한국인 제2형 당뇨병 환자의 골격근에서 인슐린 신호전달체계의 결함

최준혁,이관우,김효정,이동훈,이종우,김정은,엄현채,김경미,최성이,정윤석,김현만 대한내분비학회 2002 Endocrinology and metabolism Vol.17 No.5

연구배경: 제2형 당뇨병에서 나타나는 인슐린 저항성은 간, 지방, 근육 같은 말초조직에서 인슐린의 작용이 떨어지는 것을 말한다. 제2형 당뇨병의 발생기전에 인슐린 저항성과 인슐린 분비능 저하가 같이 관여함은 잘 알려진 사실이다. 인슐린의 세포내에서의 저항성을 규명하기 위한 인슐린의 세포내 신호전달체계에 대한 연구가 활발히 진행되고 있으며 몇몇 연구에서 인슐린 저항성을 가진 골격근육에서 IRS와 관련된 PI3-kinase의 활성감소와 Akt kinase의 활성감소를 보고하고 있으나 아직까지 명확하게 그 기작이 설명되어지지 않고 있다. 본 연구에서는 정상성인, 제2형 당뇨병 환자를 대상으로 경구당부하검사 및 인슐린 클램프검사를 시행하고 인슐린 클램프 검사시 대상인의 근육을 채취하여 인슐린 신호전달 체계(IR-β, IRS, Akt(PKB, Rac) kinase, GSK-3)를 연구하였다. 방법: 연구대상자는 한국인으로서 경구당부하검사상 정상인 및 당뇨병 환자 각각 11명, 9명을 대상으로 하였으며, 정상인은 건강인으로 과거력상 당대사에 영향을 줄 질환이 없고, 현재 당대사에 영향을 줄 약물 복용 및 다른 소견이 없는 경구당부하검사상 정성 내당능을 보이는 대상자로 하였다. 당뇨병환자는 모두 제2형 당뇨병 환자로 이환기간이 만 5년 이내인 경우로 하고, 인슐린으로 치료하는 대상자는 제외하도록 하였다. 대상자의 연령, 체질량지수, 체지방량, 공복시 총콜레스테롤, 중성지방, 고밀도지단백 콜레스테롤 및 HbA1c, 인슐린, C-peptide를 측정하였고, "HOMA model"을 이용하여 베타세포의 기능와 인슐린저항성 정도를 평가하였다. 정상혈당클램프 검사(euhlycemic hyperinsulinemic clamp test)를 시행하고 인슐린 투입 전과 인슐린 투입 30분후, 두차례에 걸쳐 대퇴부 근육생검을 실시한후 western 법으로 IR-β, IRS, Akt kinase, GSK-3의 인산화량을 측정하였다. 결과: 정상인 11명과 당뇨병 환자 9명을 대상으로 하였으며 대상자의 평균 연령, 평균 체질량지수, 체지방량, 허리/엉덩이 둘레의 비(waist hip ratio; WHR)는 두 군간에 차이가 없었다. 평균 공복혈당은 정상군 98.0±0.3㎎/dL, 당뇨병군 208.1±16.5㎎/dL(p<0.05), HbA1c는 정상군 5.4±0.5%, 당뇨병군 9.2±0.6%(p<0.05)였다. "HOMA model"을 이용한 베타세포의 기능과 인슐린저항성은 정상군 56.4±8.5%, 1.4±0.2, 당뇨병군 72.2±52.3%(p<0.01), 10.2±6.3(p<0.01)였으며 정상혈당클램프 검사상 포도당 이용률은 정상군 8.2±0.6㎎/㎏/min, 당뇨병군 3.7±1.1㎎/㎏/min(p<0.01)로 정상군과 당뇨병군간에 유의한 차이를 나타내었다. Western blot법으로 IR-β, IRS, Akt kinase, GSK-3의 인산화량을 인슐린 투입전과 인슐린 투입 30분후 측정하였을 때 IR-β에서는 정상군이 103.9±2.3에서 241.3±18.6, 당뇨군이 108.9±2.2에서 198.7±6.3으로 증가하였다(p=NS). 이는 인슐린 투입후 당뇨병군이 정상군과 비교하여 인산화량의 증가정도가 통계적인 차이가 없었다. IRS에서는 정상군이 111.6±7.3에서 295.6±17.2, 당뇨군이 114.5±6.1에서 222.0±23.2로 증가하였으며(p<0.05), 인슐린 투입후 당뇨병군이 정상군과 비교하여 인산화량의 증가정도가 약 24% 감소하였다. Akt kinase에서는 103.4±6.0에서 416.8±29.5로 증가하였으며(p<0.01), 인슐린 투입후 당뇨병군이 정상군과 비교하여 인산화량의 증가정도가 약 43% 감소하였다. GSK-3에서는 정상군이 107.7±6.7에서 595.7±28.1, 당뇨군이 104.3±4.8에서 443.3±12.9로 증가하였으며(p<0.01), 인슐린 투입후 당뇨병군이 정상군과 비교하여 인산화량의 증가정도가 약 25% 감소하였다. 