• 한국산학기술학회논문지
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• 제16권1호
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• pp.365-370
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• 2015

GM-CSF는 중요한 조혈모세포 성장인자로서 면역요법에서 중요한 기능을 한다. 본 연구의 목적은 GM-CSF가 돼지 처녀생식배아의 발달과 세포 수 및 착상관련 유전자의 발현에 관한 영향을 평가하는 것이다. 본 연구에서 돼지 처녀 활성화 배아는 GM-CSF가 5, 10, 20 ng/ml 존재 하에서 7일 동안 배양하여 배 반포의 형성율과 전 세포 수 그리고 유전자 발현을 평가하였다. 그 결과 단백질이 없는 배양액에 20 ng/ml의 GM-CSF를 첨가 하였을 때 배 반포의 형성 율이 유의적으로 증가하였으며 배반포의 세포 수 또한 GM-CSF 를 첨가한 배양액에서 증가 하였다. GM-CSF는 처녀생식 배 반포에서 interleukin-6의 mRNA 발현을 증가 시켰으나, LIF 수용체 mRNA 발현에는 영향을 주지 않는다는 것을 real time RT-PCR로 밝혀내었다. 이 결과로 GM-CSF 성분이 확인된 배양액에서 돼지 배아의 체외 발달 과 생존력을 강화 시켰음을 시사하고 있다.

• 한국발생생물학회지:발생과생식
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• 제22권3호
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• pp.235-244
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• 2018

We investigate the effect of endoplasmic reticulum (ER) stress inhibitor treatment during parthenogenetic activation of oocytes on the ER stress generation, apoptosis, and in vitro development of parthenogenetic porcine embryos. Porcine in vitro matured oocytes were activated by 1) electric stimulus (E) or 2) $E+10{\mu}M$ Ca-ionophore (A23187) treatment (EC). Oocytes were then treated by ER stress inhibitors such as salubrinal (200 nM) and tauroursodeoxychloic acid (TUDCA, $100{\mu}M$) for 3 h prior to in vitro culture. Parthenogenetic embryos were sampled to analyze ER stress and apoptosis at the 1-cell and blastocyst stages. The x-box binding protein 1 (Xbp1) mRNA and ER stress-associated genes were analyzed by RT-PCR or RT-qPCR. Apoptotic gene expression was analyzed by RT-PCR. At the 1-cell stage, although no difference was observed in Xbp1 splicing among treatments, BiP transcription level in the E group was significantly reduced by salubrinal treatment, and GRP94 and ATF4 transcription levels in EC group were significantly reduced by all treatments (p<0.05) compared to control. In the EC group, both apoptotic genes were reduced by ER stress inhibitor treatments compared to control (p<0.05) except Caspase-3 gene by TUDCA treatment. These results suggest that the treatment of ER stress inhibitor during parthenogenetic activation can reduce ER stress, and thereby reduce apoptosis and promote in vitro development of porcine parthenogenetic embryos.

• 미생물학회지
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• 제38권3호
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• pp.186-191
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• 2002

장내바이러스(enterovirus),로타바이러스(rotavirus),그리고 아데노바이러스(adenovirus)등 물을 통해 전파되는 환경바이러스의 신속한 검출 및 1 차적인 분류를 위해 올리고뉴클레오티드 DNA chip을 이용한 분석 시스템의 유용성에 대하여 연구하였다. BGM 세포배양실험에서 바이러스성 세포병변효과(cytopathic effect) 양성으로 판정된 세포단층으로부터 접종배양 3 일 후 세포내 모든 RNA를 분리하여 중합효소연쇄반응과 DNA chip으로 검출여부 및 유전형을 비교 분석한 결과 세포배양에서 양성으로 판정된 10개의 시료 중 3개가 바이러스 음성으로, 7개가 바이러스 양성으로 나타났다. 중합효소연쇄반응과 DNA chip에 의해 양성으로 나타난 7개 시료의 유전형은 두 방법에 의해 모두 장내바이러스로 동정되어 DNA chip에 의한 1차 동정의 유용성도 증명하였다. 세포배양 후 전기영동분석 없이 일차적인 동정과정까지 불과 3∼4 시간 이내에 수행해낼 수 있는 DNA chip분석은 특이성, 신속성, 및 경제성을 고루 갖추어 환경바이러스 검출방법론의 새로운 영역을 구성할 것으로 사료된다.

