Effect of Virus Infection on Insulin Secretion of Cultured Murine Pancreatic Beta Cells

정두영,차종희,고광삼,이근배,Chung, Doo-Young,Cha, Jong-Hee,Koh, Kwang-Sam,Lee, Keun-Bai 생화학분자생물학회 1985 한국생화학회지 Vol.18 No.2

SJL/J mice의 췌장베타세포를 배양하여 coxsackie $B_3$와 $B_4$, murine cytomegalovirus, mengo vrius, encephalomyocarditis virus의 M variant, mumps vrius, herpes simplex virus type I과 type II등의 virus를 감염시켜, 배양한 췌장 베타세포의 viral induction, viral antigen, viral replication 그리고 세포내와 세포의 insulin치와의 관계등을 연구 검토한 결과, encephalomyocarditis virus의 M variant로 감염시킨 경우에 high viral titer ($8.1{\times}10^5$ PFU)를 나타냈고, immunofluorescence에 의해서도 coxsackie virus로 감염시킨 경우보다 encephalomyocarditis virus의 M variant로 감염시킨 배양한 췌장베타세포에서 high viral antigen이 발견되었으며 immunoreactive insulin치도 coxsackie virus로 감염시킨 경우보다 encephalomyocarditis virus의 M variant로 감염시킨 경우가 세포내와 세포외 모두에서 현저하게 감소되어 이었다. 이를 종합하여 볼 때 배양한 췌장 베타세포의 insulin분비에 미치는 virus의 영향은 췌장베타세포서의 viral replication능력을 갖은 특정한 virus에 따라 다름을 알 수 있었다. In vitro infections of murine pancreatic beta cell cultures with coxsackie $B_3$, coxsackie $B_4$, murine cytomegalovirus, mumps virus, EMC-M virus, Herpes simplex virus type I and type II were determined. In vitro infection of the beta cell cultures with EMC-M virus resulted in higher viral titer than the beta cell cultrues infected with other viruses. Double-labelled antibody techniques showed that coxsackie viral antigens were rarely detected in the cultured beta cells which were positive in fluorescence-labelled anti-insulin antibody staining, while high per cent of EMC-M viral antigen was found in those cells. And also its intracellular and extracellular levels of immunoreactive insulin from the beta cell cultures infected with EMC-M virus were more decreased than the beta cell cultures infected with other viruses. It is concluded that the viral infection to murine pancreatic beta cell cultures is restricted to the specific virus which has the ability of replication in the beta cells.

EMC-D Virus에 대한 단Clone성항체 생산세포주의 개발에 관한 연구

고광삼,정영기,김성준,이근배,Koh, Kwang-Sam,Chung, Young-Kee,Kim, Sung-Jun,Lee, Keun-Eai 생화학분자생물학회 1984 한국생화학회지 Vol.17 No.1

EMC virus의 D variant를 항원으로 하여 단clone성항체를 생산하는 hybridoma cell을 만들때 EMC virus의 D variant로 면역시킨 mouse의 spleen cell과 myeloma cell의 융합율은 56%, 3차 cloning한 후 항체생산율은 93%이었고, 이 hybridoma cell의 supernate에서 얻은 immunoglobulin의 각 fraction은 항원에 대하여 특이성을 보였으며, 3차 cloning 한 hybridoma cell이 생산하는 항체는 단clone성항체로서 한 clone은 IgM, 또 다른 clone은 $IgG_1$을 생산하였을 뿐 아니라 항원인 EMC virus의 D variant에 대하여 monospecific하게 반응하였다. Hybridoma technique has been used to obtain monoclonal antibodies to specific antigenic determinants on complex antigens. For example, myeloma cells, which are resistant to 8-azaguanine due to deficiency of hypoxanthine phosphoribosyl transferase and can not survive in media containing hypoxanthine, aminopterine, and thymidine CHAT), is hybridized to primed spleen cells from animal immunized with specific antigens. These cells can survive in HAT media, proliferate continuously, and secrete homologous antibody to specific antigens. The M variant of encephalomyocarditis (EMC) virus contains two stable variants, one diabetogenic CD variant) and the other nondiabetogenic CB variant). When the D variant of EMC virus was inoculated into SJL/J mice, hyperglycemia developed in over 90% of the animals, while none of the mice developed diabetes when inoculated with the B variant of EMC virus. However, the D and B variants are immunologically indistinguishable from each other by standard conventional method: Antibody made against the D variant neutralized the D and B variants to about the same degree. Similarly, antibody made against the B variant neutralized the Band D variant equally well. To distinguish antigenic specificity of these two variants of EMC virus, hybridoma techniques were applied. Balb/c mice immunized intraperitoneally every week with increasing doses of D variant of EMC virus beginning with $5.0{\times}10^5$ PFU per mouse and ending with $1.0{\times}10^7$ PFU per mouse for two months. Spleen cells from these immunized mice were hybridized with myeloma cells (NS-1). The hybrid cells were selected in HAT media. The antibody secreting cells among the hybrid cells were selected by viral neutralization test (against D variant of EMC virus) and then further cloned. Several different clones from these antibody-producing hybrid cells were characterized by immunological and biochemical methods. By these methods, it was found that some hybridoma produced monospecific antibodies which enabled us to identify specific antigenic variants.

