Establishment and characterisation of ovine blood monocyte-derived cell lines

Studies of the important functions in host defense assured by macrophages, both as functional elements and as potential targets for intracellular pathogens, are often inhibited by the lack of a source of large numbers of uniform, well-characterised cells. To address this lack for ovine studies, we have established cell lines from spontaneously-proliferating adherent mononuclear cells from sheep blood. Eight such lines which have been continuously cultured for over 400 passages have phagocytic activities and cytochemical characteristics indicating that they retain the nature of mononuclear phagocytes. They display typical functional membrane proteins such as CD14, Fc receptors and MHC class II. Such cells can facilitate in vitro studies of pathogen-monocyte interactions and can furnish copious amounts of cells for transfer experiments.     

Establishment and characterization of four canine melanoma cell lines

Four canine melanoma cell lines were established from the subcutaneous, oral gingival and mucosal melanoma tissues at the primary and metastatic sites. These cell lines were designated as CMeC-1, CMeC-2, KMeC and LMeC. The cells were spindles in shape, similar to that of primary tumor cells. The doubling times of these cells ranged from 34.1 +/- 5.61 to 57.9 +/- 3.28 hr and their chromosome number ranged from 46 to 80. When transplanted into nude mice, CMeC-1 and LMeC produced tumors, whereas CMeC-2 and KMeC did not. The morphology of the tissue formed by xenotransplantation of these cells was similar to their primary tumors.     

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 eight feline mammary adenocarcinoma cell lines

Eight new feline mammary adenocarcinoma cell lines derived from either primary or metastatic lesions were established. The morphology of all the cell lines was epithelioid and round to spindle in shape, with cell growth occurring in a monolayer fashion. On immunohistochemistry, these cells reacted with anti-keratin and anti-vimentin antisera. The doubling time of these cells was between 19 and 54 hr. Tumor masses were developed in nude mice by subcutaneous inoculation of the cells that were histologically identical to their original mammary tumor lesions. Telomerase activities measured using the telomeric repeat amplification protocol assay revealed high telemetric activity in all of the cells.     

Establishment and biological characterization of canine histiocytic sarcoma cell lines

Seven novel cell lines from canine histiocytic sarcoma (HS), three of which were disseminated cutaneous HS and four of which were synovial HS, were established. All of the established cell lines had the same morphological (by light and electron microscopic findings), cytochemical (alpha-naphthyl butyrate esterase-positive), and immunohistochemical (vimentin- and lysozyme-positive, and cyto-keratin-negative) characteristics as the original HS tumor cells. All of the established cell lines injected into nude mice subcutaneously produced solid tumors. Because the established cell lines also showed phagocytic and processing activities, the HS tumor cells appear to originate from the mononuclear phagocytic system cells, despite their differences in locations or organs.     

转录靶向猪瘟病毒3''''-UTR shRNA细胞株的初步建立

利用质粒载体在细胞内转录并加工成siRNA的方法，设计3对针对猪瘟病毒3＇-UTR不同位点的干扰片段，分别与干扰载体pGenesil-1连接，转化DH5a，筛选阳性克隆得到重组干扰质粒pGene-1，pGene-2和pGene-3，脂质体介导转染重组干扰质粒于PK-15细胞，G418抗性筛选得到转录靶向猪瘟病毒3＇-UTRshRNA的PK-15细胞株，为今后应用RNAi研究猪瘟病毒Y-UTR调控病毒复制的功能以及抑制猪瘟病毒增殖的研究奠定了基础。     

