섬유아세포의 증식과 Collagen 합성에 대한 Prostaglandin E2 의 조절작용

김인산,조준승 ( In Sam Kim,Joon Seung Jo ) 생화학분자생물학회 1990 BMB Reports Vol.23 No.1

PGE₂ has been known to modulate diploid human fibroblast proliferation and collagen production by intracellular cyclic AMP mediated pathway but it still remains unclear that the suppressive effect of PGE₂ on proliferation and collagen synthesis are mediated by the elevated intracellular cyclic AMP levels. The purpose of present study was to determine the effects of PGE₂, db-cAMP or cholera toxin on cell proliferation and collagen production and to study how the temporal changes of intracellular cyclic AMP levels caused by PGE₂ are associated with cell proliferation and collagen production. And we also hypothesized that lengthy exposure of fibroblast cultures to prostaglandins could lead to overgrowth of the insensitive cells which might be responsible for fibrotic diseases. At least, 48 hours of incubation were required to suppress the cell proliferation by PGE₂, and under the same conditions there were no changes of intracellular cyclic AMP levels during 72-hour incubation of PGE₂. 48- and 72-hour incubation of PGE₂ showed dose- and time-dependent suppression of the cell proliferation. Dibutyryl cyclic AMP and cholera toxin appeared to suppress cell proliferation by even 24 hours incubation and they also showed dose- and tme-dependent suppression of the cell proliferation. Cholera toxin increased intracellular cyclic AMP levels maximally after one hour and then decreased its level but still maintained its elevated state for 24 hours. Short-term incubation of dibutyryl cyclic AMP or cholera toxin for six hours wass enough to suppress the cell proliferation. Total protein and collagen production were suppressed by PGE₂, dibutyryl cyclic AMP or cholera toxin in a dose-dependent manner and their effects seemed to be associated with intracellular cyclic AMP levels which were elevated by PGE₂ or cholera toxin. One hour incubation of PGE₂ was enough to suppress total protein and collagen production by fibroblasts. Judging by the fact that treatment of PGE₂ or dibutyryl cyclic AMP decreased the steady state levels of mRNAs for procollagens and fibronectin and that co-treatment with actinomycin D didn`t show any difference in the steady state levels of each mRNA, the inhibitory actions of PGE₂ and dibutyryl cyclic AMP on the fibronectin and collagen production seem to occur at transcriptional level. Long-term exposure for PGE₂ could result in selection of subpopulation of fibroblasts which showed less responsiveness to reexposure of PGE₂ in the cell proliferation but not in collagen production. These results indicate that prostaglandin E₂ could suppress the proliferation of human fibroblast cells by cyclic AMP-independent events and by which some subpopulations of fibroblasts might be selected to overgrow when exposed to PGE₂ for a long time and also indicate that PGE₂ could suppress total protein and collagen production at the transcriptional level by cyclic AMP-dependent events.

Collagen Dynamics and Polymorphism in Rat Liver During Fibrogenesis

조준승,류현모,은선진,김인산,Jo, Joon-Seung,Ryoo, Hyun-Mo,Eun, Sun-Jin,Kim, In-San Korean Society for Biochemistry and Molecular Biol 1991 한국생화학회지 Vol.24 No.4

