210）为优势血清型,O46、O32＆O93混合型、O60＆O93混合型为首次从鸭群中分离到。应用PCR结合核酸序列测定对210株致病性E.coli（鸭大肠杆菌病分离株）和28株自健康雏鸭泄殖腔拭子分离的E.coli（临床健康鸭大肠杆菌分离株）进行包括强毒力岛（HPI）中的鼠疫菌素受体基因（fyuA）和铁调节蛋白基因（irp2）、Ⅰ型菌毛必需蛋白基因（fimC）、P型菌毛结构基因（papA）和血清耐受基因（iss）检测,结果表明：fyuAi、rp2、fimC、papA和iss基因在鸭大肠杆菌病分离株的携带率分别为41.0%、43.3%、92.9%、97.6%和96.7%,在临床健康鸭大肠杆菌分离株的携带率分别为21.4%、25.0%、92.9%、100%和92.9%,患病鸭和临床健康鸭大肠杆菌分离株iss、fimC和papA携带率差异不显著（P〉0.05）,但papA的携带率均显著高于其他宿主（鸡、猪和人）源E.coli;HPI毒力岛在鸭源E.coli中分布较广,其携带率表现为鸭大肠杆菌病分离株极显著高于临床健康鸭大肠杆菌分离株。HPI毒力岛的携带率与菌株的致病性呈明显的正相关,与O78等特定的血清型有一定的关系。鸭大肠杆菌病分离株有37.6%（79/210）同时携带fyuAi、rp2、fimCi、ss和papA基因,极显著高于健康鸭大肠杆菌分离株的14.3%（4/28）（P〈0.01）。     

水环境中噬菌蛭弧菌的分离鉴定

噬菌蛭弧菌为一类杆形、弧形或螺旋形的革兰阴性菌,可以寄生并裂解其他细菌。本试验以禽源大肠杆菌、沙门菌以及环境水体中分离的1株大肠杆菌为宿主菌,采用双层琼脂培养法成功地从环境水体中分离到3株噬菌蛭弧菌,分别命名为BDD、BDE、BDH。电镜观察证明菌体大小约0.33μm×0.78μm,单个弧形,一端有一根长鞭毛。基因序列分析表明,分离株与已发表的噬菌蛭弧菌属其他菌株16S rRNA基因序列的同源范围分别为96.0%~99.9%。通过对不同细菌的裂解试验证明,3个分离株对大肠杆菌、沙门菌、摩根摩根氏菌、变形杆菌和阴沟肠杆菌等均具有裂解作用,但对铜绿假单胞菌、鸭疫里默氏菌和金黄色葡萄球菌不敏感。     

铜的来源和水平对生长猪生长性能禾粪铜排出量的影响

选用平均体重29．6kg的三元杂（杜×大×长）生长猪144头，按随机区组设计分为6个处理。饲粮中分别添加0、100、200mg／kg硫酸铜、200mg／kg碱式氯化铜、100mg／kg小肽铜、100mg／kg复合氨基酸螯合铜（以铜含量计）。结果表明：100mg／kg小肽铜和100mg／kg复合氨基酸螯合铜均能有效提高生长猪日增重和日采食量，其促生长效果与200mg／kg硫酸铜无显著（P〉0．05）差异，而粪中铜含量分别下降了36．8％和48．4％（P〈0．05）。     

Adeno-associated virus type 9-mediated p65 silencing inhibits angiotensin II-induced apoptosis of H9c2 cells