携带抗口蹄疫病毒ScFv基因猪成纤维细胞系的建立

根据口蹄疫病毒的感染特征及其在临床发病过程中呈现的症状,筛选抗口蹄疫病毒单链抗体基因。通过EcoRⅠ/NotⅠ双酶切中间质粒pGEX-ScFv获得单链抗体基因片段,并克隆至反转录病毒载体质粒pFB-neo,构建反转录病毒穿梭载体质粒pFB-ScFv。将pFB-ScFv与辅助质粒pVPack-GP、pVPack-10A1共转染HEK293T细胞,包装重组反转录病毒MMLV-ScFv。同时,构建含增强型绿色荧光蛋白基因的重组反转录病毒MMLV-EGFP作为对照。利用所包装重组反转录病毒感染猪成纤维细胞,通过G418筛选获得携带目的基因的单克隆细胞集落。试验结果表明,本试验在成功构建反转录病毒穿梭载体pFB-ScFv和pFB-EGFP的基础上,获得了分别携带抗口蹄疫病毒单链抗体基因和增强型绿色荧光蛋白基因的反转录病毒,并最终筛选出分别携带上述基因的猪成纤维细胞克隆,为抗口蹄疫病毒转基因动物的研究奠定了基础。     

Establishment of four pairs of canine mammary tumour cell lines derived from primary and metastatic origin and their E-cadherin expression

Four new pairs of canine mammary carcinoma cell lines derived from both primary and metastatic lesions were established. The cells were cultured in RPMI‐1640 with 10% fetal bovine serum and they showed stable growth for more than 120 passages. Using these cell lines, the expression of E‐cadherin was measured by flow cytometry and the function of E‐cadherin was evaluated by cell aggregation assay and results from the primary and metastatic lesions were compared statistically. E‐cadherin was strongly expressed in all of the cell lines, without a notable difference between cells of primary and metastatic origin. In the cell aggregation assay, the function of E‐cadherin was significantly weaker in the cells of primary origin (p < 0.05), as compared with cells of metastatic origin. The present results suggest that a reduction in E‐cadherin function may be implicated in the invasive and metastatic potential of canine mammary tumour cells; however, further study will be needed to clarify E‐cadherin function in the context of the metastasis of canine mammary carcinoma.     

Establishment and characterization of primary canine hepatocellular carcinoma cell lines producing alpha-fetoprotein

A primary cultured cell line named CHKS was established from a hepatocellular carcinoma (HCC) of a dog showing a high level of serum alpha-fetoprotein (AFP). CHKS secreted a 66 KDD AFP into the growth medium regardless of the presence or absence of fetal bovine serum (FBS). Cloning CHKS with limiting dilution produced 4 clones, CHKS-1, -2, -3, and -4, which secreted 826, 471, 70, and less than 10 ng/ml, respectively, of AFP into the culture medium. In culture, these cell lines were similar in morphology and proliferation pattern to epithelial cells and positive to periodic acid-Schiff (PAS) staining. The presence of mRNA for canine albumin was demonstrated by nested PCR. The doubling times of the clone cell lines were 21, 45, 36, and 35 h, saturation densities 34, 18, 22, and 24 x 10(4)/cm(2), and plating efficiencies 18, 45, 46, and 45%, respectively. Chromosome analysis of these cell lines showed near triploidy. These results show that CHKS and its clones have hepatic cell functions and are useful for carcinogenetic and clinical studies of canine HCC.     

分泌抗猪伪狂犬病病毒(北京株)单克隆抗体杂交瘤细胞株的建立

用纯化的猪伪狂犬病病毒免疫Ｂａｌｂ／ｃ小鼠,取脾细胞与ＳＰ２／０骨髓瘤细胞融合 ,经间接ＥＬＩＳＡ筛选,３次有限稀释法克隆,获得了２株能稳定分泌抗伪狂犬病病毒单克隆抗体杂交瘤细胞株１Ｈ７和３Ｂ５,经鉴定两株单抗均为ＩｇＧ１亚类、Ｋａｐｐａ型轻链,杂交瘤细胞的平均染色体数为９７,细胞培养液上清及腹水效价分别为１：１０２４、１：１０２４和１：１０＾８、１：１０＾７;１Ｈ７、３Ｂ５单克隆抗体不与猪繁殖－呼吸综合征病毒、猪温病毒、猪细小病毒发生交叉反应,显示良好的特异性,为进一步应用奠定了基础。     

