OBJECTIVETo set up the animal model of hemangioma by microinjecting the PyMT transgenic DNA.

METHODSConstructing the transgenic PyMT gene, and microinjecting it into fertilized embryos which were transferred to pseudopregnant recipients then. Observing the phenotype of the newborn-mice, detecting the integration of transgenic DNA by PCR, and analyzing the histological morphon of the neoplasm of the mice.

RESULTSThe transgenic DNA was proved to be right and has been microinjected into 579 fertilized embryos, 62 mice were born. Within the 62 mice, one mouse was found being the phenotype of hemangioma. PyMT gene was expressed in the total DNA of the transgenic mouse by PCR.

CONCLUSIONIt could be a good way to build animal model of hemangioma with transgenic PyMT DNA.

Animals ; Antigens, Polyomavirus Transforming ; genetics ; physiology ; Female ; Hemangioma ; etiology ; Male ; Mice ; Mice, Transgenic ; Microinjections

BACKGROUND: The association of simian virus 40 (SV40) with certain types of human cancers, including malignant lymphomas, has been a topic of interest for some time. Although the virus is distributed worldwide, its incidences vary according to the specific types of tumors, and the epidemiological areas. The aim of this study was to investigate the frequency of SV40 in malignant lymphomas among Korean patients. METHODS: One hundred seventy three cases of malignant lymphomas were evaluated by immunohistochemical staining for SV40 large T antigen (TAg), using an extremely sensitive, tyramide based, catalyzed signal amplification method. RESULTS: From 158 non-Hodgkin's lymphomas, including 115 diffuse large B-cell lymphomas, and 15 Hodgkin's lymphomas, none of the cases were positive for SV40 TAg. CONCLUSIONS: SV40 does not appear to be related to the pathogenesis of malignant lymphomas among Koreans.
Antigens, Polyomavirus Transforming ; Antigens, Viral, Tumor ; Hodgkin Disease ; Humans ; Incidence ; Korea ; Lymphoma ; Lymphoma, B-Cell ; Lymphoma, Non-Hodgkin ; Simian virus 40 ; Viruses

OBJECTIVEThe purpose of this study was to compare the morphological character between immortalized mandibular condylar chondrocyte (IMCC) and primarily cultured mandibular condylar chondrocyte (MCC).

METHODSThe phase contrast microscope, photomicroscope and transmission electron microscope were used to observe the morphological character of IMCC and MCC. The highresolution pathological image and word report system-1000 (HPIAS-1000) was used to compare the size of IMCC and MCC.

RESULTSThe phase contrast micrography showed that MCCs in primary culture underwent distinct morphological changes with respect to shape, size, and density of the cells. The majority of MCCs were in polygonal shape earlier in culture, while more fusi-form and spindle-shaped cells were found after 4-5 passages. While IMCCs were polygonal-shaped, similar to MCCs. Subculture, freezing and recovering had no effect on cellular shape of IMCC. Transmission electron microscopy indicated that MCC had chondrocyte-like phenotype, while IMCC looked like prechondroblast or immature chondrocyte. Some of IMCCs had irregular nucleus, and the proportion of nucleus/cytoplasm changed. By analysis of HPIAS-1000, the diameter and area of IMCC were obvious smaller than those of MCC (P < 0.01).

CONCLUSIONIMCC retain the main morphological character of MCC, and also keep a stable phenotype, which belong to immature chondrocytes, similar to cells in the proliferative zone.

Animals ; Animals, Newborn ; Antigens, Polyomavirus Transforming ; genetics ; Cartilage, Articular ; cytology ; virology ; Cell Line ; Cell Transformation, Viral ; Cells, Cultured ; Chondrocytes ; cytology ; ultrastructure ; virology ; Mandibular Condyle ; cytology ; virology ; Phenotype ; Rabbits

OBJECTIVETo immortalize human umbilical vein endothelial cells (HUVECs) by ectopic expression of the telomerase reverse transcriptase enzyme (hTERT), and by Simian Virus 40 Large T (SV40LT) antigen without malignant transformation.

METHODSTwo different retroviruses that contained hTERT/SV40LT cDNA fragment and drug resistance gene were constructed, and were used to transfect normal primary HUVECs. The transfected cells were screened with 500 microg/ml G418 and 4 microg/ml puromycin. Drug resistance cell clones were selected 3 days after transfection and cultured for further studies. An under inverted microscope and a scanning electron microscope were used to observe the morphology and growth of the cells. The expression of VIII factor and transfected DNA fragments were detected for identification of the endothelial origin and successful transfection. And the expression of E-selectin and endothelial lipase with or without the stimulus of TNF-alpha were also assayed to analyze the biological activity of the transfected cells.

