ICR小鼠胚胎干细胞建系初步研究

实验旨在探讨消化方式和胚胎发育阶段对ICR小鼠胚胎干细胞(ES细胞)建系效率的影响。ICR小鼠3.5 d囊胚在饲养层上贴壁后采用单一酶消化或机械化与酶消化法相结合分离隆起的细胞集落,进行传代培养;然后选择二者中较优消化方式对不同发育时期囊胚所形成的细胞集落进行处理。结果表明:采用机械化与胰酶消化相结合的方式,形成的类ES细胞超过7代的比率(85.0%)要显著高于单一的胰酶消化(15.0%)(P<0.05);当用二者相结合的方式对ICR小鼠3.5 d(早期囊胚)、4.0 d(扩张囊胚)和4.5 d(孵化囊胚)所形成的细胞集落进行消化传代培养,三者在贴壁率和形成原代细胞集落率上均无显著差别(P>0.05),但传代超过7代的效率上早期囊胚和扩张囊胚均高于孵化囊胚(P<0.05)。结果提示,采用机械化与酶消化法相结合更适合于3.5～4.0 d ICR小鼠囊胚的ES细胞建系。     

Malignant Mesothelioma in the Thoracic Cavity of a Crj:CD(SD) Rat Characterized by Round Hyalinous Stroma

Spontaneous malignant mesothelioma was found in a 104-week-old male Crj:CD(SD) rat. The tumor was scattered on the surface of the lung, heart, mediastinal pleura and thoracic wall and metastasized to the alveolar septa. Histopathologically, small flattened or cuboidal tumor cells proliferated with stroma, formed almost normal papillary structures and reacted positively to colloidal iron stain and immunohistochemical staining for mesothelin. Round hyalinous stromata were pronounced, which is a characteristic feature, and the possible reason for this is as follows; at first, a small amount of collagen fibers was formed in the center of the clusters of several tumor cells, and then the cell clusters expanded like balloons with an increase in the collagen fibers.     

ICR小鼠胚胎干细胞的分离与培养

为了研究不同条件对ICR小鼠ES细胞的影响,试验以12.5~13.5 d ICR小鼠胚胎成纤维细胞(MEF)为饲养层,以3.5 d ICR小鼠胚胎为试验材料,探讨了血清、生长因子及传代方法对ICR小鼠ES细胞分离培养的影响。结果表明:采用含15%FBS的ES细胞培养液的囊胚贴壁率及ICM增殖率(79.3%,69.0%)均比含15%KSR、5%FBS+10%KSR的细胞培养液高(42.9%,28.6%;75.0%,54.2%),ES细胞最高传至6代;培养液中添加10 ng/mL LIF+10 ng/mL SCF的效果比单独添加1种因子的效果好,最高传至6代,高于单独添加1种因子的传代数(4代,2代);用3种传代方法进行传代时,采用差异贴壁法传代效果最佳,最高传至8代,酶消化法传至4代,机械加酶消化法传至6代。     

由体外受精囊胚获得ICR小鼠ES细胞

收集鼠卵母细胞和精子，采用体外受精技术，获得早期胚胎。体外培养胚胎至囊胚，分离内细胞团，获得ICR小鼠的ES细胞。结果不但获得了来自体外受精囊胚的ICR小鼠ES细胞，而且表明用体外受精方法获得的小鼠早期胚胎的数量明显大于常规方法，且比较稳定。ICR小鼠ES细胞的获得，为研究小鼠的ES细胞分化提供了条件。     

Spontaneous ovarian choriocarcinoma in a young ICR mouse

This paper describes the spontaneous ovarian choriocarcinoma observed in a young female Crl:CD1 (ICR) mouse. The mouse was sacrificed at 8 weeks of age after oral administration of a compound for 2 weeks. The left ovary was found to be cystically enlarged with dark red hemorrhaging. The cystic mass contained abundant blood plasma and erythrocytes. At the peripheral regions of the mass, large pleomorphic tumor cells with bizarre shaped nuclei were detected. Tumor cells contained a single large nucleus and abundant eosinophilic to amphophilic cytoplasm. Histopathology of the tumor cells resembled that of trophoblastic giant cells. Therefore, the observed ovarian lesion was diagnosed as a choriocarcinoma. No microscopic lesions were observed in the right ovary or other reproductive organs. Ovarian choriocarcinoma was considered to be of non-gestational origin. This is the first report of ovarian choriocarcinoma in a young ICR mouse.     

