Immunohistochemical characterization of hepatoblastomas in B6C3F1 mice treated with diethylnitrosamine and sodium phenobarbital.

Hepatoblastomas (HBs) were induced in B6C3F1 male mice by diethylnitrosamine (DEN) and sodium phenobarbital (PB). Six-week-old mice received a single intraperitoneal dose of DEN followed by a continuous treatment with PB in diet at a concentration of 0 (group 1) or 500 (group 2) ppm for 50 weeks. HBs were observed in 13 of 21 (62%) group 2 mice, with typical histologic features as reported previously, while no such tumors were observed in group 1. Seven of 13 (54%) HBs were found in and/or adjacent to hepatocellular adenomas (HCAs) or hepatocellular carcinomas (HCCs). Immunohistochemically, all HBs were positive for S-100 protein but negative for keratin, alpha-fetoprotein (AFP), albumin (ALB) and vimentin, while HCC cells occasionally reacted positively for AFP with a mosaic pattern. HCC and HCA cells were occasionally positive for ALB. Non-neoplastic hepatocytes and normal bile ducts were positively stained for ALB and keratin/S-100 protein, respectively. S-100 protein is known to be expressed in many mesenchymal tissues and neoplasms including neuroectodermal elements but negative in cells of the hepatic lineage. Thus, the present immunohistochemical results suggested that mesenchymal differentiation occurs in mouse HB cells as observed in human HBs, one of the most frequent infant liver tumors in humans. Although the susceptibility of mouse HBs to PB-promotion suggests a hepatocytic histogenesis, the present immunohistochemical results support the hypothesis that the mouse HB is derived from pluripotent endodermal stem-like cells.     

Natural infection of nude mice with low-virulent mouse coronavirus

The outbreak of wasting disease of nude mice occurred in the laboratory colony of a Pharmaceutical Company. The viruses producing cytopathic effect with syncytium formation were isolated from the wasted nude mice by DBT cells, and were identified as mouse coronavirus by direct immunofluorescence. The nude mouse colony was closed and all the nude mice (about 500) were killed by the reason of disease control. At autopsy about 60% of nude mice showed necrotic hepatitis. By the virus isolation to see the source of contamination, viruses were isolated from the feces of apparently healthy mice of ICR, CDF1, DBA/2 and C3H, and from human cancer cell line stocked in liquid nitrogen. In experimental infection, the isolates produced only mild hepatitis in ICR mice treated with cortisone. By cross-neutralization test, the nude isolate reacted closely with the virus from C3H mice but not with the virus from cancer cell line. The isolates from nude and C3H mice produced experimentally wasting disease with necrotic hepatitis in nude mice. These findings suggest that wasting disease in nude mice might be caused by low-virulent mouse coronavirus shed in feces from C3H mice introduced before the outbreak of disease.     

Biological effects of leptospiral lipopolysaccharide to mouse B, T and NK cells

Leptospiral lipopolysaccharides (LPSs) extracted from Leptospira interrogans serovars copenhageni and hebdomadis were tested for the biological effect to mouse B, T and NK cells. Each leptospiral LPS was a potent mitogen for spleen B cells. Activation of the cells was also expressed by polyclonal B cell activation. In contrast, mitogenicity for T cells, induction of interleukin-2 (IL-2) secretion in T cells and increase of tumor-killing activity and chemiluminescence in NK cells were not observed after stimulation with leptospiral LPS. After intravenous injection of leptospiral LPS in mice, the spleen and lymphnodes were examined by histocytochemical technique. Increase of Ig-bearing lymphocytes was recognized while decrease of T cells was observed in the lymphoid organs. Mitogenic response to PHA, Con A and PWM decreased with relation to the T cell depletion. In conclusion, it is apparent that leptospiral LPS possess marked immunological potencies on B cells but not T and NK cells. The biological effects of leptospiral LPS were common ones as LPS but the level was considered to be different from classical LPS such as Escherichia coli LPS.     