RESULTSThe cells were homogenous, closely apposed, large, flat, and polygonal, displayed a characteristic ovoid nucleus with one or two nucleoli and formed monolayer with polygonal shape without overlapping. Immunocytochemical staining showed the existence of VIII factor. SV40LT/hTERT antigen expressed by the transfected cells was detected, while the contrasts had non-expression. Telomerase activity of the cell was detected in the transfected cells, which was 0.36 at 12 th passage and 0.38 at 50 th passage. However, the activity in the normal HUVECs was 1.12 at the first passage and 0.06 at the third passage assayed by PCR-ELISA. Both E-selectin and endothelial lipase were all specific in endothelial cells. The expressions of these two were also detected. And the expression of E-selectin can be up-regulated with the stimulus of TNF-alpha, while the expression of endothelial lipase was not unregulated significantly.

CONCLUSIONEctopic expression of hTERT and SV40LT can effectively immortalize HUVECs without tumorigenesis.

Antigens, Polyomavirus Transforming ; genetics ; Cell Line, Transformed ; Endothelial Cells ; cytology ; metabolism ; Humans ; Simian virus 40 ; immunology ; Telomerase ; genetics ; Transfection ; Umbilical Veins ; cytology

OBJECTIVE: To establish a cell lines for quality control of prenatal genetic diagnosis. METHODS: The recombined SV40LTag-pcDNA3.1(-) vector was constructed and transfected by lipidosome into human amniotic fluid cells with common aneuploidy. Positive clones were screened by G418, and the immortality of transfected cell line was identified. RESULTS: Cell line with karyotype of 46, XY, t(8;19)(q24.3;q13.1) from primary amniotic fluid cells was established. Karyotype analytical results indicated that the cell line at its 15th generation maintained the same karyotype of its primary cell. CONCLUSIONS Gene can lead to immortality of amniotic fluid cells, which contributes to preparing cell lines for internal and external quality control in prenatal genetic diagnosis.
Antigens, Polyomavirus Transforming ; genetics ; Cell Line ; Female ; Genetic Vectors ; Humans ; Karyotype ; Pregnancy ; Prenatal Diagnosis ; methods ; Quality Control ; Recombinant Proteins ; genetics ; Transfection

OBJECTIVE: To establish immortalized embryonic fibroblast lines in heat shock transcription factor 1 (HSF1) HSF1-/- and HSF1+/+ mice and to provide experimental models to study the function of HSF1. METHODS: A mammalian expression vector (pSV3neo) containing the SV40 large T antigen was used to transfect the HSF1-/- and HSF1+/+ mouse embryonic fibroblast using Lipofectamine 2000. Colonies were screened by G418 and expanded to immortalized cell lines. PCR was used to detect the integration of the large T antigen with genome in the mouse embryonic fibroblast. Expression of SV40 large T antigen gene in expanded cells was identified by RT-PCR. HSP70 expression was examined by Western blot in the embryonic fibroblast lines. RESULTS: The stable growth and serial propagation were observed in the HSF1-/- and HSF1+/+ cell lines for six months. The mRNA of SV40 T antigen gene expressed in the two cell lines. HSP70 expression could not be induced in the heat-treated HSF1-/- mouse embryo fibroblasts. CONCLUSION The immortalized cells of HSF1+/+ and HSF1-/- mouse embryo fibroblasts are successfully established.
Animals ; Antigens, Polyomavirus Transforming ; pharmacology ; Cell Line ; DNA-Binding Proteins ; genetics ; Embryo, Mammalian ; Female ; Fibroblasts ; cytology ; Heat Shock Transcription Factors ; Male ; Mice ; Mice, Knockout ; Transcription Factors ; genetics

MicroRNA (miRNA) levels in serum have recently emerged as potential novel biomarkers for various diseases. miRNAs are routinely measured by standard quantitative real-time PCR (qPCR); however, the high sensitivity of qPCR demands appropriate normalization to correct for nonbiological variation. Presently, RNU6B (U6) is used for data normalization of circulating miRNAs in many studies. However, it was suggested that serum levels of U6 themselves might differ between individuals. Therefore, no consensus has been reached on the best normalization strategy in 'circulating miRNA'. We analyzed U6 levels as well as levels of spiked-in SV40-RNA in sera of 44 healthy volunteers, 203 intensive care unit patients and 64 patients with liver fibrosis. Levels of U6 demonstrated a high variability in sera of healthy donors, patients with critical illness and liver fibrosis. This high variability could also be confirmed in sera of mice after the cecal ligation and puncture procedure. Most importantly, levels of circulating U6 were significantly upregulated in sera of patients with critical illness and sepsis compared with controls and correlated with established markers of inflammation. In patients with liver fibrosis, U6 levels were significantly downregulated. In contrast, levels of spiked-in SV40 displayed a significantly higher stability both in human cohorts (healthy, critical illness, liver fibrosis) and in mice. Thus, we conclude that U6 levels in the serum are dysregulated in a disease-specific manner. Therefore, U6 should not be used for data normalization of circulating miRNAs in inflammatory diseases and previous studies using this approach should be interpreted with caution. Further studies are warranted to identify specific regulatory processes of U6 levels in sepsis and liver fibrosis.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Animals ; Antigens, Polyomavirus Transforming/blood ; Case-Control Studies ; Down-Regulation ; Female ; Humans ; Liver Cirrhosis/*blood/diagnosis ; Male ; Mice ; Mice, Inbred C57BL ; Middle Aged ; RNA, Small Nuclear/*blood ; Reference Values ; Sepsis/*blood/diagnosis