Spontaneous Accumulation of Globotriaosylceramide (Gb3) in Proximal Renal Tubules in an ICR Mouse

This report describes spontaneous cytoplasmic vacuolation in the proximal renal tubules of a 7-week-old male ICR [Crlj:CD1(ICR)] mouse. The contents of vacuoles were positively stained with periodic acid-Schiff (PAS) and Sudan black, and the membranes were positive on immunohistochemical staining for lysosomal-associated membrane protein-2 (LAMP-2), a marker of lysosomal membrane. Electron microscopy revealed electron-dense lamellar bodies in the proximal tubular epithelial cells. These histopathological features are similar to those in α-galactosidase A-deficient mice, in which globotriaosylceramide (Gb3), a glycosphingolipid, accumulates in lysosomes. When we performed immunohistochemical staining for Gb3, the contents of vacuoles were positively stained. From these results, spontaneous cytoplasmic vacuolation in the proximal renal tubules in the mouse was identified as lysosomal accumulation of Gb3.     

藏药绿萝花对高血糖小鼠模型的治疗试验

研究藏药绿萝花对高血糖动物模型的治疗作用,为临床合理用药和进一步开发利用提供科学依据。采用饲喂高糖高脂饲料和注射链脲佐菌素(STZ)建立ICR小鼠高血糖动物模型,将实验动物分成对照组、模型组、二甲双胍(250mg/kg)组及绿萝花提取物组(剂量分别为50、25、12.5g/kg),分别在造模试验的第14天、第21天、第28天,以及治疗试验的第14天、第21天、第28天,以空腹血清血糖含量、血清胰岛素含量为检测指标,研究造模因素和不同剂量绿萝花对血糖的影响。连续饲喂高糖高脂饲料,结合STZ注射,造模小鼠表现为体重下降、血糖升高、胰岛素含量下降;表明3个剂量绿萝花组均具有降血糖的作用,12.5g/kg剂量组连续服用绿萝花14d后降血糖作用明显,50g/kg剂量组连续口服绿萝花7d~14d,显著提高成模小鼠血清胰岛素含量。连续饲喂高糖高脂饲料(配方:蔗糖∶猪油∶奶粉∶鸡蛋∶猪胆酸盐∶普通饲料=30∶20∶4∶2∶0.05∶63)14d~28d,结合STZ注射的造模方法成功获得了Ⅱ型糖尿病动物模型;绿萝花通过降低成模小鼠血糖含量、升高成模小鼠血清胰岛素含量而对Ⅱ型糖尿病动物模型具有治疗作用。     

Malignant Peritoneal Mesothelioma with a Sarcomatoid Growth Pattern and Signet-Ring-Like Structure in a Female F344 Rat

We report a biphasic malignant mesothelioma in an aged female F344/DuCrlCrlj rat. Macroscopically, multiple pale brown nodules were observed in the abdominal cavity with retention of bloody ascites. Histopathologically, the tumor cells spread over the peritoneum and formed masses on the surface and underlying adipose tissues. The tumor cells dominantly proliferated in a solid, nodular or nest-like pattern with modest amount of fibrillar connective tissues, which contained hyaluronan. The tumor consisted of ovoid, polygonal or spindle-shaped cells that possessed eosinophilic cytoplasms including glycogen; some tumor cells showed a signet-ring-like structure. Multinucleated cells and mitosis were found frequently, and direct invasion to intra-abdominal organs and intravascular metastasis to the liver were observed. Immunohistochemically, keratin and mesothelin were strongly positive in most of tumor cells, while vimentin was mainly positive in spindle-shaped cells. Podoplanin was also positive, particularly in the cell membrane of tumor cells. Electron microscopically, tumor cells showed an intercellular desmosome-like structure, basement membrane and microvillus. We diagnosed the case as a malignant peritoneal mesothelioma with a sarcomatoid growth pattern and signet-ring-like structure.     

使用超净工作台实施子宫摘除术净化ICR小鼠的研究

为了建立SPF级远交系ICR小鼠种群。使用超净工作台实施子宫摘除术净化小鼠,结果:使用上述方法成功剖腹孕鼠20只,得到净化小鼠140只,育成小鼠120只,检测各项指标符合SPF级标准。结论:在普通环境中使用超净工作台实施子宫摘除术净化ICR小鼠,是建立SPF级种群唯一经济有效的方法。     

Mesothelioma of the tunica vaginalis testes in a stallion

Scrotal enlargement is a problem in stallions that often leads to poor semen quality. Although there are many causes for scrotal enlargement, testicular neoplasia is often high on the differential list, especially in an aged stallion. This case report describes a stallion with scrotal enlargement caused by a rare neoplasia located in the tunica vaginalis, and not in the testicular parenchyma. Diagnosis of mesothelioma was made following hemicastration. Prognosis in other species is variable, but this stallion returned to his successful breeding career following treatment and has remained healthy one year following the diagnosis.     