결론: 제2형 당뇨병 환자에서 정상인과 비교하여 인슐린 자극후 IR-β는 인산화량의 증가정도가 큰 차이가 없었으나, IRS, Akt kinase, GSK-3에서는 인산화량의 증가 정도가 감소되는 것으로 보아 상위 인슐린 신호전단체계부터 결함이 있는 것으로 생각된다. Background: The glucose uptake rate is the limiting step in glucose utilization and storage. The failure of insulin to stimulate glucose uptake in muscle appears to be a primary defect of insulin resistance. This study was undertaken to examine the effect of physiological hyperinsulinemia on the phosphorylation of the insulin receptor (IR-β), insulin receptor substrate (IRS), Akt kinase and GSK-3 in isolated skeletal muscle, in people with type 2 diabetes(n=9) and control subjects(n=11). Methods: 75g OGTT and euglycemic hyperinsulinemic clamp test were done. And vastus lateralis muscle was obtained before and 30 min into the euglycemic clamp. Western blots were performed for tyrosine phosphroylation of insulin receptor substrate (IRS) and phosphorylation of the insulin receptor(IR-β), Akt and GSK-3. Result: There were no statistical differences in the mean age, BMI and body fat between the control subjects and diabetic patients. The fasting blood sugar and HbA_1c in controls and diabetic patients were 98.0±0.3 and 208.1±16.5ng/dl, and 5.4±0.5 and 9.2±0.6%, and 1.4±0.2 in the control subjects, and 72.2±52.3%(p<0.01) and 10.2±6.3(p<0.01) in the diabetic patients, respectively. The insulin resistance from the euglycemic hyperinsulinemic clamp test were 8.2±0.6㎎/㎏/min and 3.7±1.1ng/㎏/min in the control subjects and in the diabetic patients, respectively(p<0.01). Compared with the normal controls, insulin-stimulated IR phosphorylation was no different to that in the diabetic patients. However, insulin-stimulated IRS phosphorylation, insulin-stimulated Akt phosphorylation and insulin-stimulated GSK-3 phosphorylation were reduced in the diabetic patients compared with the normal controls by 24, 43 and 25%, respectively(p<0.05). Conclusion: In Korean type 2 diabetic patients, the insulin resistance may be due to the impairment of the upstream insulin signal molecular network. Further studies will focus on determining whether these signaling defects are the cause of the development of insulin resistance, or secondary to the altered metabolic state, associated with type 2 diabetes mellitus (J Kor Soc Endocrinol 17:685∼697, 2002).