Macromolecular collagen components in rat liver at different stage of fibrosis induced by common bile duct ligation were studied. Animals were killed on the 3rd, 7th, 14th, 21st and 29th day after bile duct ligation and liver specimens and blood samples were obtained. Hepatic collagen was fractionated into neutral salt soluble, acid soluble, pepsin soluble and insoluble fractions, and hydroxyproline content of each fraction was determined. The collagen content, especially in insoluble fraction, was increased as the duration of biliary obstruction increased. Pepsin solubilized collagens were subjected to determination of the collagen types by SDS-polyacrylamide disc gel electrophoresis. The ratio of type I to type III collagen was decreased gradually with progression of fibrosis. These results indicate that an alteration in tissue collagen polymorphism as well as variations in the collagen solubility accompany the fibrotic process, suggesting possible pathogenetic implication. 백서의 총담관을 결찰하여 간섬유화를 유발하고, 그 진행에 따른 간조직내 교원질의 양적 및 질적 변화를 조사하였다. 실험동물의 총담관을 결찰하고 각각 3,7,14,21 그리고 28일째 되는 날에 얻은 간조직과 혈액을 시료로 사용하였다. 간조직내 교원질을 neutral soluble, acid soluble, pepsin soluble 그리고 insoluble fraction으로 분획채취 하였으며, 각 분획의 hydroxyproline 함량으로 교원질을 측정하였다. 담관폐쇄 이후 간기능의 저하가 나타났고, 기간이 길어짐에 따라 간경화의 척도인 간장과 비장의 종대 및 복수의 생성을 관찰하였다. 또한 담관 폐쇄기간이 길수록 간조직의 교원질 함량은 증가하였으며, 특히 insoluble fraction의 증가가 주를 이루었다. Pepsin soluble fraction을 SDS-PAGE한 결과, type I에 대한 type III 교원질의 상대적 비율이 정상조직의 20%에서 4주군에서는 45%까지 증가하였다. 이러한 결과는 섬유화가 진행됨에 따라 교원질의 양적인 변화 뿐 아니라 용해도의 변화 혹은 결합조직을 형성하는 교원질 구성의 변화 등 질적인 변화가 발생함을 보여주고 있으며, 섬유화의 발생기작과 관계가 있을 것으로 볼 수 있다.

흰쥐의 간섬유화 과정에서의 교원질의 다형성 및 양적 변동

조준승,류현모,은선진,김인산 ( Joon Seung Jo,Hyun Mo Ryoo,Sun Jin Eun,In San Kim ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.4

Macromolecular collagen components in rat liver at different stage of fibrosis induced by common bile duct ligation were studied. Animals were killed on the 3rd, 7th, 14th, 21st and 29th day after bile duct ligation and liver specimens and blood samples were obtained. Hepatic collagen was fractionated into neutral salt soluble, acid soluble, pepsin soluble and insoluble fractions, and hydroxyproline content of each fraction was determined. The collagen content, especially in insoluble fraction, was increased as the duration of biliary obstruction increased. Pepsin solubilized collagens were subjected to determination of the collagen types by SDS-polyacrylamide disc gel electrophoresis. The ratio of type I to type III collagen was decreased gradually with progression of fibrosis. These results indicate that an alteration in tissue collagen polymorphism as well as variations in the collagen solubility accompany the fibrotic process, suggesting possible pathogenetic implication.

복수내 Fibronectin 치와 Adenosine Deaminase 치의 감별진단적 의의

권오종(Oh Jong Kwon),박승국(Soong Kook Park),박낭운(Rang Woon Park),김인산(In San Kim),조준승(Joon Seung Jo) 대한내과학회 1989 대한내과학회지 Vol.37 No.6

N/A The differential diagnosis of ascitic fluids remains to be solved. The aim of this study was to evaluate the diagnostic values of the fibronectin level and adenosine deaminase activity in differentiation of ascites. Ascitic fluid samples from 16 patients with malignancy, 5 patients with tuberculosis and 18 patients with liver cirrhosis were investigated. The mean ascitic fibronectin level was lower(p<0.01) in cirrhosis (32.8±16.7 μg/ml) than in malignancy (138.6±62.3 μg/ml) and tuberculosis (191.5±40.8 μg/ml) The mean value of ascitic adenosine deaminase activities was higher (P<0.01) in tuberculosis (68.1±37.2U/L) than in malignancy (11.4±5.9U/L) and cirrhosis (2.3±2.5 U/L) Fibronectin levels and adenosine deaminase activities did not show significant overlap among these 3 groups, while several other parameters analyzed simultaneously in these samples showed considerable overlap, By discriminant analysis, the fibronectin level was the best indicator for differentiation of tuberculous from cirrhotic ascities (% correctly classified 100) and the adenosine deaminase activity was the best indieator in the differential diagnosis of malignant and tuberculous ascities (% correctly classified=95.2). In differentiating the 3 groups, simultaneous determination of the fibronectin level and adenosine deaminase activity had the best discriminant ability (% correctly claseified=89.7) The results suggest that determination of both fibronectin level and adenosine deaminase activity in ascitic fluid may improve the accuracy of differential diagnosis of ascites.