血清学检测的雄性特异性抗原Fab抗体的特异性鉴定

旨在研究血清学检测的雄性特异性抗原噬菌体Fab抗体的特异性。以从噬菌体Fab抗体库中筛选到具有较高雄性特异性结合活性的重组噬菌体克隆为基础,构建Fab抗体的可溶性表达噬质粒,并诱导和纯化该抗体片段,利用免疫荧光FITC/DAPI共染技术和ELISA分析其特异性。结果表明,该抗体在约49 ku处有条带出现。在Fab抗体和H-Y抗血清的细胞免疫荧光比较分析试验中发现,从雌、雄鼠脾细胞的阳性细胞数量和平均荧光强度的角度分析,Fab抗体的雄性特异性强于抗血清。石蜡切片免疫荧光定量分析显示,Fab抗体与雄鼠肝脏的结合活性明显高于对应雌鼠,差异极显著(t=20.73,P=0.002 3<0.01),而H-Y抗血清与雄鼠肝脏的结合活性略高于对应雌鼠,差异显著(t=7.11,P=0.019 2<0.05)。以C57BL/6雄鼠脾细胞、睾丸细胞作为抗原的ELISA分析显示,Fab抗体具有雄性特异性,但Fab抗体的OD值低于抗血清。结果提示,Fab抗体的雄性特异性高于H-Y抗血清,但其雄性特异性结合活性低于H-Y抗血清,Fab抗体在具备雄性特异性的同时,仍有少量雌性非特异性结合,Fab抗体的体外亲和力成熟可作为后续研究工作,以获...     

鼠源性抗雄性特异性抗原噬菌体Fab抗体库的构建

利用雄鼠脾细胞免疫6~7周龄C57BL/6雌鼠,加强免疫后取脾细胞,TRIZOL法提取细胞总RNA,并逆转录合成cDNA第一条链。以其为模板,利用特异性Ig基因的引物PCR扩增出重链Fd片段和κ链基因,并将扩增出基因重组到噬质粒载体pComb3中,重组噬质粒转化大肠杆菌XL1-Blue中,分别构建κ链基因库和抗体Fab段基因库。分别经蓝白斑筛选,计算转化效率,通过PCR反应和双酶切反应鉴定抗体库的重组率,轻链、重链Fd段基因的重组率均为80%。抗体Fab段基因库经过辅助噬菌体VCSM13超感染,成功构建了鼠源性噬菌体Fab抗体库,并测定噬菌体抗体库的库容和滴度,结果分别为1.5×107,3.2×1011pfu/mL。对噬菌体抗体库的18个克隆进行ELISA分析,结果显示以雄鼠脾细胞作为抗原时,有9个克隆含有雄性特异性的噬菌体抗体,而以雄鼠睾丸细胞作为抗原时,有8个克隆与睾丸细胞呈现结合活性。噬菌体抗体库的构建为雄性特异性抗原的鼠源性噬菌体抗体Fab片段的筛选及其应用、雄性特异性抗原和抗体功能性分子基础的研究奠定了坚实基础。     

Spontaneous extraskeletal osteosarcoma with various histological growth patterns in the abdominal wall of an ICR mouse

Extraskeletal osteosarcoma is extremely rare in mice. This case report demonstrates a spontaneous murine extraskeletal osteosarcoma that exhibited various histological growth patterns in an ICR mouse. At necropsy, the tumor mass was located in the abdominal wall and was 45 × 30 × 25 mm in size. Histopathologically, the tumor showed the following four growth patterns: a solid pattern of polygonal cells embedded in an osteoid eosinophilic matrix with calcification, an irregular sheet pattern of short spindle cells accompanying some eosinophilic multinucleated cells, a fascicular pattern of spindle cells and a cystic pattern lined by short spindle cells. Immunohistochemically, most of the tumor cells were positive for vimentin, proliferating cell nuclear antigen and osterix. The multinucleated cells mentioned above were desmin positive and were regarded as regenerative striated muscles but not tumor cells. Since no clear continuity with normal bone tissues was observed, the tumor was diagnosed as an “extraskeletal osteosarcoma.”     

