• 생명과학회지
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• 제13권5호
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• pp.551-558
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• 2003

C형 간염바이러스는 간암을 야기하는 심각한 바이러스이다. C형 간염바이러스의 core 단백질의 과발현은 섬유아세포를 암화시키는 것으로 알려져 있다. Phospholipase D (PLD)의 효소활성이 세포증식 신호전달에 의해 활성화되어 있으며, 사람의 암조직에서 과발현 및 활성이 증가되어 있는 것으로 알려져 있다. 본 연구의 목적은, core 단백질에 의해 암화된 세포에서 PLD가 어떻게 조절되는지를 이해하고자 하는 것이다. 자극이 없는 상태에서뿐만 아니라 PMA에 의해 유도되는 PLD효소활성은, 암화된 세포에서 더 증가하였으며, control 세포와 core 단백질에 의해 암화된 세포에서 PLD와 PKC 단백질의 발현은 서로 유사하였다. PKC 특이적인 억제제와 PKC의 세포막으로의 이동에 관한 실험을 통해서, PKC-d가 암화된 세포에서 PMA에 의해 유도되는 PLD활성의 증가에 중요하게 관여하고 있음을 밝혔다. 이러한 결과는, PLD가 core 단백질에 의해 유도되는 세포의 암화과정에 관여하고 있을 것으로 추정된다.

• Reproductive and Developmental Biology
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• 제41권1호
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• pp.7-16
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• 2017

The knock-in efficiency in the fibroblast is very important to produce transgenic domestic animal using nuclear transfer. In this research, we constructed three kinds of different knock-in vectors to study the efficiency of knock-in depending on structure of knock-in vector with different size of homologous arm on the ${\beta}-casein$ gene locus in the somatic cells; DT-A_cEndo Knock-in vector, DT-A_tEndo Knock-in vector I, and DT-A_tEndo Knock-in vector II. The knock-in vector consists of 4.8 kb or 1.06 kb of 5' arm region and 1.8 kb or 0.64 kb of 3' arm region, and neomycin resistance gene(neor) as a positive selection marker gene. The cEndo Knock-in vector had 4.8 kb and 1.8 kb homologous arm. The tEndo Knock-in vector I had 1.06 kb and 0.64 kb homologous arm and tEndo Knock-in vector II had 1.06 kb and 1.8 kb homologous arm. To express endostatin gene as transgene, the F2A sequence was fused to the 5' terminal of endostatin gene and inserted into exon 7 of the ${\beta}-casein$ gene. The knock-in vector and TALEN were introduced into the bovine fibroblast by electroporation. The knock-in efficiencies of cEndo, tEndo I, and tEndo II vector were 4.6%, 2.2% and 4.8%, respectively. These results indicated that size of 3' arm in the knock-in vector is important for TALEN-mediated homologous recombination in the fibroblast. In conclusion, our knock-in system may help to create transgenic dairy cattle expressing human endostatin protein via the endogenous expression system of the bovine ${\beta}-casein$ gene in the mammary gland.

• Animal cells and systems
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• 제1권3호
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• pp.521-526
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• 1997

We isolated a mouse nck cDNA from the thymus cDNA expression library. The cDNA encodes a 377 amino acid protein and displays 97% amino acid sequence identity to human oncogenic protein nck, which is composed almost exclusivelv of three src homology 3 (SH3) domains and one SH2 domain. The sequence analysis also showed that the isolated cDNA is the mouse counterpart of the human nck and different from the mouse grb4, which has been reported to be highly similar to the human nck and, therefore considered as a mouse nck, Northern blot analysis showed that the transcript of the gene was 1.8 kb and was highly expressed in the testis, thymus, and brain but moderately in the liver and lymph node. Western blot analysis showed that the size of the protein was about 47 kDa. Overexpression of the mouse Nck transformed a mouse fibroblast cell line, NIH3T3. The results clearly indicate that normal nck gene has transforming ability and provide an argument against a suggested possibility that the transforming ability of the human nck gene is due to a mutation(s) in the gene.

• Journal of Oral Medicine and Pain
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• 제24권4호
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• pp.361-374
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• 1999