EMC - D Virus 에 대한 단 Clone 성항체 생산세포주의 개발에 관한 연구

고광삼,정영기,김성준,이근배 ( Kwang Sam Koh,Young Kee Chung,Sung Jun Kim,Keun Bai Lee ) 생화학분자생물학회 1984 BMB Reports Vol.17 No.1

Hybridoma technique has been used to obtain monoclonal antibodies to specific antigenic determinants on complex antigens. For example, myeloma cells, which are resistant to 8-azaguanine due to deficiency of hypoaanthine phosphoribosyl transferase and can not survive in media containing hypoaanthine, aminopterine, and thymidine (HAT), is hybridized to primed spleen cells from animal immunized with specific antigens. These cells can survive in HAT media, proliferate continuously, and secrete homologous antibody to specific antigens. The M variant of encephalomyocarditis (EMC) virus contains two stable variants, one diabetogenic (D variant) and the other nondiabetogenic (B variant). When the D variant of EMC virus was inoculated into SJL/J mice, hyperglycemia developed in over 90% of the animals, while none of the mice developed diabetes when inoculated with the B variant of EMC virus. However, the D and B variants are immunologically indistinguishable from each other by standard conventional method: Antibody made against the D variant neutralized the D and B variants to about the same degree. Similarly, antibody made against the B variant neutralized the B and D variant equally well. To distinguish antigenic specificity of these two variants of EMC virus, hybridoma techniques were applied. Balb/c mice immunized intraperitoneally every week with increasing doses of D variant of EMC virus beginning with 5. 0×10^5 PFU per mouse and ending with 1.0×10^7 PFU per mouse for two months. Spleen cells from these immunized mice were hybridized with myeloma cells (NS-1). The hybrid cells were selected in HAT media. The antibody secreting cells among the hybrid cells were selected by viral neutralization test (against D variant of EMC virus) and then further cloned. Several different clones from these antibody-producing hybrid cells were characterized by immunological and biochemical methods. By these methods, it was found that some hybridoma produced monospecific antibodies which enabled us to identify specific antigenic variants.

배양한 췌장베타세포의 Insulin 분비에 미치는 virus 감염의 영향

정두영,차종희,고광삼,이근배 ( Doo Young Chung,Jong Hee Cha,Kwang Sam Koh,Keun Bai Lee ) 생화학분자생물학회 1985 BMB Reports Vol.18 No.2

遺傳工學的 利用을 위한 人體細胞培養에 대한 硏究(Ⅰ) : 人體包皮細胞培養에 影響을 주는 因子에 대하여 Human Foreskin CellCulture

高光三,金盛植,車鍾希,李根培,金成原 조선의대 1983 The Medical Journal of Chosun University Vol.8 No.1

쥐췌장 베타세포 소배양법의 생리적 검정

고광삼,김성식,김양균,이근배 朝鮮大學校 附設 醫學硏究所 1984 The Medical Journal of Chosun University Vol.9 No.1