• Journal of Applied Biological Chemistry
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• 제48권4호
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• pp.170-177
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• 2005

Oriental medicinal herb extracts (OHE) showing anticancer activities were investigated for effectiveness as antiviral drugs. Infection of MuLV to cell line resulted in formation of giant syncytia. Number of giant syncytia in culture treated with OHE decreased by 40% compared to that of non-OHE-treated cell culture. To determine OHE effects on progeny release, RT-PCR was performed. In vivo animal studies demonstrated effectiveness of OHE as antiviral drug when administered orally. After OHE administration, viral cytopathic effects decreased. Infected mice showed splenomegaly and over-proliferation of lymphocytes with decreased CD4+ cell counts. These symptoms decreased in OHE-treated mice, indicating OHE maybe useful therapeutics against MuLV/MAIDS as Human Immunodeficiency Virus (HIV)/AIDS animal model. Results show XC plaque assay and in vivo MAIDS model using MuLV are suitable tools for screening anti-retroviral drug candidates.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.60-60
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• 2003

Embryonic stem (ES) cells have property of self-renewal and can differentiate into the cells of all three primary germ layers. Recently, many growth factors, alteration of culture condition and gene modifications have been used to differentiate mouse and human ES cells into specific cell types. This study was performed to evaluate the differentiation protocol for human ES cells to the endodermal lineage cells. Human ES cells (Miz-hESl ) were cultured on STO feeder layer mitotically inactivated with mitemycin C, and embryoid bodies (EBs) were formed by suspension culture. Differentiation protocol of EBs consisted of three steps: stage I, culture of EBs for 6 days with ITSFn medium; stage II, culture of stage I cells for 8 days with N2 medium ; stage III, culture of stage II cells for 22 days with N2 medium. mRNA levels of the endodermal lineage differentiation genes were analyzed by semi- quantitative RT-PCR. The Oct-4 expression, a marker of the pluripotent state, was detected in undifferentiated human ES cells but progressively decreased after EBs formation. Differentiating human ES cells expressed marker genes of endodermal differentiation and pancreatic islet cells. GATA4, a-fetoprotein, Glut-2, and Ngn3 were expressed in all stages. However, albumin and insulin were expressed in only stage III cells. The human ES cells can be differentiated into endodermal lineage cells by multiple step culture system using various supplements. We are developing the more effective protocols for guided differentiation of human ES cells.

• IMMUNE NETWORK
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• 제10권1호
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• pp.15-25
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• 2010

Background: Rat mast cells were regarded as a good model for mast cell function in immune response. Methods: Rat bone marrow mast cells (BMMC) were prepared both by recombinant rat IL-3 (rrIL-3) and by recombinant mouse stem cell factor (rmSCF), and investigated for both proliferation and differentiation in time course. Rat BMMC was induced by culture of rat bone marrow cells (BMCs) in the presence of both rrIL-3 (5 ng/ml) and rmSCF (5 ng/ml). Culture media were changed 2 times per week with the cell number condition of $5{\times}10^4/ml$ in 6 well plate. Proliferation was analyzed by cell number and cell counting kit-8 (CCK-8) and differentiation was by rat mast cell protease (RMCP) II and histamine. Results: Cell proliferation rates reached a maximum at 8 or 11 days of culture and decreased thereafter. However, both RMCP II production and histamine synthesis peaked after 11 days of culture. By real time RT-PCR, the level of histidine decarboxylase mRNA was more than 500 times higher on culture day 11 than on culture day 5. By transmission electron microscopy, the cells were heterogeneous in size and contained cytoplasmic granules. Using gated flow cytometry, we showed that cultured BMCs expressed high levels of $Fc{\varepsilon}RI$ and the mast cell antigen, ganglioside, on culture day 11. Conclusion: These results indicate that rat BMMCs were generated by culturing BMCs in the presence of rrII-3 and rmSCF and that the BMMCs have the characteristics of mucosal mast cells.

• Journal of Korean Dental Science
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• 제11권2호
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• pp.62-70
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• 2018

Purpose: Bone marrow has long been a source of primary cells. This study was performed to evaluate the effects of age and sex on the cellular viability and expression of stem cell markers of mRNA and on the protein expression of bone marrow stem cells (BMSCs) derived from healthy donors. Materials and Methods: Stem cells were isolated from human bone marrow and plated in culture plates. The shape of the BMSCs was observed under inverted microscope. Quantitative cellular viability was evaluated using a Cell-Counting Kit-8 assay. The expression of stem cell surface markers was tested and a series of quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot was performed to evaluate the expression in each group. Result: The shapes of the cells at 20s, 30s, and 50s were similar to each other. No significant changes in cellular viability were noted among different age groups or sex groups. The BMSCs expressed CD44, CD73, and CD90 surface markers but did not express CD14 and CD34. There were no noticeable differences in CD surface markers among the different age groups. The expressions of CD surface markers were similar between men and women. No significant differences in the secretion of vascular endothelial growth factors (VEGFs) were noted at Day 3 between different age groups. qRT-PCR regarding the expression showed differences between the age groups. However, Western blot analysis showed a decrease in expression but did not reach statistical significance (P>0.05). Conclusion: This study clearly showed no significant differences in shape, cell viability, expression of stem cell surface markers, or secretion of human VEGF among different age groups. However, western blot analysis showed a tendency of age-related decrease which did not reach statistical significance. Collectively, autologous or allogeneic BMSCs should be meticulously applied to obtain optimal results regarding age and sex.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.22-22
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• 2003