猪瘟与猪圆环病毒2型混合感染检测方法的建立与初步应用

根据GenBank上发表的猪瘟（classical swine fever virus,CSFV）和猪圆环病毒2型（porcine circovirus type2,PCV2）的核苷酸序列,应用NCBI/Primer-Blast设计合成分别针对CSFV和PCV2的1对特异性引物,建立了分别用于检测CSFV和PCV2的RT-PCR和PCR方法,并对2008年4月～8月采自昆明8个可疑发病猪场的19份病料进行了CSFV和PCV2检测。结果显示,来自3个猪场的4份检测样品为CSFV阳性,猪场的CSFV阳性率为21.05%;来自2个猪场的2份检测样品为PCV2阳性,猪场的PCV2阳性率为10.53%。CSFV和PCV2混合感染率为10.53%。表明所建立的CSFV和PCV2混合感染检测方法具有特异性,可用于CSFV和PCV2混合感染的辅助诊断和流行病学调查,同时也为CSFV和PCV2混合感染的防制提供科学的依据。     

Inflammation of the cardiac coronary artery in ICR mice

Inflammation of the cardiac coronary artery in ICR mice is occasionally observed in toxicity studies; however, this has not been well explored histologically. Herein, we investigated the detailed histology of the associated lesions in 6–8-week-old ICR mice. Coronary artery inflammation in the right ventricular wall was observed in 10 of 142 mice (7.0%). Histopathological examination revealed hypertrophy of the vascular smooth muscle cells and perivascular infiltration of macrophages in mild cases. In moderate to marked cases, single-cell necrosis of vascular smooth muscle cells, hemorrhage of the tunica media, and fibrinoid necrosis of the vessel wall were observed, in addition to the changes seen in mild cases. Electron microscopic examination of moderate cases revealed a discontinuous internal elastic lamina suggestive of rupture, and vascular smooth muscle cells beneath the elastic lamina showed degeneration and necrosis. These findings suggest that the lesions developed as a rupture of the internal elastic lamina and necrosis of vascular smooth muscle cells, while leaked plasma components caused vascular and perivascular inflammation. In ICR mice, dystrophic calcinosis (DCC) is known to occur rarely in the right ventricle. DCC is defined as focal calcification in necrotic myocardial fibers, the pathogenesis of which is considered to involve ectopic calcification. Since calcification was not observed in any part of the heart, including the inflammation region, the pathophysiology of cardiac arterial inflammation seen in our ICR mice was considered to differ from that of DCC.     

口蹄疫A型油佐剂和leo佐剂弱毒灭活疫苗免疫持续期的研究

用实验室配制的口蹄疫油佐剂和leo佐剂A型弱毒灭活疫苗各免疫6月龄健康敏感牛（乳鼠中和抗体滴度小于1：4）5头，免疫后每月采血一次，分离血清；采用固定病毒稀释血清的方法，通过乳鼠中和试验检测免疫牛血清中的口蹄疫A型中和抗体的产生与消长情况，用karber法计算中和效价，绘制动态曲线图。结果显示，口蹄疫弱毒灭活疫苗免疫牛，可以产生高滴度的血清中和抗体，免疫牛血清中和抗体效价均在免疫后1个月左右达到高峰。油佐剂疫苗免疫的牛，血清中和抗体效价比较高，但下降比较迅速；leo佐剂疫苗免疫的牛，血清中和抗体效价比较低，但下降比较平缓。     

藏系绵羊公羔大肠杆菌和球虫混合感染的诊治

因试验需要,从西藏山南地区措美县购买了41只实验用藏系绵羊公羔,抵达拉萨1 d后羔羊陆续发生腹泻,排黑色恶臭粥样粪便,个别羔羊排血便,病情严重并有死亡情况发生。经临床诊断、剖检及实验室检查,确诊为大肠杆菌和球虫混合感染。同时,对分离培养的大肠杆菌进行药物敏感试验,结果发现,其对庆大霉素、阿米卡星、先锋霉素Ⅴ、头孢他啶4种药物敏感。以抗菌消炎、补充体液、防止电解质紊乱、防止自体酸中毒、抗休克为治疗原则,采取一系列综合治疗措施,2周后病羊痊愈。     

猪圆环病毒2型和猪链球菌混合感染的诊治

2009年6月,河南省许昌某猪场出现猪圆环病毒2型和猪链球菌混合感染的疑似病例,临床主要表现为体温升高、呼吸困难、体表瘀血等症状;剖检变化为淋巴结水肿、脑膜炎和关节炎、急性炎性脾肿等。笔者通过对其临床症状、剖检变化和实验室检测结果进行综合分析,确诊为猪圆环病毒2型和猪链球菌的混合感染。笔者对防制措施也进行了初步阐述,希望为今后该类疾病感染的诊断与治疗提供借鉴。     

小鼠体内雌二醇残留量测定方法研究

建立了反相高效液相色谱法测定小鼠体内雌二醇含量，能快速、简便、灵敏地测定小鼠肌肉、肾脏及肝脏中的雌二醇残留。本方法最低检出量为0.1 μg /kg，雌二醇浓度在1.00～10.00 μg/ ml范围内具有良好的线性关系，RSD为2.17%。