稳定表达T7 RNAP的BHK-21细胞系的建立

为建立能稳定表达T7RNA聚合酶(T7RNAP)的BHK-21细胞系以研究RNA重组疫苗,本研究从BL21(DE3)大肠杆菌中扩增T7 RNAP基因,定向克隆于质粒pcDNA3.1(+)中,经双酶切及测序鉴定,获得阳性重组质粒pcDNA3.1-T7 RNAP。用该质粒转染BHK-21细胞,经G418筛选14d后获得阳性细胞株。RT-PCR和western blot检测结果表明,建立了稳定表达T7 RNAP的BHK-21细胞系,并且在不同代次的阳性细胞中能稳定表达目的基因和蛋白,该研究为RNA病毒感染性质粒在细胞内转录及病毒拯救技术平台的建立奠定了基础。     

Establishment of two vaccine-associated feline sarcoma cell lines and determination of in vitro chemosensitivity to doxorubicin and mitoxantrone

OBJECTIVE: To establish 2 vaccine-associated feline sarcoma (VAFS) cell lines and to determine their in vitro sensitivity to the chemotherapeutic agents doxorubicin and mitoxantrone. SAMPLE POPULATION: Tumor specimens collected from 2 cats undergoing surgery for removal of vaccine-associated sarcomas. PROCEDURES: Tumor specimens were minced and treated with trypsin under aseptic conditions to obtain single-cell suspensions, which were then cultured in vitro in medium supplemented with 5% heat-inactivated fetal bovine serum. Growth rates and sensitivity after 24 hours of exposure to various concentrations (0.1 to 100 microg/ml) of doxorubicin and mitoxantrone were assessed for each cell line. Survival of cells was estimated 3 days after exposure to the 2 agents, and the concentration of each drug that resulted in a 50% reduction in the number of viable cells (IC50) was calculated. RESULTS: Two tumor-derived cell lines (FSA and FSB) were successfully established and determined to be sensitive to doxorubicin and mitoxantrone. Under the conditions tested, the IC50 of doxorubicin were 0.6 and 1.5 microg/ml for cell lines FSB and FSA, respectively. The IC50 of mitoxantrone was 0.4 microg/ml for both cell lines. CONCLUSIONS AND CLINICAL RELEVANCE: The establishment of VAFS cell lines provides a tool for the in vitro screening of antitumor drugs. Doxorubicin and mitoxantrone were effective in decreasing the number of viable cells in the 2 cell lines tested. Both of these anthracycline antibiotics have been used to treat various neoplasias in cats, and their efficacy for adjuvant treatment of vaccine-associated sarcomas should be further evaluated.     

高羊茅悬浮细胞系的建立及绿色植株的高频再生

将5个高羊茅品种的成熟种子经灭菌后，接种于含有不同浓度2，4—D和ABA的N4培养基上进行愈伤组织诱导。结果表明，不同浓度2，4—D和ABA对5个品种的愈伤组织诱导率有显著影响，以培养基中附加9mg/L 2，4—D和2mg/L ABA的诱导率最高。将经过数次继代改造后获得的高质量胚性愈伤置于AA液体培养基中进行悬浮培养，建立起来自3个品种的8个悬浮细胞系，其中6个悬浮细胞系经高渗预处理后能高频再生出绿色植株，基本克服了以往研究中再生白化植株的现象。     

Establishment and characterization of dengue virus type 2 nonstructural protein 1 specific T cell lines

Immunity against dengue viruses (DENV) infection may include cellular immune responses which involve in the immunopathology of DENV infection hosts. This study was to establish short-term dengue virus type 2 (DENV2) nonstructural protein 1 (NS1) specific T cells from splenocytes from BALB/c mice immunized with DENV2 NS1 in vitro, which may be used to identify immunopathologic mechanism of dengue. Nine DENV2 NS1 specific T cell lines were successfully established by using limiting dilution methods and maintained for 20 weeks by re-stimulated with DENV2 NS1, recombinant mouse IL-2 and antigen presenting cell weekly. Phenotypically, these cells were mainly composed of CD3⁺CD4⁺ T cells. The culture supernatants of these cells contained large amounts of TNF-α and IFN-γ. Vascular tissue pathological change could be found in the mice adoptive transferred with DENV2 NS1 specific T cells. The results indicate that DENV2 NS1 specific T cells could be established and maintained with syngeneic T cell growth factors in vitro. Meanwhile, DENV2 NS1 specific T cells might contribute to the immunopathology of vascular leakage of dengue.     