쥐 췌장 베타세포의 소 배양법에 대한 생리적 반응을 알아보기 위하여 cell density를 측정하는 방법으로 DNA 농도를 측정하였고, cell density가 intracellular와 extracellular immunoreactive insulin (IRI) 농도에 미치는 영향을 검토하였으며, glucose, arginine, isoleucine 및 leucine이 insulin 분비에 미치는 영향을 알아보았다. 그 결과 DNA 농도는 배양후 2일째에 가장 높았으며, intracellular IRI은 배양후 6일째까지 점차적으로 증가되었고, extracellular IRI은 배양시작후 8일째까지 증가되었으나 그후 감소되었다. Cell density는 intracellular IRI와 extracellular IRI 농도에 영향을 주지 않았으며, glucose는 0~300㎎/100㎖ 농도에서 점차적으로 IRI 분비를 증가시켰으나 300~500㎎/100㎖ 농도에서는 변화가 없었다. Leucine은 IRI 분비를 3배증가시켰으며, arginine과 isoleucine은 2배 증가시켰다. 이로 미루어 보아 본 연구에 이용한 쥐 췌장 베타세포와 소 배양법으로 배양한 쥐 췌장 베타세포는 적절한 생리적 반응을 나타내고 있음을 알 수 있었다. Physiological responses of monolayered mouse beta cell vary according to culture conditons. These cells maintained normal morphological characteristics when cultured in a microculture system. Following determination of DNA concentration as a means of measuring cell density, the effect of cell density on intracellular and extracellular immunoreactive insulin (IRI) conccntration was determmed. Then the effect of glucose, arginine, isoleucine and lencine on insuline secretions was evaluated. When 2 davs old cultures with cell densities between 10^(3) and 10^(6) were compared. the increase of DNA concentration was highest in the 2 days old cultures and no further change by the 14th days. The intracellular IRI through the increase of cell population increased progressively up to culture day 6, and then slowly decreased. Extracellular IRI increased until day 8 and then decreased thereafter regardless cell density. The production of either intracellular or extracellular IRI was therefore independent from the density of cultured cells, Exposure to glucose in the range of 0 to 300㎎/100㎖ resulted in a progressive increase of IRI secretions, but from 300 to 500㎎/100㎖ there was no change. Exposure to leucine resulted in a three-fold increase of IRI secretions, whereas arginine and isoleucine caused these microcultured mouse beta cells to double their IRI secretions. This invetigation, therefore, not only demonstrated that cultured cells in the microculture system were morphologically normal, but also the system was suitable for studies of physiological responses to various culture conditions of murine pancreatic beta cells.

肝組織에서의 Glucagon의 Cholesterol合成 抑制機轉에 對한 硏究

李根培,車鐘希,朴悅,高光三 朝鮮大學校 附設 醫學硏究所 1983 The Medical Journal of Chosun University Vol.8 No.1

The inhibitory mechanism of glucagon on hepatic cholesterol biosynthesis in rat was studied by the measurement of incorporation of tritium from ^(3)H₂O into cholesterol, and intermediary metabolite of lipids. But the rate of cholesterol synthesis was not affected. The hepatic acetyl-CoA carboxylase activity and malonyl-CoA concentration were decreased to 25~35% of control value during the same period after glucagon injection. These data indicates that one site of action of glucagon in regulating fat biosynthesis is at the acetyl-CoA carboxylase reaction.

방사선을 조사한 생쥐 장기에서의 Superoxide Dismutase 분포에 대한 연구

김양균,전현우,차종희,고광삼,이근배 朝鮮大學校 附設 醫學硏究所 1986 The Medical Journal of Chosun University Vol.11 No.1

The cytosolic and mitochondrial supreoxide dismutases (EC 1.15.1.1) were investigated in various organs, thyroid gland, lung, heart, liver, spleen and kidney, of normal and irradiated mice. The mouse organs were fractionated into cytosolic and mitochondrial fractions by differential centrifugation, and the supsroxidp dismutase was measured by the method of McCord and Fridovich. The mouse organs contain two types of superoxide dismutase ; one is localized in the cytosol and contains cupper and zinc, the other in the mitochondria and contains manganese. Superoxide dismutasc activity was found in all organs of mouse investigated, and maximal activity of enzyme was observed in liver. most of superoxide dismutase activity, 79-86%, was present in the cytosolic fraction, and the rest was associated with mitochondria. The activity of cytosolic superoxide dismutase was most prominent in the liver among those tissues assayed. However, that of mitochoadrial enzyme was highest in the heart. Thc specific activity of superoxide dismutase in mitochondria was found about 2-5 folds greater than that of the enzyme in cytosol and cyanide inhibit the activity of cytosolic enzyme but not mitochondrial enzyme, In irradiatcd mice, the enzyme activity and the specific activity of superoxide dismutase in cytosol and mitochondna of various organs were decreased with minimal value on 7th and l4th day after exposured by whole body-irradiation.

Phenylhydrazine으로 處置한 토끼 赤血球의 Lactate Dehydrogenase Isozymes의 活性度에 關하여

金良均,朴在允,車鍾希,高光三,李根培 조선의대 1983 The Medical Journal of Chosun University Vol.8 No.1