• Clinical and Experimental Reproductive Medicine
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• 제25권1호
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• pp.77-86
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• 1998

The uptake of glucose for metabolism and growth is essential to most animal cells and is mediated by glucose-transporter (GLUT) proteins. The aim of this study was to determine which class of glucose transporter molecules was responsible for uptake of glucose in the mouse early embryo and at which stage the corresponding genes were expressed. In addition, co-culture system with vero cell was used to investigate the effect of the system on GLUT expression. Two-cell stage embryos were collected from the superovulated ICR female and divided into 3 groups. As a control, embryos were cultured in 0.4% BSA-T6 medium which includes glucose. For the experimental groups, embryos were cultured in either co-culture system with vero cells or glucose-free T6 medium supplemented with 0.4% BSA and pyruvate as an energy substrate. 2-cell to blastocyst stage embryos in those groups were respectively collected into microtubes (50 embryos/tube). Total RNA was extracted and RT-PCR was performed. The products were analysed after staining ethidium bromide by 2% agarose gel electrophoresis. Blastocysts were collected from each group at l20hr after hCG injection. They were fixed in 2.5% glutaraldehyde, stained with hoechst, and mounted for observation. In control, GLUT1 was expressed from 4-cell to blastocyst. GLUT2 and GLUT3 were expressed in morula and blastocyst. GLUT4 was expressed in all stages. When embryos were cultured in glucose-free medium, no significant difference was shown in the expression of GLUT1, 2 and 3, compared to control. However GLUT4 was not expressed until morular stage. When embryos were co-cultured with vero cell, there was no significant difference in the expression of GLUT1, 2, 3 and 4 compared to control. To determine cell growth of embryos, the average cell number of blastocyst was counted. The cell number of co-culture ($93.8{\pm}3.1$, n=35) is significantly higher than that of control and glucose-free group ($76.6{\pm}3.8$, n=35 and $68.2{\pm}4.3$, n=30). This study shows that the GLUT genes are expressed differently according to embryo stage. GLUTs were detectable throughout mouse preimplantation development in control and co-culture groups. However, GLUT4 was not detected from 2- to 8-cell stage but detected from morula stage in glucose-free medium, suggested that GLUT genes are expressed autocrinally in the embryo regardless of the presence of glucose as an energy substrate. In addition, co-culture system can increase the cell count of blastocyst but not improve the expression of GLUT. In conclusion, expression of GLUT is dependent on embryo stage in preimplantation embryo development.

• Journal of Veterinary Science
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• 제21권5호
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• pp.64.1-64.14
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• 2020

Background: Canine distemper virus (CDV) infection results in high morbidity and mortality in dogs. There has been no report about Isolation of Korean CDV since 1980 in Korea. Objectives: To investigate the biological properties and the genetic characterization of Korean CDV. Methods: Vero cells expressing dog signaling lymphocyte activation molecule (dSLAM) gene named as Vero/dSLAM were used to isolate CDV using 17 samples. Diagnostic methods such as cytopathic effects, immunofluorescence assay, peroxidase linked assay, electron microscopy, rapid immunodiagnostic assay, and reverse transcription polymerase chain reaction were used to confirm the Korean CDV isolate as a CDV. The genetic analysis was performed through cloning and sequencing of hemagglutinin gene of CDV isolate. Results: A virus propagated in Vero/dSLAM cell was confirmed as CDV (CD1901 strain) based on the above methods. The CD1901 strain showed the highest viral titer (10^5.5 50% tissue culture infectious dose [TCID₅₀]/mL) in the Vero/dSLAM cells at 4 days post inoculation, but did not form a fork on chorioallantoic membrane of 7-day-old egg. Ribavirin, a nucleotide analogue anti-viral agent, inhibits moderately the Korean CDV propagation in the Vero/dSLAM cells. The nucleotide and amino acid sequences of the H gene of CD1901 strain were compared with those of other CDV strains. The CD1901 strain belonged to Asia 1 group and had the highest similarity (99.9%) with the BA134 strain, which was isolated in China in 2008. Conclusions: We constructed successfully Vero/dSLAM and isolated one Korean CDV isolate (CD1901 strain) from a naturally infected dog. The CD1901 strain belonged to Asia 1 genotype.

• 미생물학회지
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• 제33권2호
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• pp.97-104
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• 1997

인간을 비롯하여 여러 동물의 생식기와 구강. 안구 점막에 수포성, 괴양성 병변을 일으키는 Herpes simplex 2형 바이러스(HSV-2)에 대한 단클론항체를 하이브리도마 기술을 이용해 생사하였다. 생산된 단클론항체 C-2는 western blotting에서 134, 86 그리고 43 kDa의 분자량을 갖는 항원을 인식하였다. C-2의 isotype은 IgM이었다. SDS-PAGE를 이용하여 HSV-2에 감염되어 원형의 다핵 거세포를 형성한 vero 세포주를 인식하였으며, 이는 HSV-2 항원이 숙주세포에서 발현되고 있음을 알 수 있다. 이 결과는 HSV-2 백신개발의 목표항원이 되는 항원검출과 HSV-2 감염 진단법 개발에 기초가 될 수 있을 것이다.