지속성 각막상피결손 환자에서 autologous fibronectin 국소점안의 효과

남문진(Moon Jin Nam),김기산(Ki San Kim),오준섭(Joon Sup Oh),박낭운(Rang Woon Park),김인산(In San Kim),조준승(Joon Seung Jo) 대한안과학회 1989 대한안과학회지 Vol.30 No.1

肺纖維化 過程中 Collagen 및 Fibronectin의 遺傳子 發現과 Collagen 分劃의 變動

조준찬,김인산,조준승 慶北大學校 醫科大學 1990 慶北醫大誌 Vol.31 No.3

Bleomycin-induced lung damage is a well-characterized experimental model for studying pulmonary fibrosis. This model was used to examine α1(Ⅰ), α1(Ⅲ) procollagen and fibronectin mRNA levels and changes of collagen fractions during the development of pulmonary fibrosis. Rats were given 1 unit of bleomycin intratracheally and sacrificed at 4 days, 8 days, and then every week for 4 weeks. The lung tissue was assayed for collagen content by measuring total hydroxyproline content. Characteristics and specific increase of procollagen mRNAs and fibronectin mRNA were determined by northern blot and dot blot hybridization. There was a significant increase of hydroxyproline content of lung tissue following 2 weeks of bleomycin treatment. Northern blot analysis identified a major(5.3kb) and a minor (7.1kb) α1(Ⅰ) procollagen mRNA species and a single fibronectin mRNA species of 8.8kb in size. There was a significant difference among the three mRNAs in the relative increase and the time of maximum accumulation. While the accumulation of procollagen mRNAs was maximum between the 8 days and 14 days, that of fibronectin mRNA peaked in the first week after bleomycin treatment. The procollagen α1(Ⅰ) mRNA accumulated most dramatically(tenfold above the levels in the control lungs) compared with fourfold increases of mRNAs coding α1(Ⅲ) procollagen and fibronectin. In conclusion, specific increases in procollagen mRNAs in lung fibrosis precede increase of tissue collagen content. Type Ⅰ collagen may accumulate much more than type Ⅲ collagen, and the metabolism of tissue fibronectin may be perturbed during pulmonary fibrosis.