Establishment and characterization of a canine sebaceous epithelial cell line derived from an eyelid mass

Little is known about the pathological roles of sebaceous glands in canine skin diseases, as most examinations have been conducted with cultured human sebaceous epithelial cell lines. To our knowledge, there is no available canine sebaceous epithelial cell line. The purpose of this study was to establish a canine sebaceous epithelial cell line and characterize it. An eyelid mass in a dog was surgically resected for treatment, and it was histologically diagnosed as sebaceous epithelioma. Collected tissue was conducted for culture, and the growing epithelial-like cells were passaged. The cells showed continuous proliferation for over 6 months. After 40 passages, the cells were named CMG-1. Lipid droplets in the cytoplasm of CMG-1 cells were confirmed by Oil Red O staining. As reported in studies with human sebaceous epithelial cell lines, lipogenesis in CMG-1 cells was promoted by linoleic acid, whereas transforming growth factor-β (TGF-β) suppressed it. Additionally, real-time PCR revealed that the expression levels of chemokines and cytokines, including CC chemokine ligand (CCL)-2, CCL-20, CXCL-10, Tumor necrosis factor-α (TNF-α), Interleukin (IL)-1α, IL-1β, and IL-8, were significantly increased in CMG-1 cells following treatment with lipopolysaccharide. In conclusion, we successfully established a new canine sebaceous epithelial cell line. Our data indicated that lipogenesis and inflammatory responses were quantitatively evaluable in this cell line. CMG-1 cells could be useful for the pathological analysis of sebaceous gland diseases in dogs.     

稳定表达犬细小病毒VP2结构蛋白的CHO-K1细胞系的建立

为构建犬细小病毒VP2基因分泌性表达细胞系,通过酶切将人CD5信号肽序列从质粒中切出,将其连接到真核表达载体pcDNA3.1A的多克隆位点上,构建成pcDNA3.1-CD5sp质粒。然后再通过PCR方法扩增犬细小病毒VP2基因,并将其插入到pcDNA3.1-CD5sp载体中CD5信号肽的下游,使其与CD5信号肽序列融合,构建成VP2基因的真核分泌型表达载体pcDNA-CD5sp-VP2。经脂质体介导转染细胞,后通过G418筛选,建立出稳定表达VP2蛋白的CHO-K1细胞系。测序结果表明,构建的犬细小病毒VP2基因的分泌型表达载体结构正确,表达载体经脂质体介导转染CHO-K1细胞,通过G418加压,筛选出稳定转染VP2基因的细胞株,经PCR检测证明VP2基因已经整合到细胞的染色体中;经RT-PCR、Westernblot分别检测VP2基因表达的mRNA和VP2蛋白,证明犬细小病毒VP2基因能够在CHO-K1细胞进行稳定性表达。这为下一步研究犬细小病毒VP2蛋白与宿主细胞的相互作用及VP2DNA疫苗奠定了基础。     

Spontaneous infection of cell lines with mycoplasmas and their detection using the fluorescence method

The continual cellular lines of bovine kidneys MDBK and AUBEK and of monkey kidneys VERO were infected spontaneously in the course of long-range cultivation by the mycoplasmas M. arginini and A. laidlawii. The mycoplasmic infection reduced significantly the growth activity of the cells and influenced negatively their morphology. The method of an indirect proof of mycoplasmas by bisbenzimid Hoechst suited well to demonstrate the infection; it had been modified to a simple and rapid test. The starting infection could be demonstrated already at the time when the amount of infected cells in the culture was relatively low.