Selection of foot-and-mouth disease viruses by passage in bovine and swine kidney cell cultures

Two uncloned populations of foot-and-mouth disease virus, one pathogenic for adult mice and the other nonpathogenic, were passaged in cultures of primary bovine kidney (BK) cells and a line of pig kidney (MVPK) cells. Within 10 passages in MVPK cells, the nonpathogenic virus became pathogenic for adult mice, but similar passages of this virus in BK cells did not affect its pathogenicity. In contrast, passage of the pathogenic virus in MVPK cells resulted in a decrease in pathogenicity, but again passage in BK cells had no effect on this characteristics. Neither of the viruses changed in pathogenicity for infant mice during the four passages that were tested. The nonpathogenic virus passaged in BK cells was more infectious for BK than for MVPK cells, but after passage in MVPK cells, this virus was about equally infectious for the two types of cells. Infectivity of the pathogenic virus was relatively unchanged by passage in either type of cell. The parent nonpathogenic virus and the 10th BK cell passage of this virus were much more resistant to adsorption with homogenized mouse kidney than were the MVPK cell passages of nonpathogenic virus. The parent and passaged pathogenic viruses were readily adsorbed. The results demonstrated that passage of the two viruses in MVPK cells had a pronounced selective effect.     

Evaluation of immunogenicity and protective efficacy of Actinobacillus pleuropneumoniae HB04C(-) mutant lacking a drug resistance marker in the pigs

Previously, we reported the construction and characterization of a genetically defined Actinobacillus pleuropneumoniae (A. pleuropneumoniae) apxIIC gene mutant, HB04C(-), which conferred protection to mice against infection with A. pleuropneumoniae. In this study, we further evaluated HB04C(-) for safety and its ability to elicit protective immunity in pigs. It was demonstrated that a dose of 2 x 10(8) CFU HB04C(-) was safe to the pigs via intranasal or intramuscular injection. Immunization with a dose of 2 x 10(8) HB04C(-) by both intranasal and intramuscular routine could yield equal protective efficacy and elicited significant protection against experiment challenge with homologous or heterologous serotypes of a virulent A. pleuropneumonia. Taken together, HB04C(-) might serve as a promising vaccine candidate against infection with A. pleuropneumoniae.     

一种犬细小病毒基因工程抗体的制备

本研究旨在利用HEK-293细胞系制备鼠源犬细小病毒（canine parovirus，CPV）基因工程抗体并检测其生物活性。通过抗体亚型检测试剂盒检测CPV单克隆抗体亚型；采用间接ELISA检测CPV单克隆抗体的亲和力和特异性；经RACE-PCR获得CPV单克隆抗体的可变区序列，将可变区序列与鼠源抗体恒定区序列连接；分别构建真核表达载体pcDNA3.1（+）-L和pcDNA3.1（+）-H，将载体共转染HEK-293细胞，采用血凝抑制与中和试验的方法检测鼠源CPV基因工程抗体生物活性；采用HEK-293F细胞悬浮表达并用间接ELISA方法检测鼠源CPV基因工程抗体的表达量；用Protein A亲和层析柱纯化鼠源CPV基因工程抗体后进行SDS-PAGE鉴定；间接免疫荧光检测纯化后鼠源CPV基因工程抗体的活性。结果显示，CPV单克隆抗体亚型为IgG_2b，亲和力常数6个Ka平均值为1.02×10¹¹ L/mol，只与CPV VLPs发生反应。琼脂糖凝胶电泳结果显示，试验成功构建真核表达载体pcDNA3.1（+）-L和pcDNA3.1（+）-H；HEK-293和HEK-293F细胞培养上清液血抑效价分别为1∶2⁴和1∶2⁶，中和试验结果显示，HEK-293和HEK-293F细胞培养上清液中和效价分别为1∶152和1∶1 290；鼠源CPV基因工程抗体在HEK-293F细胞中的表达量为5.97 mg/L，SDS-PAGE分析在55和25 ku处出现条带，表明鼠源CPV基因工程抗体成功在HEK-293F细胞中表达并纯化。间接免疫荧光检测结果表明，纯化后鼠源CPV基因工程抗体具有良好的生物活性。本研究在HEK-293F细胞中成功表达具有中和活性、纯度较高的鼠源CPV基因工程抗体，为今后CPV基因工程抗体药物的研发奠定基础。     