흰쥐의 Bleomycin 誘發 肺纖維化 過程에서의 Collagen과 Glycosaminoglycan의 變動

金仁山,鄭泰浩,曺準承 慶北大學校 醫科大學 1990 慶北醫大誌 Vol.31 No.3

폐섬유화 과정에서 collagen과 GAG의 변동을 조사하기 위하여 bleomycin을 기관지를 통하여 흰쥐에 투여하여 폐섬유증을 유발한 후 경시적으로 collagen과 GAG의 변동을 조사하였다. 총 collagen량은 7일군에서는 변화가 없었으며 15일군에서부터 증가하여 계속 증가를 나타내었다. 총 GAG량은 7일군에서부터 유의성있게 증가하여 15일군에서는 약 100%의 증가율을 보였으며 30일군에서는 50%의 증가율을 나타냄으로써 감소경향을 보였다. 총 GAG에 대한 HA의 백분률은 전실험기간 동안 별다른 변화가 없었으며 Chs-A와 Chs-C의 백분률은 초기에 증가하였고 DS는 15일군부터 유의성있는 증가를 나타내어 폐섬유화 과정에서 collagen 증가시기와 비슷함을 보여줬다. H/HS의 백분률은 초기부터 감소하였다. 이상의 결과로써 폐섬유화 과정중 collagen과 GAG는 현저한 변화를 보였으나 그 의미와 상호관계는 알 수 없었으며 단지 GAG의 증가가 collagen의 증가보다 선행하는 것으로 보아 GAG가 collagen의 침착에 영향을 끼칠 것으로 사료되며 특히 DS는 collagen의 증가 시기와 비슷한 것으로 미루어 collagen과 밀접한 관계를 가질 것으로 사료된다. In order to determine the changes of collagen and glycosaminoglycans during the course of development of interstitial pulmonary fibrosis, a single intratracheal injection of 1.5 unit of bleomycin was given in rats. Total lung collagen contents were increased at 15 days after bleomycin administration and this increase was continued to 60 days. Total lung glycosaminoglycan contents were significantly increased at 7 days, maximally increased at 15 days and then declined. Chondroitin sulfate A and C fractions and dermatan sulfate fraction were both increased in fibrotic lungs but the former at an early stage and the latter at a later stage. On the contrary, heparin/heparan sulafe fraction was decreased in fibrotic lungs at an early stage, and hyaluronic acid fraction remained unchanged during the fibrotic changes of lung. The results indicate that the changes of glycosaminoglycans precede those of collagen, so glycosaminoglycans may ffect the accummulation of collagen fibrils.

血淸 PZ-Peptidase의 酵素 活性度 測定法

曺永燮,金仁山,曺準承 慶北大學校 醫科大學 1987 慶北醫大誌 Vol.28 No.4

혈청 PZ-peptidase 활성도의 비색측정법을 개선하기 위해 여러가지 측정조건을 검토하였다. 기본원리는 PZ-peptide을 기질로 하여 효소작용 후 생긴 PZ-Pro-Leu을 benzene으로 추출하여 320㎚에서 흡광도를 잼으로써 효소 활성도를 측정하는 것이다. 10mM의 염화칼슘과 0.2mM의 dithithreitol을 기질액에 첨가시킴으로써 29%의 효소 활성도 증가를 얻었으며, 반응시간과 혈청량도 각각 30분과 100㎕로 감소시켰다. 유리된 PZ-Pro-Leu의 흡광도는 2시간까지 변하지 않는 안정된 값을 보여주었으며 본 방법의 정밀도 측정에서는 변이계수가 1.5%에서 5.1% 사이에 있었다. 건강한 남녀에서 채취한 혈청 PZ-peptide의 평균편차는 14.70이었다. 본 방법은 수기가 간단하고 시간이 오래 걸리지 않으며 정밀도도 우수함으로 임상에서 질병의 진단 및 예후와 치료효과 판정에 필요한 PZ-peptidase 활성도 변화 측정에 보다 적합한 방법이라 사료된다. The appropriate conditions for determining PZ-peptide in human serum are described. The method is based on monitoring the absorption at 320㎚ of PZ-Pro-Leu enzymatically formed from the substrate. PZ-I-Pro-l-Leu-Gly-1-Pro-d-Arg. We tested two enzyme activators, calcium chloride and dithiothreitol to obtain higher enzyme activity and found that 10 mM calcium chloride and 0.2mM dithiothreitol was suitable for maximal activity. This method requires only thirty minutes of incubation time and 100 ㎕ of serum. The normal level of serum PZ-peptidase from healthy donors was 53.05±14.70 The within-day and CV% ranged from 1.5 to 5.1%. Its simplicity, rapidity, rapidity and precision make the proposed method very suitable for routine work and clinical investigation.