F. nucleatum is a gram-negative obligate anaerobe which is the principal and most frequent cause of gingival inflammation and is the predominant pathogen isolated in subsequent periodontal breakdown. It is also one of the most numerous bacteria found in subgingival plaque samples from healthy sites; its numbers are about 10-fold greater in plaque from periodontally diseased sites. The purpose of this study is to examine the effects of outer membrane(OM), outer membrane vesicle(OMV), and lipopolysaccharide(LPS) from F. nucleatum ATCC 25586 strain on the growth of human gingival fibroblasts and HOS 941 cells, and on the $TNF-{\alpha}$ production / $TNF-{\alpha}$ mRNA expression of mouse splenocytes. For the examination of cytotoxic effects, $TNF-{\alpha}$ production and $TNF-{\alpha}$ mRNA expression, the MTT assay, the ELISA and the RT-PCR were performed, respectively. All extracts of F. nucleatum tested were cytotoxic to both of human gingival fibroblasts and HOS 941 cells, and the significant difference of cytotoxic activity among the extracts was not observed. In the effects of these extracts on the $TNF-{\alpha}$ production / $TNF-{\alpha}$ mRNA expression of mouse splenocytes, all extracts of F. nucleatum tested also stimulated the $TNF-{\alpha}$ production / $TNF-{\alpha}$ mRNA expression, but the effects of the OM extracts on the $TNF-{\alpha}$ production / $TNF-{\alpha}$ mRNA expression were higher than those of the OMV and the LPS extracts. The pattern of the $TNF-{\alpha}$ mRNA expression was similar to that of the $TNF-{\alpha}$ production. These results indicate that F. nucleatum seems to contribute to the pathogenesis of periodontal diseases at least by its cytotoxicity, directly and its $TNF-{\alpha}$ production, indirectly.

• BMB Reports
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• 제34권2호
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• pp.176-181
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• 2001

Using a retrovirus, foreign genes can be introduced into mammalian cells. The purpose of this study is to produce a retrovirus that can make the infected cells express two genes; the human multidrug resistance gene (MDR1) and the HLA-B7 gene, which is one of the major human histocompatibility complex (MHC) class I genes. For the expression of these genes, the internal ribosome entry site (IRES) was used, which was derived from the encephalomyocarditis (EMC) virus. In order to produce retroviruses, a retroviral vector was transfected into a packaging cell line and the transfected cells were treated with vincristine, which is an anti-cancer drug and a substrate for the MDRI gene product. This study revealed that two genes were incorporated into chromosomes of selected cells and expressed in the same cells. The production of the retrovirus was confirmed by the reverse transcription (RT)-PCR of the viral RNA. The retrovirus that was produced infected mouse fibroblast cells as well as the human U937. This study showed that packaging cells produced the retroviruses, which can infect the target cells. Once the conditions for the high infectivity of retrovirus into human cells are optimized, thus virus will be used to infect hematopoietic stem cells to co-express MDRl and HLA-B7 genes, and develop the lymphocytes that can be used for the immnogene therapy.

• 대한화장품학회:학술대회논문집
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• 대한화장품학회 2003년도 IFSCC Conference Proceeding Book I
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• pp.352-373
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• 2003

Rhamnose-rich (RROP-s) and fucose-rich (FROP-s) oligo-and polysaccharides were prepared and extensively characterised by physical and chemical procedures [1,2] and compared to L-fucose. Their biological properties were then studied on human skin fibroblast cell cultures, human skin explant cultures and on hairless rat skin, using a variety of cell-biological, biochemical and computerised morphometrical procedures. Among the most important properties we could establish, the following are of particular interest for the tretment and prevention of age-dependent modifications of human skin (loss of skin-tissue, cells and matrix, wrinkle formation and others) : stimulation of cell proliferation (by $^3$[H]-thymidine incorporation and the MTT test), scavenging of reactive oxygen species (ROS) using several different procedures, and protease (MMP-2 and MMP-9) down-regulation. A topical preparation, using RROP-s and FROP-s, and/or L-fucose, was shown to increase cell proliferation, dermal matrix synthesis, efficient scavenging of ROS-s and to increase also the thickness of dermal tissue when applied for 4 weeks on hairless rat skin, accompanied by the densification of collagen bundles as well as by an increase of elastin synthesis. Using fluorescent labeled FROPs, it could be shown that these oligosaccharides react with cell-membrane receptors and especially with the elastin-laminin-receptor and the fucose-mannose receptor, but they penetrate also in the cell nucleus, suggesting the possibility of a direct action on the regulation of gene expression. When applied to the human skin of a team of voluntary women encompassing all age-groups, the efficiency of FROP-containing preparation could be confirmed using indentometry and computerised evaluation of skin micro-relief, as well as evaluation of periorbital wrinkles. It appears therefore that these preparations correspond to all the requirements of active anti-aging principles.

• Journal of Korean Dental Science
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• 제8권2호
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• pp.57-64
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• 2015

Purpose: This study was conducted to determine cell viability and differentiation capability of human periodontal ligament (PDL) cells and to elucidate the effects of cryopreservation on the activity of human third molar PDL cells by comparing PDL cells with and without cryopreservation. Materials and Methods: Human PDL fibroblasts obtained from immature third molars were cultured and divided into two groups. The experimental group was cryopreserved with a slow freezing rate of $0.5^{\circ}C/min$ from $4^{\circ}C$ to $-35^{\circ}C$ followed by plunging in liquid nitrogen at $-196^{\circ}C$ and cultured after fast thawing. The control group was cultured without cryopreservation. Cell viability, growth capacity and morphology were evaluated in both groups. Bivariate statistics were used to compare 2 groups and linear mixed model analysis was used to investigate the growth trends difference over time. Result: Cell viability and growth capacity were not significantly different between the 2 groups (P>0.05). Cultured cell of both groups showed fibroblast-like in appearance, and there were no significant differences in morphology between 2 groups. The mixed model analysis revealed no significant difference of growth capacity between 2 groups over time (${\beta}=-0.0009$; P=0.138). Conclusion: This study demonstrates that cryopreservation under control does not affect the biological properties of PDL cells, supporting the feasibility of autotransplantation of cryopreserved impacted third molars.