This study was conducted to determine effects of polyvinyl alcohol (PVA), fetal bovine serum (FBS) and bovine serum albumin (BSA) on blastocoel formation, cell number, apoptosis and apoptosis-related gene expression of porcine diploid parthenotes developing in vitro. Embryos were collected from 2-cell or late 4-cell diploid parthenotes that activated with electro pulse, and in vitro cultured in the NCSU 23 medium supplemented without or with 0.1% PVA, 10% FBS or 0.4% BSA for day 7. The morphological analysis of apoptosis in embryos was carried out using propidium iodide staining and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling. The expressions of Bcl-xL, Bak and P53 in blastocyst stage parthenotes and in vivo-derived blastocysts were determined using semiquantitative RT-PCR. The addition of 0.4% BSA to the culture medium enhanced the development of 2- or late 4-cell stage parthenotes to the blastocysts stage (P < 0.01) while FBS decreased the incidence of blastocoel formation. FBS also reduced cell numbers of blastocysts developed from both 2- (P < 0.001) and late 4-cell (P < 0.05) embryos and increased percentage of apoptosis in the blastocysts (P < 0.001). The relative abundance of Bcl-xL mRNA in diploid parthenotes cultured from 2-cell stage in the presence of BSA is similar with that in in vivo derived embryos, but is significantly higher than in parthenotes cultured with FBS, PVA or none protein supplement control. Bak mRNA showed a significant increase at the blastocyst stage in FBS supplement medium. This result suggests that apoptosis related gene expression is significantly affected by protein supplements, which may result in alteration of apoptosis and embryo viability of porcine embryos developing in vitro.

• 대한미생물학회지
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• 제34권5호
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• pp.453-459
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• 1999

Astrovirus is frequently associated with diarrhea in children. It can not be readily isolated by cell culture, and an electronmicroscope is usually used for detection of this agent. Recently in 1995 a combined method of reverse transcription-polymerase chain reaction (RT-PCR) was designed for easier detection of astrovirus, which is based on the conserved sequence in 3'-end of genomes of the 7 known serotypes of human astrovirus. As of yet there has not been any report of astrovirus data in Korea using the RT-PCR methods. The purpose of this study was to detect astrovirus incidence, severity of symptoms, seasonal variation and co infection rate with rotavirus in Korean children inpatients with diarrhea. Fecal specimens from 61 young children hospitalized with gasteroenteritis Korea from Jan. 1996 through Mar. 1997. They were examined for astroviurs infection by RT-PCR method. Results are as follows:1. Astrovirus was detected at 9.8% (6/61) from fecal specimens of children with severe diarrhea by EIA using monoclonal antibody coated plates. 2. Astorvirus was detected at 29.5% (18/61) from fecal specimens of children with severe diarrhea by RT-PCR. 3. The age of the 18 children affected by astrovirus ranged from 2 monthes to 7 years with mean of 3.0 years. 4. Mean hospital stay of the 18 children was 6.1 days. 5. Five (27.8%) astrovirus RT-PCR positive strains were confirmed in November and in December, respectively out of 18 specimens in total. 6. Astrovirus coinfection with rotavirus type G1 was confirmed in 15/16 specimens (93.8%), and with type G2 was in 1/16 specimens (6.3%).

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.71-71
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• 2003

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multifunctional cytokine that has been implicated in the regulation of pre-implantation embryo development across several species. The aim of this study was to determine the effects of mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) on development of porcine parthenotes and nuclear transferred embryos, and on their expression of implantation-related genes. In the presence of bovine serum albumin, mGM-CSF did not increase the percentage of oocytes that developed to the blastocyst stage and at day 7 did not increase oocyte cell number. Addition of 10 mM GM-CSF to protein-free culture medium significantly increased the compaction and blastocoel formation of 1- to 2-cell parthenotes and cloned embryos developing in vitro. However, cell number was not increased when they were cultured in the presence of GM-CSF. Semi-quantitative reverse transcripts polymerase chain reaction (RT-PCR) revealed that mGM-CSF enhances mRNA expression of the leukemia inhibitory factor receptor, but does not influence interleukin-6 or sodium/glucose co-transporter protein gene expression in blastocyst stage parthenotes. These results suggest that mGM-CSF may enhance viability of porcine embryos developing in vitro in a defined culture medium.