비만 펩티드 백신의 효능에 영향을 미치는 구조적 요소

김형진,이희종,김효준 한양대학교 이학기술연구소 2007 이학기술논문지 Vol.10 No.-

Apo B-100 is a constitutive component of low density lipoprotein (LDL) of which functions in packaging, transport, and absorption of lipids. Extortive binding of anti B4-antibody onto Apo B-100 should inhibited the functions of Apo B-100. Previously, we had showed that the peptide B4 induced antibodies recognized Apo B-100 and thus elicited anti-obesity effect. In this study, we compared the efficacy of peptide vaccine against obesity and of antibody inducing immune response by introducing additional peptides fused to B₄ Peptide. Previously, we had constructed B₄T, introducing additional peptides fused to the C-terminal of B₄ peptide, and BTB, fused to the middle of B₄ peptide, with preS2 of HBV. Then we manufactured vaccines, and vaccinated on C57BL/6 mouse. We measured weight of mice 3 days a week and, after vaccinating, we gathered blood and performed ELISA in interval 2 weeks for indicating antibody inducing immune response. Among these two artificial vaccines, B₄T was better Vaccine against obesity than another. But BTB was batter efficient inducer of the antibody against B₄. These result explains that B₄ tetramer is very crucial structural factor to operate as an obesity vaccine. Apo B-100은 low-density lipoprotein의 구성 요소로서 지방의 패키징, 운송, 흡수를 담당한다. App B-100에 anti B4-antibody의 인위적 결합은 이러한 Apo B-100의 기능을 저해할 것이다. 선행 연구에서 B4 펩티드에 의해 유도된 antibody가 Apo B-100을 인지하고 그에 따라 비만억제 효과를 나타내는 것을 확인하였다. 본 연구는 비만에 대한 B cell epitope을 4번 반복한 B4 펩티드에 추가적인 펩티드를 연결한 두 가지의 백신을 사용하여 효능을 비교하였다. HBV의 preS2를 B4의 C-terminal에 연결한 B₄T와 중간에 삽입시킨 BTB를 제조하여 C57BL/6 마우스에 주사하였다. 일주일에 3회 마우스의 체중을 측정하였고, 백신 주사는 2주 간격으로 4회 실시하였으며, 백신 주사 후 2주 간격으로 혈액을 채취하여 면역 반응에 의해 유도된 antibody를 indirect Enzyme -Linked Immunosorbent Assay에 의해 측정하였다. 두 가지 백신 사이에서 체중 증가 억제율로 본 비만에 대한 백신효과는 B₄T가 더 우수하였다. 그러나 B₄T에 비해 BTB가 유도된 antibody의 titer와 혈중 지속 시간이 더 우수했다. 이런 결과는 B펩티드가 항체유도에 있어서 중요하지만, 비만억제 기능에 있어서 B₄ tetramer가 매우 중요한 인자임을 입증하였다.

면역증강제로서의 Phellinus 屬菌의 항암 면역활성

김호정,김효준 한양대학교 이학기술연구소 1999 이학기술연구지 Vol.1 No.-

버섯류의 약리효과에 관한 연구로 버섯 중에 함유되어 있는 다당체들의 항암 및 면역 조절 작용 등과 관련하여 많은 연구가 진행되고 있으며, 최근에는 상황(桑黃)버섯으로 알려진 목질진흙버섯(Phellinus linteus)의 항암 약리작용이 관심의 대상이 되고 있다. 본 연구에서는 국내에서 채집한 P. igniarius(P-1), P. peni(P-2), P. robustus(P-3), P. linteus(p-5), P. B(P-8)의 5종류의 Phellinus 속균에 대하여 위암세포인 DLD-1 및 KATO Ⅲ, 대장암세포인 AGS와 같은 고형암 및 혈액암이 Jurkat T lymphma에 대하여 현미경적 관찰에 의한 약물효능 경시효과 및 효소분광학적 방법에 의한 유효농도(ED_(50))를 조사하여 항암효과를 평가하였다. 그 결과, DLD-1 및 DATO Ⅲ에 대하여 P. robustus(P-3), P. linteus(p-5), P. B(P-8)의 세 종류만이 40 ㎍/㎖의 범위에서 ED_(50)가 인정되었으며, AGS 및 Jurkat T cell의 경우에는 5종의 Phellinus spp. 모두에 있어서 각각 750, 180 ㎍/㎖의 높은 농도로 확인 되었다. 한편, 암세포 생자에 미치는 Phellinus spp.의 투여 효과를 검토한 결과, 처리하지 않은 대조군 암세포의 경우 30일 이상 생장을 지속하는 것에 비하여, Phellinus spp.를 200, 1000 ㎍/㎖ 처리한 경우 5가지 속균 모두에 있어서 암세포의 종류에 무관하게 3-7일의 단기간에 90%이상의 암세포가 사멸되는 것을 확인하였다. 이상의 결과로부터 Phellinus spp.는 위암세포에 특이적 효과를 보이는 것으로 나타났으며, 생체 내 함유량이 높은 경우에는 전반적으로 항암 효과를 나타내는 것을 확인하였다. Medicinal efficacy of mushrooms were focused on the anti-tumor immune regulatory effects of polysaccharieds in the pilcus of fruiting bodies. Recently, the Phellinus linteus is one of the most highlighted fungi for its remedial results. Five isolates in Phellinus spp., namely P. ingiarius, P. peni, P. robustus, P. linteus, and P. B were studied to evaluate the anti-tumor efficacy against several tumor cell lines including stomach cancer derived DLD-1 and KATO Ⅲ, colon cancer derived AGS and a blood lymphoma Jurkat T cells by microscopic observations of the cell viability and by the MTT and SRB enzyme-linked chromophoric assays. Two stomach cancer cells DLD-1 and KATO Ⅲ were arrested in its proliferation and lysed by three isolates of P. robustus, P. linteus, and P. B with the ED_(50) ranges of 40 ㎍/㎖ of hot-water extractives. Other carcinomas AGS and Jurkat T cells were suppressed by all of the isolates in Phellinus spp. tested, with the 750, 180 ㎍/㎖ of high ED_(50) concentrations, respectively. Also, time-course dose effects on the survival of cancer cells gave the same results with all the five isolate extractives. Tumor cells tested were survived over 30 days in vitro. However, those treated with Phellinus spp. extractives were extipated over 90% of cancer cells within a week. These results insisted that the Phellinus spp. has general carcinostatic effects and especially alimentary canal cancer cells are more susceptible than other tissues.

항AK3 단클론 항체의 single chain fragment variable(ScFv) 제작 및 생화학적 특성 연구

김백길,김효준 한양대학교 이학기술연구소 2002 이학기술연구지 Vol.5 No.-

단클론 항체는 항원에 대한 높은 특이성을 갖는다. 이러한 특성은 혈액이나 조직에 존재하는 특정 항원을 검출하기 위해 널리 이용되고 있다. 그러나 혈액 내에 존재하는 human anti mouse antibody (HAMA)와 같은 plasma protein 에 의한 단클론 항체와의 결합에 의해 특이성이 심하게 저하되는 현상이 나타난다. 최근 Fc 부분이 제거된 단클론 항체가 혈액내 비특이적 결합을 줄일 수있음이 ㅂ조고되고 있으며, 이러한 목적으로 phage display system을 이용한 항체공학이 Fc부분을 제거하기 위해 방법론적으로 활용되고 있다. 본 연구에서는 phage display system을 이용하여, 항 adenylate kinase isoenzyme 3(AK3) 단클론 항체 SJA3-86으로부터 single chain fragment variable gene(ScFv)을 cloning하였고, E.coli expression system을 통해 재조합 항체 ScF86을 발현시켰다. ScF86의 cDNA 염기배열로부터 유추한 CDR1, 2 및 3는 negative charge의 amino acid가 다수 포함되어 염기성 효소인 AK3를 전기적으로 효율적인 인식이 이루어짐을 확인하였고, parental antibody인 SJA3-86이 conformational epitope보다 denatured epitope을 효과적으로 인식하는 항원-항제 반응서으을 유지하고 있었다. 그러나 Fc 부분이 절단되고, 아미노산10개로 구성된 linker peptide가 도입된 결과 항원에 대한 Kd값은 1.1×10-9으로 원래의 약60%의 친화력을 단백공학에 의한 형태변화에도 불구하고 항원의 인식능력은 크게 변하지 않았다. 이러한 결과로 ScF86은 혈액내 비특이적 결합을 제거한 항체공학적 단클론 항체로서 임상적으로 유용하게 활용될 수 있을 것으로 사료된다. Abstract : As monoclonal antibody has high specificity for a antigen, it is widely used for the detection of spexific antigen in blood or tissues. however, non-specific binding of monoclonal antibody in blood is still troublesome. Recently, it is reported that antibody without Fc reactivity can decrease non-specific binding in blood. To remove Fc reactivity antibody engineering technique using phage display system has been used developed. By utilizing phage display system we constructed ScFv(Single chain Fragment variable)fragment of monoclonal antibody against human adenylate kinase isozymes 3(AK3) expressed as a single peptide in E.coli. From the analysis of amino acids seuuence we verified that the CDRs of ScF86 contained many negatively charged amino acids, which were reasonable to interact with AK3 by charge interaction. However the deletion of Fc region and addition of linker peptide caused the lower 1.I× 10-9 M of ScF86 which is the affinity decrease by that of 60% of parentalantibody. This deviation is very small one compaired with other general protein engineering on receptor-ligand reactivities and the lowered affinity is not socntical in the further usage of the antibody. The refgre, we successfully constructed there combinant antibody ScF86 against AK3 that eliminate the Plasma-derivated non-specific bindings in immunochemical studies.

