당뇨병을 유발하는 Virus 에 대한 Monoclonal Antibody 의 생산과 검정

고광삼 한국유전학회 1983 Genes & Genomics Vol.5 No.2

1975년 영국의 Medical Research Council에서 일하던 Ko¨hler와 Milstein이 sheep RBC(SRBC)로 면역시킨 Balb/c mouse의 spleen cell과 myeloma cell을 융합시켜, SRBC에 특이하게 결합하는 항체를 생산할 뿐만 아니라 세포배양에서 영구히 살 수 있는 잡종세포를 만들어 냄으로써, hybridoma cell의 개념이 확립되게 되었다. Hybridoma란 특이항체를 생산하는 plasmacyte와 영구히 세포배양이 가능한 plasmacytoma간의 세포융합에 의해 만들어진 잡종세포로서, 양쪽 세포의 유전자를 다 갖게되어 plasmacyte의 특이항체를 생산하는 성질과 plasmacytoma의 세포배양에서 영구히 분열증식하는 성질을 동시에 갖는 세포를 말한다. Hybridoma cell이 생산하는 항체는 monospecific한 특징을 갖는 monoclonal antibody로서 영구히 세포배양을 통해서 다량으로 얻을 수 있는데, 이 항체는 immunoglobulin이 Fab sequence가 동일하여 항원과의 결합력이 일정하며, 특이성이 매우 높다. 본인은 encephalomyocarditis virus의 M variant 중에서 당뇨병을 일으키는 것으로 알려진 D variant와 당뇨병을 일으키지 않는 것으로 알려진 B variant를 일반적인 방법으로 구별할 수 없으므로, 이를 hybridoma technique을 이용하여 구별하고자 D variant를 항원으로 하여 Balb/c mouse를 일주일 간격으로 처음에는 5.0×10^(5)PFU로 시작하여 마지막에는 1.0×10^(7)PFU의 용량으로 2개월간 면역시킨 다음, 이 mouse의 spleen cell을 myeloma cell(NS-1)과 Polyethylene glycol(PEG)을 이용하여 융합시킨 다음, HAT(Hypoxanthine, Aminopterine, Thymidine) media 중에서 잡종세포만을 선별하였다. 이를 cloning한 다음, ELISA법을 이용하여 monoclonal antibody를 생산하는 세포와 생산하지 않는 세포를 분리한 다음, 이들 clone들을 면역학적, 생화학적, 세포생물학적 방법으로 검정하였다. 그 결과 이 hybridoma cell은 특정한 항원인 encephalomyocarditis virus의 D variant를 동정(identify)하는 monospecific antibody를 생산하였으며, monoclonal antibody를 생산하는 hybridoma cell은 이를 생산하지 않는 세포보다 RNA-dependent DNA polymerase 활성도가 높음을 알 수 있었고, 이 효소의 활성도가 높은 hybridoma cell에서는 특이하게 많은 수의 extracellular C-type virus particle을 볼 수 있었으며, 반대로 이 효소의 활성도가 낮은 세포에서는 많은 수의 intracellular C-type virus particle을 볼 수 있었다.

韓國人의 赤血球 G-6-PD에 關한 硏究

高光三,金昌世 최신의학사 1973 最新醫學 Vol.16 No.6

1. On the studies of hemolyzing solution we were obtained such optimum concentration of enzyme G-6-PD activity as followings: 2.7mM of EDTA, 5mM beta-mecrcaptoethanol, and 10 pM NADP. 2. Many different experimental conditions with relation to the measurement of enzyme activity were studied and obtained as following results: a) The production rate of NADPH was constant during first 30 minutes after adding G-6-P into the reaction mixture. b) Enzyme concentration, 1 to 6 volumes of usual concentration, obeyed by Beer's law. c) Influence of temperature was so significant that we used to a temperature of 25°C throughout the experimeatal procedures. d) The reaction mixture except G-6-P was stable on 8 days or more in deep freezer (-45°C) and about 5 days in refrigerator (5°C). e) The samples to be examined were stable on 3 hours at room temperature in case of intact whole blood and on 1 hour or less in case of hemolysates. 3. Erythrocyte G-6-PD values in Korean was such as follows: The distribution of 294 males was 7.82±4.77, and 286 females 7.24±4.23 IU per gm of Hb. Age distributions of both groups, male and female, were 1 year or less to 75 years old which contained 5-10 healthy individuals in each age groups. 4. There was a groups strongly suggested such as G-6-PD Hektoen those enzymic value is shown about three times of mean value on the distribution curve.

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

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.

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.