• BMB Reports
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• 제36권5호
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• pp.514-519
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• 2003

The ribonucleotide reductase (RR) 2 gene of the HSV-2 strain G was cloned, sequenced, and expressed in an E. coli cell. The RR2 gene was located on the PstI 2.4 kb fragment, which was cloned and sequenced. The ORF of the gene was 1,011 bp and its termination codon was TAG; also, the CATATAA sequence was present in the promoter of the RR2 gene. A Poly A signal sequence (AATAAA) was found in the 3'-noncoding region. The RR2 proteins that were produced in the E. coli and Vero cells were confirmed using a Western blot analysis. SDS-PAGE revealed that the molecular weights of the fusion-RR2 that was produced in the E. coli cells were approximately 24 kDa and 38 kDa in the Vero cells. The RR2 proteins were soluble. The differences in the molecular weights might be due to modifications in the Vero cells.

• Asian Pacific Journal of Cancer Prevention
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• 제13권5호
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• pp.2095-2100
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• 2012

Sulforaphane (SFN) an isothiocyanate formed by hydrolysis of glucosinolates found in Brassica oleraceae is reported to possess anticancer and antioxidant activities. In this study, we isolated SFN from red cabbage (Brassica oleraceae var rubra) and evaluated the comparative antiproliferative activity of various fractions (standard SFN, extract and purified SFN) by MTT assay in human epithelial carcinoma HEp -2 and and Vero cells. Probable apoptotic mechanisms mediated through p53, bax and bcl-2 were also examined. The SFN fraction was collected by HPLC, enriched for its SFN content and confirmed. Expression of apoptosis-related proteins was detected by western blotting and RT PCR. Results showed that Std SFN and purified SFN concentration found to have closer $IC_{50}$ which is equal to 58.96 microgram/ml (HEp-2 cells), 61.2 microgram/ml (Vero cells) and less than the extract which is found to be 113 microgram/ml (HEp-2 cells) and 125 microgram/ml (Vero cells). Further studies on apoptotic mechanisms showed that purified SFN down-regulated the expression of bcl-2 (antiapoptotic), while up-regulating p53 and Bax (proapoptotic) proteins, as well as caspase-3. This study indicates that purified SFN possesses antiproliferative effects the same as Std SFN and its apoptotic mechanism in HEp-2 cells could be mediated through p53 induction, bax and bcl-2 signaling pathways.

• KSBB Journal
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• 제10권3호
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• pp.292-297
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• 1995

Vero 세포를 이용한 뉴캐슬병 바이러스 생산에서 pH, 온도, 혈청농도, M.O.I. 등의 최적조건을 구하고 polycation, 항산화제, 그리고 DMSO 등의 첨가제가 바이러스 생산에 미치는 영향에 대한 연구를 수행하였다. 뉴캐슬병 바이러스의 최적 배양조건은 초기 p pH 7.2, 혈청농도 2 % FBS, 바이러스 접종량 0.1 M.O.I. 및 바이러스 증식온도 $34^{\circ}C$로 확인하였다. Polycation 인 DEAE-dextran, poly-L-Iysine과 항 산화제인 ascorbic acid는 각 $15\mu\textrm{g}/ml, 3\mu\textrm{g}/ml$그리고 0.1mM의 최적농도로 첨가했을 때 대조군에 비해 상당히 증대된 최대 바이러스 수율을 얻을 수 있었다.

• Molecules and Cells
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• 제21권1호
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• pp.112-120
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• 2006

It has been suggested that defective interfering (DI) RNA contributes to the persistence of Japanese encephalitis virus (JEV). In this study, we characterized molecular and biological aspects of the DI RNA and its relation to viral persistence. We identified a homologous DI virus intimately associated with JEV persistence in Vero cells. The production of DI RNA during undiluted serial passages of JEV coincided with the appearance of cells refractory to acute infection with JEV. We also established a Vero cell clone with a persistent JEV infection in which the DI RNA coreplicated efficiently at the expense of helper virus. The infectious virus yield of the clone fluctuated during its growth depending upon the amount of DI RNA accumulated in the previous replication cycle. Identification of the corresponding negative-sense RNA of the DI RNA indicated that the DI RNA functioned as a replication unit. Most of the DI RNA molecules retained their open reading frames despite a large deletion, encompassing most of the prM, the entire E, and the 5' half of the NS1 gene. Taken together, these observations suggest that the generation of homologous DI RNA during successive JEV acute infections in Vero cells probably participates actively in persistent JEV infection.