SV40 large T antigen, a viral oncoprotein, is known to immortalize human diploid fibroblast by soaking up cellular RB and p53, but its frequency is extremely low. Additional genetic alteration is necessary for single-step immortalization. We attempted to find out what this alteration is by overexpressing cellular signal mediator genes; c-myc and cyclin D frequently amplified in many cancer cells. Overexpression of cyclin D did not affect the immortalization, but, overexpression of c-myc along with T antigen could immortalize normal human diploid fibroblast. Several cellular markers tested during immortalization process showed that p21, a cyclin-dependent kinase inhibitor and a marker of cellular senescence, disappeared in the life span-extended cells by T antigen and in the immortalized cells by c-myc. p21 was, however, elevated in the senescent cells and in the cells of crisis. Interestingly, p16 was upregulated whenever T antigen is overexpressed. Telomerase activity was also activated only in the immortalized cells. These results suggest that overexpression of c-myc contributes to immortalization of human diploid fibroblast by activating telomerase activity and suppressing p21 activity.
Antigens, Polyomavirus Transforming/genetics/*metabolism ; Biological Markers ; Cell Aging/*genetics ; Cell Transformation, Viral ; Cells, Cultured ; Cyclins/metabolism ; Diploidy ; Fibroblasts/*metabolism ; Genes, myc/*genetics ; Human ; Protein p16/metabolism ; Simian virus 40/genetics ; Support, Non-U.S. Gov't ; Telomerase/metabolism

SV40 large T antigen, a viral oncoprotein, is known to immortalize human diploid fibroblast by soaking up cellular RB and p53, but its frequency is extremely low. Additional genetic alteration is necessary for single-step immortalization. We attempted to find out what this alteration is by overexpressing cellular signal mediator genes; c-myc and cyclin D frequently amplified in many cancer cells. Overexpression of cyclin D did not affect the immortalization, but, overexpression of c-myc along with T antigen could immortalize normal human diploid fibroblast. Several cellular markers tested during immortalization process showed that p21, a cyclin-dependent kinase inhibitor and a marker of cellular senescence, disappeared in the life span-extended cells by T antigen and in the immortalized cells by c-myc. p21 was, however, elevated in the senescent cells and in the cells of crisis. Interestingly, p16 was upregulated whenever T antigen is overexpressed. Telomerase activity was also activated only in the immortalized cells. These results suggest that overexpression of c-myc contributes to immortalization of human diploid fibroblast by activating telomerase activity and suppressing p21 activity.
Antigens, Polyomavirus Transforming/genetics/*metabolism ; Biological Markers ; Cell Aging/*genetics ; Cell Transformation, Viral ; Cells, Cultured ; Cyclins/metabolism ; Diploidy ; Fibroblasts/*metabolism ; Genes, myc/*genetics ; Human ; Protein p16/metabolism ; Simian virus 40/genetics ; Support, Non-U.S. Gov't ; Telomerase/metabolism

OBJECTIVETo establish immortalized epiphysis cartilage cell strains in order to provide a stable cell resource for cell substitution and gene therapies of growth retardation.

METHODSPlasmid pEGFP-IRES2-SV40LTag containing simian virus 40 large T antigen gene was transfected into primarily cultured epiphysis cartilage cells of the newborn rat using the lipofectin transfection method. Colonies were isolated by G418 selection and cultured to immortalized cell strains. Fibroblast growth factor receptor-3 (FGFR-3), anti-collagen type II and type X antibodies were used to identify cultured cells and to investigate the capability of differentiation of the transfected cells. SV40LTag expression in expanded cell strains was identified by RT-PCR, Southern blot and immunocytochemistry method.

RESULTSAnti-G418 cell clone was obtained, which was confirmed as FGFR-3 positive epiphysis cartilage cells with the capability of stable proliferation. mRNA and protein of SV40LTag were expressed in transfected cells after stable transfection. The transfected cells were expanded to immortalized cell strains and named as immortalized epiphysis cartilage cells. The immortalized cells were elliptic or triangular, with two or three short axons. The immortalized epiphysis cartilage cell strains had stable biological characters.

CONCLUSIONSSV40LTag gene transfection can immortalize epiphysis cartilage cells. The establishment of FGFR-3 positive immortalized epiphysis cartilage cell strains may provide a stable cell resource for cell substitution and gene therapies of growth retardation.

Animals ; Antigens, Polyomavirus Transforming ; genetics ; Cartilage ; cytology ; Cell Proliferation ; Epiphyses ; cytology ; Immunohistochemistry ; Rats ; Rats, Sprague-Dawley ; Receptor, Fibroblast Growth Factor, Type 3 ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection