• Restorative Dentistry and Endodontics
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• v.26 no.3
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• pp.191-199
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• 2001

목적 - 기질금속단백분해효소(matrix metalloproteinase)는 조직의 염증 및 치유과정에서 숙주세포에서 생성, 분비되어 세포외기질(extracellular matrix)의 분해에 작용한다. 다양한 염증반응에 기질금속단백분해효소가 중요한 역할을 하는것으로 보고되고 있으나 치수 및 치근단 질환에서의 그 역할은 거의 알려져 있지 않은 상태이다. 본 연구에서는 염증이 있는 사람의 치수 및 치근단 조직을 채취하여 Enzymeimmunoassay 및 면역조직화학적 검색을 통해 제1형, 2형, 3형 기질금속단백분해효소의 수준 및 그 분포를 측정하여 치수 및 치근단 병소에서 이 효소의 작용을 알아보는 것을 목적으로 한다. 방법 - 연구재료는 근관치료를 위해 서울대학교 병원 치과 진료부 보존과에 내원한 환자를 대상으로 34개의 치아에서 통상의 근관치료 중 발수한 치수조직과 치근단 수술중 얻은 치근단 병소(n=10)를 이용하였다. 치수는 발수 전에 임상진단을 통해 급성 치수염(n=12), 만성 치수염(n=12), 정상 치수(n=10)로 구분하고 정상치수로 진단된 것을 대조군으로 설정하였다. 채취된 표본은 둘로 나누어 절반은 30분 이내에 5$\mu\textrm{m}$ 두께로 동결절단을 시행하여 조직표본을 제작하였고 deep freezer에 보관하였다가 헤마톡실린-에오신 염색 및 면역조직화학적 검색을 시행하였다. 나머지 조직은 ELISA를 위해 액체 질소에 보관하였다. ELISA를 시행하기전 표본의 단백질 정량을 시행하여 모든 표본의 단백질 양을 50mg/$\mu\textrm{l}$로 일치시키고 Amersham사의 ELISA kit를 사용하여 제1형, 2형, 3형의 기질금속단백분해효소의 양을 측정하였으며 그 결과를 Mann-Whitney U test를 사용하여 각 군간의 통계학적 유의성을 검증하였다. 결과 1. ELISA의 결과 제1형 기질금속단백분해효소의 농도는 모든 실험군에서 대조군보다 유의성있게 높게 나타났다.(p<.05). 또한 급성치수염군의 제1형 기질금속단백분해효소의 농도가 다른 실험군보다 유의성있게 높았다(p<.05). 2. 제2형 기질금속단백분해효소의 경우 급성치수염군과 대조군에서만 유의성있는 차이를 보였다(p<.05). 3. 제3형 기질금속단백분해효소의 경우 급성치수염군에서 대조군이나 만성치수염군보다 유의성 있는 높은 수치를 보였다(p<.05). 4. 면역조직화학검색 결과 염증성 치수에 존재하는 급성 및 만성염증세포 주위로 기질금속단백분해효소에 대한 면역 반응이 존재하였으며 주로 제1형과 제3형 기질금속단백분해효소의 경우 대식세포 및 림파구 주위로 강한 발색제의 침윤양상이 관찰되었다. 5. 치근단병소의 면역조직화학적 검색 결과 만성염증 세포 주변으로 미약한 발색제의 침윤양상이 관찰되었다.

• Journal of Periodontal and Implant Science
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• v.36 no.3
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• pp.579-590
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• 2006

1. 연구배경 교원질 분해작용을 하는 호중구의 세포질 효소인 기질금속단백분해효소-8은 치주질환, 류마티스 관절염, 그리고 궤양결장염과 같은 염증성 질환에서 농도가 증가한다고 알려져 있다. 최근에는 A. actinomycetemcomitans의 leukotoxin이 사람호중구에서 기질금속단백분해효소-8의 분비를 유도하는 것이 보고되었다. 이 연구의 목적은 선천면역 체계에서 세포표면 항원무리14, Toll-like 수용기, 그리고 $NF-{\kappa}$ B경로를 통하여 A. actinomycetemcomitans의 지질다당질로 유도된 기질금속단백분해효소-8의 분비 여부와 세포기전을 알아보고자 하였다. 2. 연구재료 및 방법 건강한 개인 제공자(남자 13명, 여자 3명)로부터 얻은 개개인의 20ml 말초혈액을 제조사의 지침에 따라 호중구를 추출한 후 항세포표면 항원무리14와 함께 $4^{\circ}C$에서 30분간 전배양 한 후, $37^{\circ}C$에서 9시간 동안 배양시켰다. 추출한 호중구에 Toll-like 수용기 억제제 또는 $NF-{\kappa}$ B억제제인 TPCK를 첨가한 후 $37^{\circ}C$에서 1시간 동안 전배양하고 $37^{\circ}C$에서 9시간 동안 배양시켰다. 호중구에 세포뼈대 억제제인 cholchicine, nocodazole, demecolcine, 그리고 cytochalasin B를 A. actinomycetemcomitans의 지질다당질과 함께 $37^{\circ}C$에서 9시간 동안 배양시켰다. 기질금속단백분해효소-8 분비량은 효소면역측정법을 통해 결정하였다. 통계처리는 일원배치 분산분석법을 이용하였다(p<0.05). 3. 결과 A. actinomycetemcomitans 지질다당질은 기질금속단백분해효소-8의 분비를 증가시켰다. 기질금속단백분해효소-8의 분비는 항세포표면 항원무리14에 의해서 억제되었지만, 항 Toll-like 수용기2, 항 Toll-like 수용기4 항체는 억제시키지 못했다. $NF-{\kappa}$ B 억제제는 A. actinomycetemcomitans의 지질다당질로 유도된 $NF-{\kappa}$ B 결합 활성도와 기질금속단백분해효소-8 분비를 억제하였다. 미세섬유 중합반응 억제제는 A. actinomycetemcomitans의 지질다당질로 유도된 기질금속단백분해효소-8의 분비를 억제시켰으나, 미세관 중합반응억제제는 억제시키지 못했다. 4. 결론 위의 연구결과를 종합하여 볼 때, 기질금속단백분해효소-8은 A. actinomycetemcomitans의 지질다당질로 유도되며, 세포표면 항원무리-$NF-{\kappa}$ B 경로를 통하여 분비되고, 이 분비 과정은 미세섬유 계통이 관여하는 것으로 보인다.

• Radiation Oncology Journal
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• v.21 no.1
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• pp.66-74
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• 2003

Purpose : The matrix metalloprotelnases (MMPs) are a family of enzymes whose main function is the degradation of the extracellular matrix. Several studies have revealed that MMPs and TIMPS are related to the wound heating process and in photoaging caused by ultraviolet Irradiation. However, the expressions of MMP and TIMP after irradiation have not, to the best of our knowledge, been studied. This study investigates the expressions of MMP-2 and TIMP-2 in rat Intestinal mucosa following irradiation. Materials and Methods : The entire abdomen of Sprague-Dawley rats was irradiated using a single dose method. The rats were sacrificed on day 1, 2, 3, 5, 7 and 14 following irradiation. Histopathological observations were made using hematoxilin & eosin staining. The expressions of MMP-2 and TIMP-2 were examined using immunohistochemistry, Irnrnunoblotting and ELISA. Results : Radiation induced damage associated with atrophic villi, and infiltration of inflammatory cell was observed from the first postirradiation day, and severe tissue damage was observed on the second and the third postirradiation days. An increase in mitosis and the number of regenerating crypts, as evidence of regeneration, were most noticeable on the fifth postirradiation day. From the immunohistochemlstry, the MMP-2 expression was observed from the first postirradiation day, but was most conspicuous on the third and the fifth postirradiation days. The TIMP-2 expression was most conspicuous on the fifth postirradiation day. From the irnrnunoblotting, the MMP-2 expression was strongly positive on the third postirradlatlon day, and that of TIMP-2 showed a strong positive response on the fifth postirradiation day. In ELISA tests, the expressions of MMP-2 and TIMP-2 were increased in the postirradiation groups compared to those of the normal controls, and showed a maximum increase on the fifth postirradiatlon day. These results were statistically significant. Conclusion : The expressions of MMP-2 and TIMP-2 were increased in the intestinal mucosa of the rats following irradiation, and these results correlated with the histopathological findings, such as tissue damage and regeneration. Therefore, this study suggests that MMP-2 and TIMP-2 play roles in the mechanisms of radiation-induced damage and regeneration of intestinal mucosa of rats.

• Tuberculosis and Respiratory Diseases
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• v.59 no.5
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• pp.517-521
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• 2005

Background : The balances of the proteinases and antiproteinases system have been implicated in the pathogenesis of various exudative pleural effusions. The aim of this study was to examine the matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) levels in exudative pleural effusions. Methods : The study included 33 tuberculous effusions, 17 malignant, and 5 transudates. The pleural levels of MMP-1 and TIMP-1 were determined using a commercially available ELISA assay. Results : The group of tuberculous effusions showed higher pleural MMP-1 levels than the malignant and transudates. The pleural TIMP-1 levels of the tuberculous and malignant effusions were higher than the transudates. Conclusion : Elevated pleural MMP-1 and TIMP-1 levels were found in tuberculous effusions.

• The Journal of Natural Sciences
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• v.14 no.2
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• pp.55-65
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• 2004

A thiol-specific antioxidant(TSA) protein was purified from Earthworm, Lumbricus terrestris by DEAE-Cellulose, Phenl sepharose, Sephacryl S-200 gel filtration and HPLC S-300 Column Chromatography. This protein showed a thiol-specific antioxidant activity against inactivation of glutamine synthetase by a metal-catalysed oxidation system capable of generation reaction oxygen species. The molecular mass of the protein was determinated to be 51-kDa by SDS-polyacrylamide gel electrophores. Taken together, the purified TSA protein could be a new member of TSA family.

• The Journal of Internal Korean Medicine
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• v.33 no.4
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• pp.405-417
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• 2012

Objectives : In the present study, anti-metastatic properties of the methanol extract of L. obtusiloba (MLO) were evaluated. Methods : To determine the effect of MLO on cancer metastasis, we checked matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) activities and expressions in B16F10 melanoma cells. In addition, we performed cell migration assay as well as invasion assay using Matrigel. Finally, we used an in vivo lung metastasis model to confirm the anti-metastatic activity of MLO. Results : 1. MLO showed potent inhibitory effects on MMP-2 and MMP-9 activities and expressions via down-regulation of activation of NF-${\kappa}B$ in B16F10 melanoma cells. 2. Melanoma cell migration and invasion were down-regulated by MLO treatment. 3. Not only in vitro model, but MLO also significantly suppressed lung metastasis in vivo. Conclusions : The present results indicate that MLO has strong inhibitory effect on cancer metastasis. Therefore, L. obtusiloba could be a valuable anti-metastatic agent.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.31 no.5
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• pp.631-636
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• 1998

Candida lipolytica(C. lipolytica) FM5 was selected as one of the strong saprophytic yeasts isolated from mackerel (Scomber japonicus). The selected strain could produce extracellular proteolytic enzyme. The effective medium for production of proteolytic enzyme by C. lipolytica FM5 was TPPY broth containing Bacto-tryptone $0.5\%$, proteose peptone $0.5\%$, yeast extract $0.25\%$, NaCl $0.5\%$ and $CaCl_2\;0.2\%$. The pattern of proteolytic enzyme production by C. lipolytica FM5 was the almost same as that of growth curve of the strain. Namely, the enzyme production was begun from the early stage of exponential phase and it was reached the highest at the begining of the stationary phase of the yeast growth. The optimum toeperature of the produced proteolytic enzyme was $35^{\circ}C$ and its activity was not significantly changed by pH between 6.5$\~$9.0 and also it was not significantly affected by several kinds of cations such as $Ca^{2+},\;Cu^{2+},\;Fe^{2+}$ and $Mg^{2+}$ but it was affected negatively by some cations such as $Zn^{2+},\;Mn^{2+}$ and $K^+$.

• Journal of Life Science
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• v.15 no.2 s.69
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• pp.231-235
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• 2005

Matrix metalloproteinases (MMPs) are a family of structurally and functionally related zinc-dependent enzymes responsible for proteolytic degradation of extracellular matrix components such as base membrane or interstitial stroma. MMPs play an important role in a variety of physiological and pathological tissue remodeling processes, including wound healing, embryo implantation, tumor invasion and metastasis. Since MMP-9 (gelatinase B) has unique ability to cleave type IV collagen, gene expression of MMP-9 has been focused on as a pharmacological target. Flavonoids are a class of compounds that are widely spread in plants. In the coures of screening for the suppressors of MMP-9 gene expression from natural products, Metasequoia glyptostroboides was selected. Six flavonoids, sciadopitysin, isoginkgetin, bilobetin, 2,3-dihydrohinokiflavone, luteolin and apigenin were purified as suppressors of MMP-9 gene expression from M. glyptostroboides. The suppressing activity of the isolated flavinoids on the MMP-9 gene expression was measured by gelatin zymography and Nothern blot analysis.

• Korean journal of food and cookery science
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• v.17 no.5
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• pp.497-502
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• 2001

능이버섯 〔Sarcodon aspratus(Berk.) S.Ito〕으로부터 단백질 가수분해 효소를 추출하여 75%(NH$_4$)$_2$SO$_4$ 염석과 DE52 anion exchange column chromatography 와 sepharyl-S 200 column 및 Mono s column chromatography 에 의해 정제하였는데 조 효소의 특이 활성은 55.2U/mg protein으로 조효소액에 비하여 11.26배 증가하였고 수율은 49.5%로 나타났다. 정제된 효소는 전기영동을 행한 결과 단일 band를 나타내었으며 분자량은 29,300으로 추정되었다. pH의 안정성은 4$^{\circ}C$에서 48시간 보존하였을 때 pH 8.5에서 가장 안정하였고, pH 5.5~10.5까지 높은 활성을 유지하였다. 온도에 대한 안정성은 30분간 보존 한 후 활성을 검토한 결과 단백분해능은 5$0^{\circ}C$까지 비교적 처음활성을 유지하다가 6$0^{\circ}C$에서는 53%정도의 활성이 유지되었으나 그 이상의 온도에서는 급격히 실화하여 7$0^{\circ}C$에서는 완전히 실화하였다. 금속 이온에 의해서는 크게 저해를 받지 않았으나 PMSF 저해제에 대하여 저해되어 본 효소가 serine protease임을 시사하였다.

• Biomedical Science Letters
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• v.4 no.1
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• pp.1-9
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• 1998

A Corynebacterium diphtheriae iron-repressible gene dirA, that was homologous to TSA of Saccharomyces cerevisiae and AhpC subunit of Salmonella typhimurium alkyl hydroperoxide reductase, was amplified with PCR and expressed in E. coli. The DirA purified from the transformed E. coli crude extracts prevented the inactivation of enzyme caused by metal-catalyzed oxidation (MCO) system containing thiols but not by ascorbate/Fe$^{3+}$/$O_2$ MCO system. The DirA concentration, which inhibited the inactivation of glutamine synthetase by 50% (IC$_{50}$) against MCO system, was 0.12 mg/ml. The multimeric forms of DirA were converted to the monomeric form in SDS-PAGE under the thioredoxin system comprised of NADPH, Saccharomyces cerevisiae thioredoxin reductase, and thioredoxin. Also, DirA showed thioredoxin dependent peroxidase activity. All of these results were consistent with the characteristics of a thiol specific antioxidant (TSA) protein having two conserved cysteine residues.