• 한국해양바이오학회지
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• 제12권2호
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• pp.108-114
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• 2020

A variety of shellfish species sold for human consumption are available for purchase in the domestic fish market. The microalgae families inhabit the ocean, where planktons supply the main nutritional resource for the growth of shellfish. Some phytoplanktons produce toxic compounds that are accumulated in shellfish and ultimately cause toxicity in humans. This article reports the cytotoxicity of commercially available shellfish species. Accordingly, hot water extract (HWE) and an aqueous fraction of 50% methanol extract (MEE-AF) showed no significant cytotoxicity on the two cell lines (i.e., HL-60 and Vero cell lines), but 50% methanol extract (MEE) in 3, 6 samples showed 50% cytotoxic effects on HL-60 cells, and 1, 4 samples showed 40%, 20% cytotoxic effects on Vero cells, respectively. In addition, their consequential dichloromethane fractions (MEE-DF) exhibited significant toxicities at the highest concentration (1,000 ㎍/ml) on HL-60 and Vero cells. Since the shellfish samples showed cytotoxicity in the dichloromethane fraction, it is possible that the dichloromethane fraction contains marine toxins. Further research will be needed to identify the toxic components from each sample.

• Radiation Oncology Journal
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• 제6권1호
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• pp.1-11
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• 1988

방사선 조사 후 발생한 잠재치사손상의 회복(PLDR)에 있어 조사선량 및 시간에 따른 환경변화가 회복의 동적양상에 미치는 영향을 Vero 세포계를 이용하여 실험하였다. 배양액을 교환시키지 않고 배양하여 평형기에 도달한 세포에 동물실험용 세시움 조사기로 $1Gy\~9Gy$의 감마선을 조사하고 각 조사조건에서 $O\~6$ 및 24시간동안 정치시킨 후 Agarose가 포함된 새로운 배양액에서 배양하였다. 16Gy를 조사한 동종의 세포를 feeder세포로 첨가하여 배양액내의 전 체세포수를 일정하게 한 조건에서 형성된 세포집락수에 따라 세포의 생존을 정하였다. 잠재치사손상의 회복은 $2\~4$시간 정치후에 포화수준에 도달한 빠른 회복이었다. 방사선량이 증가함에 따라 회복속도는 증가하였고, 포화수준의 회복량도 증가하였다. Linear-quadratic mode에 의한 "방사선량-생존분획" 분석 결과 잠채치사손상이 회복됨에 따라 일차 비활성계수 $\alpha$는 급속히 감소하여 $\beta$에 접근하였고 이차 비활성계수 $\beta$는 미미하게 증가하여 PLDR은 $\alpha$로 표시되는 손상에 주로 영향을 주었다. Multitarget model에 따라 분석한 결과 Do는 변화가 없고 Dq가 증가하였다. 세포 생존분획 이 높은 3Gy 이하의 저선량 영역에서 dose modifying factor가 높아 잠재 치사손상의 회복에 의한 영향이 저선량 영역에서 상대적으로 크게 나타났다.

• KSBB Journal
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• 제8권5호
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• pp.465-472
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• 1993

현재 상품화되어 시판되고 있는 4가지의 미립담체를 이용해 각각의 성능을 비교하기 위하여 부착성동물세포배양 실험을 수행한 결과를 요약하면 다음과 같다. Cytodex 3의 경우 세포의 초기 접종농도를 약 $2.0{\times}10^5$ cells/ml로 하였을 때, 3g/l는 약 $1.4{\times}10^6$cells/ml, 5g/l는 약 $2.0{\times}10^6$cells/ml의 최종세포밀도를 얻을 수 있었다. 또한 bead-to-bead trandsfer 실험을 한 결과 3g/l를 간헐적으로 첨가하였을 때는 약 $1.9{\times}10^6$cells/ml, 5g/l 첨가하였을 때는 약 $3.0{\times}10^6$cells/ml까지 최종세포밀도가 증가하였다. Cultispher-G, 3g/l를 이용해 초기 접종농도를 약 $2.0{\times}10^6$cells/ml로 하여 배양하였을 때 약 $1.3{\times}10^6$cells/ml까지 세포농도가 증가했고, 5g/l를 이용해 초기 접종농도를 $4.0{\times}10^5$cells/ml로 접종하였을 때 최종세포농도가 약 $3.2{\times}10^6$cells/ml까지 증가하는 것을 확인할 수 있었다. 그리고 bead-to-bead transfer배양 실험결과에서 3g/l의 미립담체를 간헐적으로 첨가해 약 $1.8{\times}10^6$cells/ml까지 최종세포밀도가 올라갔고 5g/l를 간헐적으로 첨가하였을 때 $2.5{\times}10^6$cells/ml까지 세포밀도를 얻었다. VX-100을 사용하여 세포를 배양하였을 때 초기 접종농도가 약 $2.0{\times}10^5$cells/ml에서 최종세포밀도가 약 $4.4{\times}10^6$cells/ml까지 증가하는 것을 알게 되었다. 따라서 실험에 사용한 다른 종류의 다공성 젤라틴 bead보다 성능이 우수함을 알 수 있었고 Cytodex-3보다는 최종세포농도가 약 2배이상 증가한 결과를 얻었다. Informatrix bead는 초기 접종농도를 약 $3.0{\times}10^5$cells/ml로 하였을 때 최종세포밀도가 약 $2.1{\times}10^6$cells/ml까지 증가하였다. Collagenase효소를 이용하여 젤라틴 bead를 녹인 후 회수한 세포는 대부분 viable하였고 새로 도입된 bead에 성공적으로 부착하여 성장하였다. 따라서 담체 내부에서 자라는 세포도 회수하여 재사용 할 수 있게 되었다.