효율적인 보전업무 수행을 위한 Y-Cycle의 개발

김영인,최병관,함효준 대한설비관리학회 2004 대한설비관리학회지 Vol.9 No.2

Whenever we execute the work, the work success or failure depend on what kind of method use and how it appled. If the aim is not to be sure and good method is be used, the conclusion not gain our want. When we want to success the work, then we have to check the aim continually during our work. This paper present Y-Cycle that execute more effective work method that combine with Deming cycle and System cycle. If we combine each cycle's characteristic and merit and execute the work using right order, and adjust feedback in each step, then we can success the aim using less cost and endeavor.

설비보전 자격검정을 위한 직무분석 모형 구축

김만균,함효준 대한설비관리학회 2003 대한설비관리학회지 Vol.8 No.4

Job analysis is essential in the development of vocational education and hiring course as well as developing guidelines for vocational qualification exams. However, due to inadesuacy in systematic job analysis in the past, development and revision of educational programs, vocational training standards and guidelines for examination questions not only lack efficiency and Connectivity, but are also becoming a waste of resources. The purpose of the research lies in developing methods of job analysis and procedure models for establishment of a system that efficiently links education, training and qualification by understanding the job analysis. The purpose also lies in developing basic materials for vocational education and training course as well as developing guidelines for examination questions through job analysis on plant engineering in the field. Job analysis was conducted according to the newly established job analysis procedure and form for plant engineering mentioned above, and as a result, knowledge, function and tool were obtained and vocational education and training course as well as guidelines for examination questions were developed.

설계공정과 제조공정 연동을 위한 PDM/CPC 통합 인터페이스 시스템 개발

김만균,함효준 대한설비관리학회 2004 대한설비관리학회지 Vol.9 No.4

Recently, the situation of the new product development has been drastically changed. Also, the analysis of PDM(Product Data Management) has long been interested by researchers. PDM system is the system which collects and manages the information of the products in the products development process, especially in the design stages. They are hastening the implementation of PDM and CPC(Collaborative Product Commerce) for getting market initiatives and to get an advantage in competition through Product Collaboration. Industries begin to require the PDM which integrated design and manufacturing information concurrently to reduce the delivery time to market of new products. Finally from this study, I compared with the deduced model of methodology and the mod-el of ERP Systems implementation. Additionally, PDM/CPC System as decision making needs more to be provided the stage of system product collaboration process to get easy-of-use for diverse user than verified the model of ERP System implementations by many users

부비동 악성 종양의 임상적 분석

백병준,유명상,신재민,김효진,박훈,김장묵,오천환 순천향의학연구소 2003 Journal of Soonchunhyang Medical Science Vol.9 No.2