催眠鎭靜劑가 白鼠의 血淸脂質量에 미치는 影響

高光三,金吉雄,趙萬熙 中央醫學社 1974 中央醫學 Vol.27 No.4

Administration of phenobarbital, amobarbital and chloropromazine to rats fed a standard laboratory diet produced the change in levels of serum lipid concentration. In phenobarbital and amobarbital administration group, triglyceride level was elevated, phospholipid and cholesterol levels were decreased. Especially, phospholipid and cholesterol Ievels were remarkable decreased in amobarbital administration group. Triglyceride, phospholipid and cholesterol levels were decreased in chloropromazine administration group. As a result of hypnotic sedatives, phenobarbital, amobarbital and chloropromazine administration, serum lipid levels were markedly influenced in rats.

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.

名種藥物의 G-6-PD 缺乏誘發로 인한 溶血性貧血 및 染色體不活性化에 關한 硏究

高光三,梁南吉,朴順化,車種希 中央醫學社 1976 中央醫學 Vol.30 No.2

Glucose 6 phosphate dehydrogenase (G-6-PD) deficiency was found to be the main genetic cause for the participation of acute hemolysis. Since the gene determining the structure of G-6-PD molecule is located on the X chromosome, the ingestion of certain drugs can be induce the X chromosomal inactivation. In this study, an attempt has been made to investigate the drug-induced hemolytic anemia, erythrocyte G-6-PD deficiency and X chromosomal inactivation ;after the ingestion of certain drugs in vivo. The ingestion of quinine, primaguine and methylene blue has been induced the severe decreased hemoglobin and erythrocyte G-6-PD values, the recovery of these decreased values was not achieved. And acetylsalicylic acid, sulfadimethoxine and ascorbic acid has been induced -the decreased hemoglobin value and erythrocyte G-6-PD activity, but the recovery was slightly achieved. The effect of the ingestion of chloramphenicol, menadione and diphenhydram?ine on the hemoglobin value and erythrocyte G-6-PD activity were slightly decreased, and the recovery was achieved. And sodium nitrite has been not affected to the hemoglobin value and eryth?rocyte G-6-PD activity. The X chromosomal inactivation was not induced by the ingestion of these drugs which was prolonged for 30 days.

韓國産 괭이밥의 低血糖?果에 對한 硏究(Ⅰ)

高光三 中央醫學社 1977 中央醫學 Vol.33 No.4

Since 1963 to 1977, the Neuroleptanesthesia and the Neuroleptanalgesia has been used. A mixture of the phenopheridine and haloperidol with inhalation of nitrous oxide-oxygen has been used to provide Neuroleptanesthesia for gynecologic surgery in 109 cases, 7 cases of laryngoscopic biopsy and larygoscopic microsurgery, with Neuroleptanalgesia was performed in 1965. Since 1965 to 1977, 29 cases Neuroleptanesthesia has been performed with an intravenous administration of Innovar with nitrous oxide-oxygen inhalation requiring muscular relaxation had to be added. Neuroleptanalgesia for laryngeal microsurgery, an anesthetic technic consists of intravenous administration of Innovar with 1% Tetracaine spray into the larynx. Total doses administered were droperidol 7.7mg (0.14mg/kg) and fentanyl 0.3mg (0.005mg/kg) with pethidine and chloropromazine, respectively. Arterial blood samples showed clinically insignificant PCO2 rise (5rmnHg up) and PO2 decrease (7mmHg down). Advantages of this anesthetic technic includes: 1. Lack of endotracheal tube produces unobstructed wide surgical fields. 2. Patients are conscious during the procedure and the surgeon is able to observed the movement of the vocal cords by the phonation of patients. 3.Anesthetic technic is simple 4. Time for surgery is not limited Disadvantages fort he technic are: 1. The degree of respiratory depression is slight difficuties encountered to obtain adequate depth of anesthesia to control cough reflex. 2. There are high incidences (80%) of hypertension, was used to control high systemic blood pressure.

各種睡濟의 脂肪成肝誘發에 關한 硏究

高光三,趙萬熙,高宇永,金明聖 최신의학사 1974 最新醫學 Vol.17 No.11

As a result of phenobarbital, amobarbital and chloropromazine administration to rats fed a standard laboratory diet, the levels of triglycerides phospholipids and total cholesterol in the livers and serum and pancreatic and serumlipase activity were changed in these animals. Phenobarbital and amobarbitalproduced elevated levels of triglycerides, phospholipids and total cholesterol in the livers and level of triglycerides in the serum, but lowered levels of phospholipids and total cholesterol in the serum were produced. Chloropromazine produced slightly lowered levels of triglycerides, phospholipids and total cholesterol in the serum, but levels in the livers were not influenced. The level of pancreatic lipase was not affected by admininstration of phenobarbitai and amobar?bital, but the level of serum lipase was elevated in twofold. Administration of chloropromazine produced elevated level of serum lipase and lowered level ' of pancreatic lipase.