섬유아세포의 증식과 Collagen 합성에 대한 Prostaglandin E2 의 조절작용

조준승,김인산 생화학분자생물학회 1993 BMB Reports Vol.17 No.3

PGE₂ has been known to modulate diploid human fibroblast proliferation and collagen production by intracellular cyclic AMP mediated pathway but it still remains unclear that the suppressive effect of PGE₂ on proliferation and collagen synthesis are mediated by the elevated intracellular cyclic AMP levels. The purpose of present study was to determine the effects of PGE₂, db-cAMP or cholera toxin on cell proliferation and collagen production and to study how the temporal changes of intracellular cyclic AMP levels caused by PGE₂ are associated with cell proliferation and collagen production. And we also hypothesized that lengthy exposure of fibroblast cultures to prostaglandins could lead to overgrowth of the insensitive cells which might be responsible for fibrotic diseases. At least, 48 hours of incubation were required to suppress the cell proliferation by PGE₂, and under the same conditions there were no changes of intracellular cyclic AMP levels during 72-hour incubation of PGE₂. 48- and 72-hour incubation of PGE₂ showed dose- and time-dependent suppression of the cell proliferation. Dibutyryl cyclic AMP and cholera toxin appeared to suppress cell proliferation by even 24 hours incubation and they also showed dose- and tme-dependent suppression of the cell proliferation. Cholera toxin increased intracellular cyclic AMP levels maximally after one hour and then decreased its level but still maintained its elevated state for 24 hours. Short-term incubation of dibutyryl cyclic AMP or cholera toxin for six hours wass enough to suppress the cell proliferation. Total protein and collagen production were suppressed by PGE₂, dibutyryl cyclic AMP or cholera toxin in a dose-dependent manner and their effects seemed to be associated with intracellular cyclic AMP levels which were elevated by PGE₂ or cholera toxin. One hour incubation of PGE₂ was enough to suppress total protein and collagen production by fibroblasts. Judging by the fact that treatment of PGE₂ or dibutyryl cyclic AMP decreased the steady state levels of mRNAs for procollagens and fibronectin and that co-treatment with actinomycin D didn't show any difference in the steady state levels of each mRNA, the inhibitory actions of PGE₂ and dibutyryl cyclic AMP on the fibronectin and collagen production seem to occur at transcriptional level. Long-term exposure for PGE₂ could result in selection of subpopulation of fibroblasts which showed less responsiveness to reexposure of PGE₂ in the cell proliferation but not in collagen production. These results indicate that prostaglandin E₂ could suppress the proliferation of human fibroblast cells by cyclic AMP-independent events and by which some subpopulations of fibroblasts might be selected to overgrow when exposed to PGE₂ for a long time and also indicate that PGE₂ could suppress total protein and collagen production at the transcriptional level by cyclic AMP-dependent events.

肝纖維化過程의 血淸 N-Acetyl-β-D-Glucosaminidase 活性化變化와 肝膠原質含量과의 關係

趙鎭煥,金仁山,孫健永,曺準承 慶北大學校 醫科大學 1990 慶北醫大誌 Vol.31 No.3

We evaluated serum N-acetyl-β-D-glucosaminidase and PZ-peptidase activities as possible markers reflecting fibrotic activity in fibrotic rat liver induced by common bile duct double ligation and excision and we also studied the correlation of liver collagen content and both enzymes activity. Serum NAG activity gradually increased as post-operation days increased, but activity at 28 days after operation showed abruptly increased level, 38.33 nmole/min/ml which was significantly higher than that of control, 21.95 nmole/min/ml (p<0.001). Liver collagen content changes during fibrogenesis also showed a pattern similar to that of serum NAG activity changes. Therefore we tested correlation coefficiency between serum NAG activities and liver collagen contents, and found that they were highly correlated with correlation coefficient value, r=0.90 (p<0.001). On the other hand, we concluded that serum PZ-peptidase activity would not be suitable for a marker because of its great individual variations and its lack of constant trend among groups. Conclusively, we confirmed that measurement of serum NAG activity be very useful for the early diagnosis of liver cirrhosis and monitoring its fibrotic activity.