转肌细胞特异表达MSTN基因RNA干涉序列的小鼠成纤维细胞株的筛选

为了筛选肌细胞特异表达MSTN基因RNA干涉序列的小鼠成纤维细胞株,研究利用人工合成的小鼠肌肉启动子SP,连接前期工作中获得带有GFP基因的小鼠MSTN基因RNA干涉载体pR-NAT-M1,构建真核表达载体pRNAT-SP-M1,将pRNAT-SP-M1线性化后转染小鼠成纤维细胞并通过300μg/mL G418筛选获得转基因阳性细胞。结果表明:获得的转基因阳性细胞呈现正常的小鼠成纤维细胞的梭形,且在荧光显微镜下呈现出表达绿色荧光蛋白的绿色;转基因阳性细胞的生长曲线呈"S"形;转基因阳性细胞经冷冻复苏后,仍具有正常的细胞形态和增殖特性;PCR鉴定结果显示转基因阳性细胞基因组DNA中整合有pRNAT-SP-M1序列。说明成功获得了转肌细胞特异表达的MSTN基因RNA干涉序列小鼠成纤维细胞株。     

Bovine herpesvirus-1 (BHV-1) recombinant expressing pseudorabies virus (PrV) glycoproteins B and C induces type 1 immune response in BALB/c mice

Bovine herpesvirus 1 (BHV-1) attached poorly and penetrated into a mouse cell line, BALB 3T3/A31, but a recombinant BHV-1/TF7-6, which expresses pseudorabies virus (PrV) gB and gC genes, did attach and penetrated into cells more efficiently. In this study the gene green fluorescent protein (GFP) has been integrated into genome of BHV-1/TF7-6 and its parental line of BHV-1. When the mouse mesenteries were incubated in vitro and infected with BHV-1/TF7-6/GFP, strong fluorescence was observed while BHV-1/GFP infection hardly demonstrated fluorescence, suggesting that BHV-1 recombinant expressing PrV gB and gC can infect mouse tissue cells more efficiently than the parental BHV-1 does. When BALB/c mice were inoculated with purified BHV-1/TF7-6 or its parental BHV-1, the former induced lower level of anti-BHV-1 immunoglobulin G (IgG) than the latter did. When sub-classes of anti-BHV-1 IgG were analyzed, it was found that mice immunized with BHV-1/TF7-6 or the parental BHV-1 demonstrated the same level of IgG2a. Since anti-BHV-1 IgG1 level was lower in mice inoculated with BHV-1/TF7-6, the IgG2a:IgG1 ratio was higher in BHV-1/TF7-6 inoculated mice than in the parental BHV-1 inoculated ones. These results indicate that BHV-1/TF7-6 induces type 1 predominant immune to BALB/c mice.     

Biological potentials for a family of disintegrin and metalloproteinase (ADAMDEC)-1 in mouse normal pregnancy