AIM: To study the effect of p65 gene silencing by adeno-associated virus type 9 (AAV9)-mediated RNA interference on angiotensin Ⅱ (Ang Ⅱ; 10^-6 mol/L for 24 h)-induced apoptosis of rat ventricular H9c2 myocytes, and to elucidate the possible mechanism. METHODS: The H9c2 cells were transfected with rAAV9-eGFP and rAAV9-eGFP-NF-κB p65-siRNA at multiplicity of infection (MOI)=4×10⁶ vg/cell. eGFP expression in the cells was observed under an inverted fluorescence microscope, and the percentage of eGFP positive cells was determined by flow cytometry. The expression of p65 was determined by Western blot. CCK-8 assay was used to measured the viability of transfected H9c2 cells. The apoptosis of the cells transfected with the virus and with Ang Ⅱ stimulation was analyzed by flow cytometry. RESULTS: The cells began to exhibit eGFP expression on the 2nd day after transfection. The fluorescence intensity was increased over the time of transfection. eGFP expression reached the maximum on the 5th day, and the transfection efficiency was (52.7±1.9)% at this time point. Compared with blank control group, no significant effect of AAV9 on the viability of H9c2 cells was observed. In resting state, p65 in the H9c2 cells had a certain activity. After Ang Ⅱ stimulation, the activity of p65 was obviously increased, while transfection of rAAV9-eGFP-NF-κB p65-siRNA effectively inhibited the expression of p65. The apoptosis of H9c2 cells in Ang Ⅱ stimulation group was significantly higher than that in blank control group, while transfection of rAAV9-eGFP-NF-κB p65-siRNA effectively inhibited apoptosis of H9c2 cells. CONCLUSION: Transfection of rAAV9-eGFP-NF-κB p65-siRNA effectively inhibits the expression of p65 gene of NF-κB pathway in the H9c2 cells without causing cell growth inhibition, and reduces the apoptosis induced by Ang Ⅱ.     

六个八仙花品种气孔特性研究

对6个品种八仙花的叶片气孔特性进行了研究。结果表明:气孔器长轴平均长度在27~40μm之间;气孔器短轴平均长度在18~25μm之间;气孔长轴平均长度在11~24μm之间;气孔短轴平均长度在5~10μm之间。气孔密度最大的是B,为550.87个/mm2,最小的是A,为295.06个/mm2。气孔面积最大的是G,为745.31μm2,最小的是E,为425.92μm2。各个品种气孔器的形状均为椭圆形或近圆形。     

鸡柔嫩艾美尔球虫病抗性主成分分析评估模型的建立

为建立鸡活体可测球虫病抗性综合评估模型,探索鸡球虫病抗性选育新方法,以京海黄鸡为试验材料,根据国内外文献选择15个球虫抗性评价指标,通过比较鸡柔嫩艾美尔球虫感染组和非感染组抗性指标的差异,进一步筛选活体可测指标,并进行主成分分析,通过各主成分和综合主成分与所有抗性指标的相关分析,筛选最优抗性综合评估模型,并进行模型有效性筛选。在所选的14个活体可测抗性指标中,有6个指标在感染和非感染组间存在显著或极显著的差异(P0.05或P0.01),他们分别是感染后第3—5天体增重、感染后第8天血浆中CAT、GSH-Px、MDA、SOD和IFN-γ的浓度;主成分分析建立了6个单主成分和累计贡献率大于80%的综合主成分的抗性评估模型,各抗性评估模型计算的综合选择指数与15个抗性指标相关分析的结果表明,第一主成分模型和7个抗性指标相关极显著(P0.01),2个相关显著(P0.05),特别是与盲肠病变记分相关极显著(P0.01),其余6个主成分评估模型只与15个抗性指标中的2~3个相关显著或极显著(P0.05或P0.01),且与盲肠病变记分相关均不显著(P0.05);经检验第一主成分模型选择指数值的分布为正偏态分布,排序后其大小与盲肠病变表现一致。因此,第一主成分模型fi1=-0.64Zxi1+0.31Zxi2+0.80Zxi3-0.05Zxi4-0.08Zxi5+0.59Zxi6可以作为鸡柔嫩艾美尔球虫抗性选择的最佳综合选择指数用于鸡的抗病育种。     

策勒黑羊选育现状及其对策

策勒黑羊是以生产羔皮为主的多胎优良地方品种,深受当地群众的喜爱。作者从育种的角度阐述了策勒黑羊选育现状及存在的问题,提出了今后的选育方向和对策。