• 한국수정란이식학회지
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• 제28권3호
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• pp.289-295
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• 2013

Xenotransplantation of pig organs into primates results in fatal damage, referred as hyperacute rejection (HAR), and acute humoral xenograft rejection (AHXR), to the organ graft mediated by antibodies pre-existing and newly-producing in primates against their cognate pig antigens. Functional ablation of ${\alpha}1$,3-galactosyltransferase (Gal-T KO) of pig which is an enzyme involved in synthesis of Gala1-3Galb1-4GlcNAc-R antigen is essentially required to prevent HAR. Moreover, additional genetic modification under Gal-T KO background for enforced expression of human complement regulatory proteins which can inhibits complement activation is known to effectively imped HAR and AHXR. In this study, we constructed a membrane cofactor protein (MCP) expression cassette under control of human $EF1{\alpha}$ promoter. This cassette was inserted between homologous recombination regions corresponding to Gal-T locus. Subsequently this vector was introduced into ear skin fibroblasts of female pig by nucleofection. We were able to obtained 40 clones by neomycin selection and 4 clones among them were identified as clones targeted into Gal-T locus of MCP expression cassette by long-range PCR. Real time RT-PCR was shown to down-regulation of Gal-T expression. From these results, we demonstrated human $EF1{\alpha}$ promoter could induce efficient expression of MCP on cell surface of fibroblasts of female pig.

• 미생물학회지
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• 제38권4호
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• pp.255-255
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• 2002

For rapid and sensitive measurement of antiviral activities, application of a recombinant virus containing firefly luciferase gene was attempted. Recombinant human cytomegalovirus (HCMV) containing luciferase gene driven by HCMV late gene pp28 promoter (HCMV/pp28-luc) was used to test the antiviral activities of three known compounds and the result was compared with results from the conventional plaque assay for measuring the production of infectious viruses. When human fibroblast cells were infected with HCMV/pp28-luc, luciferase activity was observed at 2 days after infection and reached maximum at 6 days after infection, whereas the production of infectious virus was maximal at 4 days after infection. The antiviral activities of ganciclovir, acyclovir, and papaverine were measured in HFF cells infected with HCMV/PP28-luc and the luciferase activity was compared with the infectious virus titers. Luciferase activity decreased as the concentration of ganciclovir or papaverine increased, while there was a slight decrease in luciferase activity with acyclovir. The level of the decrease in Luciferase activity was comparable to the level of decrease in the production of infectious virus. Therefore, the antiviral assay using recombinant virus HCMV/pp28-luc resulted in sensitivity similar to the conventional plaque assay with a significant reduction in assay time.

• 대한치과보철학회지
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• 제45권3호
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• pp.375-381
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• 2007

Statement of problem. Prior to determining an optimal width of micropatterned grooves provided on titanium substrata, we have done a pilot study using surface topographies in combined microm and submicrom levels. Purpose. The purpose of this study was twofold 1) to assess the proliferation and 2) to analyze the expression of genes encoding the intracellular signaling proteins involved in cell-substratum adhesions and adhesion-dependent G1 phase cell cycle progression of human gingival fibroblasts plated on smooth and microgrooved/acid-etched titanium substrata. Material and methods. Three groups of titanium discs as NE0 (smooth Ti substrata), E15 (Ti substrata with microgrooves of $15{\mu}m$ of spacing and $3.5{\mu}m$ in depth and with further acidetching), and E30 (Ti substrata with microgrooves of $30{\mu}m$ spacing and $3.5{\mu}m$ in depth and with further acid-etching) served as the human gingival fibroblasts' substrata. Viability and proliferation of fibroblasts were determined using an XTT assay. Gene expressions of fibronectin, ${\alpha}5$ integrin, CDK4, and $p27^{kip}$ were analyzed in RT-PCR. Cell-substratum interactions were analyzed in SEM. Results. From the XTT assay at 24 h incubation, the mean optical density (OD) value of E15 was significantly greater than the values of E30 and NE0. At 48 and 96 h however, the mean OD values of E30 were significantly greater than the values of E15 and NE0. No differences in the expression of PCR transcripts at 96 h incubations were noted between groups, whereas at 48 h, an unexpected increase in the expression of all the transcripts were noted in E15 compared with other two groups. Fibroblasts were observed to orient and adhere inside the microgrooves. Conclusion. Micropatterned grooves and acid-etching on Ti substrata alter viability and gene expression of adhered human gingival fibroblasts.