Our previous research has indicated local expression of ADAMDEC-1, a family of disintegrin and metalloproteinase, was confirmed in the mouse placentas and enhancement was found in the sites for spontaneous abortion. Present study was aimed to identify biological effects of ADAMDEC-1 in pregnancy process. Syngeneic pairs of C57BL/6J mice and heterogenic mating pairs of CBA/J and DBA/2 mice were used. Pregnant mice were treated with recombinant ADAMDEC-1 protein. Vasculogenesis effects was evaluated using the Matrigel plugs including vascular endothelial growth factor singularity or combination with ADAMDEC-1. ADAMDEC-1 single effects were evaluated by tubal formation and proliferation assays using HuEht-1 endothelial cells. Expression of ADAMDEC-1 was not exactly corresponded with the time periods for miscarriage initiation. ADAMDEC-1 was distributed in normal placentas and fetuses, especially at extraembryonic ectoderm, decidua cells, uterine natural killer (uNK) cells in decidua, trophoblasts in labyrinthine zone, and hematopoietic cells in umbilical blood and fetal liver. ADAMDEC-1 treatment did not affect reproductive performances, while it elevated uNK cell recruitment in placenta and enlarged lumen sizes of the intraplacental vessels. In vitro analysis also indicated ADAMDEC-1 promoting effect on tubal formation and cell length of HuEht-1. qPCR analysis showed that ADAMDEC-1 modified placental gene expression especially for linkage of actin filament rearrangement. Our findings suggested that ADAMDEC-1 is correlated on cell shape, stability, and movement via modification of actin cytoskeleton. ADMADEC-1 suspected to regulate cellular activity of endothelial cells, trophoblasts, and uNK cells and may support normal developing of mouse placentas.     

马身猪耳缘组织成纤维细胞的体外培养与鉴定

为研究马身猪体细胞的生物学特性,试验以3日龄马身猪耳缘组织为材料,采用组织块贴壁法建立马身猪耳缘组织成纤维细胞系,并对体外获得的细胞系进行生物学特性检测。结果表明,获得的马身猪耳缘组织成纤维细胞系符合成纤维细胞的基本特征,细胞贴壁生长,呈典型的梭形、星形和多边形,细胞汇合成单层的时间为10~13 d,生长曲线呈S型,中期染色体二倍体（2n=38）占主体,约占细胞总数的84%,符合细胞建系的要求;脂质体（Lipofectamine^TM 2000）转染绿色荧光蛋白质粒（pEGFP-C1）的转染效率为25%。马身猪耳缘组织成纤维细胞系的成功建立为地方猪种遗传资源的保护开辟了新的方法。     

Mice cloned by nuclear transfer from somatic and ntES cells derived from the same individuals

The current success rate of cloned mice from adult somatic cell nuclei is very low, whereas it is relatively high for cloned mice from ES cell nuclei. In this experiment, we examined whether the success rate of cloning from somatic cells could be improved via nuclear transfer embryonic stem cells (ntES cells) established from somatic cell nuclei. We obtained 11 cloned mice and 68 ntES cell lines from the somatic cell nuclei of 7 mice, and cloned 41 mice were cloned from the ntES cell nuclei. Unexpectedly, the overall success rate of cloning from ntES cell nuclei in this series was no better than when using somatic cell nuclei. Interestingly, full-term cloned mice were produced only via ntES cells from two individuals, but not by direct nuclear transfer from the somatic cells, and vice versa. Ultimately, we were able to obtain clone mice from 6 out of 7 individuals using either somatic cells or ntES cells. Thus, although ntES cells as donor nuclei do not absolutely assure a better success rate for mouse cloning than somatic cells, to preserve and clone valuable individuals, we recommend that ntES cell lines be established. These can then be used as an unlimited source of donor nuclei for nuclear transfer, and thus complement conventional somatic cell nuclear transfer cloning approaches.     

Monoclonal antibodies directed towards the two major cell populations in the bursa of Fabricius of the chicken

Two mouse monoclonal IgM antibodies, B.1 and B.2, have been produced using the mouse myeloma cell line Sp2/0-Ag 14 and spleen cells from mice immunized with chicken bursa cells. The binding of the monoclonal antibodies to cells in suspension or tissue sections was demonstrated by means of the unlabeled peroxidase-antiperoxidase method. B.1 recognizes 61% of the bursa cells, 10-14% of the cells of spleen and of the peripheral mononuclear blood leukocytes and 1% of the thymus cells. The B.1+ cells are regarded as B cells. Their location in tissue sections corresponds with the known B-dependent areas of lymphoid organs. Competitive binding and double marker experiments proved that the B.1 antigen is distinct from surface immunoglobulin (Ig). In the bursa all B.1+ cells are also Ig+, whereas in the thymus, spleen and blood only about 90% of the B.1+ cells show this conformity. B.2 mainly recognizes so called reticular epithelial and reticular cells of the bursa (36%), thymus (20%) and spleen (13%). The B.2+ cells represent the second major cell population of the bursa.     

In vitro Culture and Identification of Ear Marginal Fibroblast Cell Lines Derived from Mashen Pigs

In order to study the biological characteristics of somatic cells of Mashen pig, the ear marginal fibroblast cell lines of Mashen pig were established from 3 days old Mashen pig by direct explant method,and the biological characteristics of cell lines were detected. The results showed that the cell lines obtained from Mashen pig were in conformity with basic properties of typical fibroblasts, the shape of adherent cells were typical spindle, star and polygon, the time of grew into monolayer cells was 10 to 13 d, the growth curve was typical S shaped. The frequency of metaphase chromosome number (2n=38) was about 84% of the total number of cells, and it had met the establishment standard of fibroblast cell line. The transfection efficiency of green fluorescent protein(pEGFP-C1)transfected by lipofectamine-mediated method was 25%. The successful establishment of the fibroblast cell lines of Mashen pig had opened up a new method for the conservation of genetic resources.     

鸡CD4基因片段原核表达及其单抗制备

将PCR扩增的鸡CD4基因片段克隆到pET-32a原核表达载体,构建获得原核表达重组载体pET-32a-CD4.重组载体导入大肠杆菌BL21(DE3),经过IPTG诱导表达,Ni-NTA亲和树脂纯化获得融合蛋白His-CD4.以此融合蛋白为抗原免疫BALB/c小鼠,用杂交瘤细胞技术制备了1株能够稳定传代并分泌抗CD4多肽单抗的杂交瘤细胞株,命名为3E12.经检测该抗体亚类为IgG2b,单抗腹水的间接ELISA效价为1:105,Western blot结果显示单抗与CD4多肽能特异性地结合.     

Establishment of a canine lens epithelial cell line

Some immortalized lens epithelial cell lines have been established and are useful for molecular analysis. The establishment of additional cell lines must, however, enable a variety of in-vitro examinations. The objective of this study was to establish a new canine lens epithelial cell line by isolating CLC-1 cells from the lens tissue of a dog with cataracts. In CLC-1 cells, transforming growth factor beta (TGF-β) treatment significantly decreased gene expression of an epithelial marker and elevated that of mesenchymal markers; these characteristics are similar to those of a human lens epithelial cell line. Interestingly, CLC-1 cells exhibited lower expression of an epithelial marker and higher expression of mesenchymal markers than an anterior lens capsule. These results suggest that CLC-1 cells were derived from a cell population that was committed to epithelial-mesenchymal transition in cataract lens tissue. In conclusion, CLC-1 cells could be useful for analyzing molecular pathogenesis in canine cataracts.     

Establishment and characterization of a cell line, MCO-Y4, derived from canine mammary gland osteosarcoma

A cell line, MCO-Y4, was established from a mammary gland osteosarcoma of a 16-year-old female mongrel dog. Histopathologically the tumor was composed of osteoblastic cells with an osteoid meshwork and chondroid matrix. The mean doubling time of the cells at the 93rd passage was 32.39+/-4.66 hr. Immunohistochemically, the osteoblastic and chondroblastic cells were positive for bone morphogenetic protein (BMP)-2/4 and BMP receptor (BMPR) II. The cultured cells were spindle in shape during the growth and the confluent phases. No tumor matrix was detected in the culture dish by alcian blue staining or von-Kossa silver impregnation. MCO-Y4 cells on the chamber slides showed intense immunoreactivity for BMP-2/4 and BMPR II. Noggin, an antagonist for BMP-2/4, showed the growth inhibition on MCO-Y4 cells. In addition, fibronectin might be potential for stimulating growth of MCO-Y4 cells. When transplanted into severe combined immunodeficiency mice, the cells formed tumors consisting of solid proliferation of osteoblastic and fibroblastic cells with woven-bone trabeculae. These tumor cells were intensely positive for BMP-2/4 and BMPR II. Our results suggested that the cell line might be useful for studying the role of BMPs in canine osteosarcoma and the mechanism of ossification.     

Establishment and characterization of a thymidine kinase deficient avian fibroblast cell line derived from a Japanese quail cell line, QT35.

An avian thymidine kinase deficient (TK-) fibroblast cell line (QTTK-) was established from a Japanese quail cell line, QT35, and characterized the biological properties. QTTK- could grow in the presence of 5-bromo-2'-deoxyuridine (BUdR, 100 micrograms/ml) and not in the growth medium with hypoxanthine-aminopterin-thymidine. Compared to QT35 cells, the 3H-thymidine incorporation of the QTTK- cells and the TK activity of the cell extract significantly decreased to 0.3% and 0.5%, respectively. In the thymidylate synthetase activity, the karyotype, and the sensitivities to either fowlpox virus (FPV) or herpesvirus of turkeys (HVT), OTTK- cells were similar to the parental QT35 cells. Since QTTK- cells were permissive to FPV and HVT infections and these viruses could not grow in the presence of 50 to 75 micrograms/ml of BUdR, QTTK- cells may be useful for the construction of recombinant FPV and HVT that have foreign genes within the TK gene of the virus genome.     

Establishment of Vero Cell Line Expressing Goat SLAM Receptor Stably

Signalling lymphocyte activation molecule (SLAM,also called CD150) serves as a main cell receptor for PPRV (peste des petits ruminants virus).This study was aimed to establish a cell line,using Vero cells as the parental cell,to express goat SLAM stably,which could be used to isolate and propagate PPRV.The gene encoding goat SLAM in vitro was synthesized and cloned into eukaryotic expression vector pIRES2-GFP,and the recombinant expression plasmid pIRES2-gSLAM was obtained.The positive stably transfectant Vero-gSLAM cells were screened by G418 and identified by immunofluorescence(IF) and RT-PCR.The result of virus titration by Vero-gSLAM cell line showed that PPRV strain N75/1 had a titre of 10^-4.65 TCID₅₀ per 0.1 mL in Vero cell at 5 day after infection and the titre of PPRV N75/1 strain was 10^-5.75 TCID₅₀ per 0.1 mL in Vero-gSLAM cells.The cell line would play an active role in virus isolation,biological characteristics study and vaccine virus production of PPRV.     

稳定表达山羊SLAM受体的Vero细胞系的建立

信号淋巴激活分子（signalling lymphocyte activation molecule, SLAM）又称CD150,是小反刍兽疫病毒（peste des petits ruminants virus, PPRV）和犬瘟热病毒（canine distemper virus, CDV）等麻疹病毒属病毒感染淋巴细胞的主要受体,在病毒侵入细胞中发挥着重要作用。为了建立稳定表达山羊SLAM真核细胞系,本研究将全基因合成的gSLAM基因克隆至真核表达质粒pIRES2-GFP中,构建了重组质粒pIRES2-gSLAM。将该重组质粒转染非洲绿猴肾细胞（Vero）,经G418筛选后,筛选到稳定表达gSLAM基因的细胞系Vero-gSLAM,该细胞系在传代至第10代,仍能稳定表达gSLAM基因,PPRV N75/1病毒株可以感染且能形成明显的细胞病变（CPE）,相比在Vero细胞上10^-4.65 TCID₅₀/0.1 mL的毒价,在Vero-gSLAM上为10^-5.75 TCID₅₀/0.1 mL,其毒价有所提高。该细胞系可用于PPRV强毒分离和致弱机制等相关研究。