Background and Objectives : Paranasal sinus cancer is rare when compared with cancers of other sites. Because paranasal sinus is anatomically located adjacent to the orbit and skull base, it is difficult to remove the tumor completely with tumor free margin in advanced paranasal sinus cancer. This report was conducted to investigate clinical characteristics, histologic type and treatment outcome of paranasal sinus cancer. Material and Methods : Twenty-six cases with paranasal sinus cancer diagnosed at the Department of otolaryngology-Head and Neck Surgery, Soonchunhyang Cheonan Hospital from January 1991 to September 2000 were studied by chart review, telephone interview and letters retrospectively. Results : The male to female ratio was 4.2 : 1. Most patients were 6th and 7th decade. The maxillary sinus was the most commonly affected site(61.5%), followed by the ethmoid sinus(34.6%). The most common histologic type of malignancy was squamous cell carcinoma(57.6%), followed by adenocarcinoma(19.2%). Most patients presented with T_(3)/T_(4) or locally advanced disease, N_(0)(91.3%), and M_(0)(100%) Sixtynine pertcent of this study group underwent sugery as part of a mutimodality curative treatment plan or alone as curative treatment. Eighteen patients(69.2%) developed recurrent disease at a median time of 278 days after initial treatment. Kaplan-Meier analysis revealed the 5-year disease specific survival was 34.6%. Conclusion : Most patients with paranasal sinus cancer presented with locally advanced disease. Adanced T stage was highly predictive of poor prognosis. Recurrence rate was high and mostly occured within the second year after treatment.

편심하중을 받는 상자형의 단면변형에 관한 연구

鄭熺孝,安光列,金鎬洙,黃俊碩 경상대학교 생산기술연구소 2001 工學硏究院論文集 Vol.1 No.-

Eccentric load acts on box girder is divided into bending and torsional load and deforms cross section. Distortion that is resulted from torsional load is largely different according to tickness of each plate or stiffener. According to with and without regard of distorsion effect, it is possible to divide analysis of box gilder into two. First, analysis of box gilder can be able to calcurate with rigid box girder in assumption that there is no distorsion when the girder is well stiffened with diaphragms or cross bracing and plate thickness of box section is thick. Second is the analysis that must be considered effect of distorsion when the girder has not sufficient stiffener or plate thickness of box section is not well thick. In numerical example of this study, normal stress of rigid cross section analysis is compared with that of deformable cross section analysis without interior diaphragms. Then, it was studied that stresses of the deformable cross section analysis can be near rigid analysis when box girder was stiffened with interior diaphragms.

돼지에서 정맥, 근육 그리고 경구 투여시의 enrofloxacin의 약물동태학

윤효인,김무열,박승춘,조준형,박병권,이내경,노상석,장범수,신광순,조명행 충남대학교 수의과대학 동물의과학연구소 1996 動物醫科學硏究誌 Vol.4 No.-

In order to characterize pharmacokinetic profiles according to route of a new enrofloxacin salt form (Enrotil®), it was given to 4 healthy pigs via oral (p.o.), intramuscular (i.m.) or intravenous (i.v.) administrations at a dose rate of 5 ㎎/㎏ body weight. Enrofloxacin (ENFX) in serum was detected by bioassay using E. coli BE1186 as a test organism. The biological elimination half-lives (t_1/2(β)) of ENFX were 6.76±0.99 h (i.v.), 7.16±2.30 h (i.m.) and 11.45±3.90 h (p.o.), Volume of distribution (Vd) of enrofloxacin was 2.20±0.31 L/㎏ (i.v.), 2.52±0.60 L/㎏ (i.m.) and 1.88±0.33 L/㎏ (i.m.). Mean residence time (MRT) was 8.77±1.26 h after i.v. injection and the maximal concentration time (Tmax) following p.o. and i.m. administration was 0.76±0.09 h and 0.60±0.12 h, indicating a rapid absorption from these routes. Bioavailibility (F) was calculated as 64.1% for p.o. administration and 59.71% for i.m. injections. In summary, the newly formulated enrofloxacin salt form has shown a high water solubility, rapid absorption and large tissue distribution, suggesting a potential antibacterials for oral application on a large scale in veterinary sectors.