전후(戰後)의 불모지에 피워낸 전인교육(全人敎育)의 꽃: 호재(岵在) 함재홍(咸載弘) 선생님의 헌신적 사도(師道)의 향기

야, 선생님 오신다. 선생님……돌멩이가 듬성듬성 튀어나와 있고 아직 잘 다듬지 않아 학교 운동장이라고 하기에는 너무 작은 공터. 한 겨울 오전 이곳에 모여 놀던 어린이들이 브라운 색의 낙타 외투를 걸치고 성큼 성큼 철조망 교문으로 들어서는 선생님을 보고 외치며 달려간다. 선생님과 어린이들이 함께 맨손체조를 하고서 가볍게 달리기를 하며 몸을 데운 뒤 들어서는 판자 집 가교실. 추운 겨울방학 난로도 없는 맨 흙바닥의 판자 집 교실에 옹기종기 모여 무언가 열심히 하고 있는 해맑은 눈의 초등학교 4학년 꼬맹이들. 한편에선 도화지에 크레용으로 그림을 그리고, 다른 쪽에선 글짓기를 하고, 몇몇은 한 구석에서 웅변연습을 하는 꼬맹이들의 열기로 가득 찬 교실. 이들 어린이 하나하나를 보살펴 고쳐 주고 가르치며 전인교육에 몰두하는 20대 후반의 영화배우처럼 잘 생긴 열정에 넘친 청년교사. 바로 이 청년교사가 필자의 초등학교 4학년 1반부터 졸업 때까지 3년간 담임을 한 호재(岵在) 함재홍(咸載弘) 선생님이시다. 한국전쟁의 휴전협정이 체결된 뒤 2년 후인 1955년 12월 하순경, 부산 동구 수정동의 수정산 중턱에 새로 세워진 수성초등학교 4학년 1반의 겨울방학 자율특별활동 시간 모습이다

약물 전달에 응용하기 위한 30Kc19를 이용한 단백질 나노입자의 제조 및 특성 연구

학위논문 (박사)-- 서울대학교 대학원 : 화학생물공학부, 2015. 2. 박태현.30Kc19 protein is a member of the 30K protein family from silkworm, having molecular weights of around 30 kDa. 30Kc19 protein is the most abundant among 30K proteins in the hemolymph. In previous studies, 30K proteins exhibited anti-apoptotic effect in various cells by gene expression or addition of 30K proteins in recombinant form produced from Escherichia coli. 30Kc19 also enhanced productivity and glycosylation by expression of a 30Kc19 gene or supplementation with a recombinant 30Kc19 protein. Additionally, 30Kc19 exhibited enzyme-stabilizing and cell-penetrating abilities in vitro. In this study, it was hypothesized that supplemented 30Kc19 penetrated into the cell and enhanced the stability of the cellular enzyme, and investigated this using in vitro and cellular assessments. The activity of isolated mitochondrial complex I / III was enhanced with 30Kc19 in dose-dependent manner while initial reaction rate was unchanged, suggesting that 30Kc19 enhanced enzyme stability rather than specific activity. For intracellular enzyme activity assessment, mitochondrial complex II activity in HeLa cells increased more than 50% with 30Kc19. The enhanced mitochondrial complex activity increased mitochondrial membrane potential and ATP production in HeLa cells with 30Kc19, by over 50%. Then cell penetrating and enzyme stabilizing effect of 30Kc19 was exploited to efficient drug delivery. 30Kc19 and HSA were used as building block of protein nanoparticles to exploit both beneficial effect of 30Kc19 protein stability of HSA nanoparticles. 30Kc19-HSA nanoparticles were successfully prepared using the desolvation method, with uniform spherical morphology and stable dispersion. 30Kc19-HSA nanoparticles showed negligible toxicity when treated to cells, and 30Kc19-HSA nanoparticles also exhibited increase in cellular uptake compared with HSA nanoparticles. Because stable 30Kc19-HSA nanoparticles were successfully synthesized and characterized, nanoparticles loaded with model enzyme cargo to investigated effect of 30Kc19 on cargo enzyme. 30Kc19-HSA nanoparticles loaded with β-galactosidase were uniformly spherical in shape, dispersed evenly in phosphate buffered saline and cell culture media, and released β-galactosidase in a sustained manner. The 30Kc19-HSA nanoparticles had negligible toxicity to animal cells and exhibited enhanced cellular uptake and intracellular stability of β-galactosidase in HeLa and HEK293 cells when compared with those of HSA nanoparticles. These results suggest that 30Kc19-HSA protein nanoparticles could be used as a versatile tool for drug delivery to various cells. Next, 30kc19-HSA nanoparticles were used to deliver actual therapeutic protein to cells. Fabry disease is a genetic lysosomal storage disease caused by deficiency of α-galactosidase, the enzyme that degrades neutral glycosphingolipids transported to lysosomes. Enzyme replacement therapy (ERT) using recombinant α-galactosidase is the only treatment available for Fabry disease. Because enhancing cellular delivery and enzyme stability is a challenge of ERT using α-galactosidase to maximize treatment efficacy, 30Kc19-HSA protein nanoparticles were used to enhance delivery and intracellular α-galactosidase stability. The 30Kc19-HSA nanoparticles had a uniform spherical shape and were well-dispersed. The 30Kc19-HSA nanoparticles had negligible toxicity to human cells. The nanoparticles exhibited enhanced cellular uptake and intracellular stability of the delivered α-galactosidase in human foreskin fibroblasts. Additionally, the nanoparticles enhanced globotriaosylceramide degradation in fibroblasts from a patient with Fabry disease. It is expected that 30Kc19-HSA protein nanoparticles will be used as an effective tool for efficient delivery and enhanced stability of drugs.Contents Chapter 1. Research background and objectives 1 Chapter 2. Literature review 5 2.1 Nanoparticles for drug delivery 6 2.1.1 Terminology of nanoparticles 6 2.1.2 Classification of nanoparticles 7 2.2 Albumin nanoparticles 10 2.2.1 Albumins for nanoparticle production 10 2.2.2 Preparation of albumin nanoparticles 12 2.2.3 Surface modification of albumin nanoparticles 14 2.2.4 Uptake of albumin nanoparticles 18 Chapter 3. Experimental procedures 20 3.1 Production of recombinant 30Kc19 protein 21 3.2 Cell culture 21 3.3 Mitochondria isolation 22 3.4 Western blotting 22 3.5 Mitochondrial activity assay 22 3.6 Estimation of mitochondrial membrane potential and ATP generation 23 3.7 Preparation of nanoparticles 24 3.8 Size and zeta potential of the nanoparticles 24 3.9 Scanning electron microscopy (SEM) analysis of the nanoparticles 25 3.10 β-galactosidase loading efficiency of the nanoparticles 25 3.11 In vitro β-galactosidase release from nanoparticles 26 3.12 Cell viability assay 27 3.13 Cellular uptake of nanoparticles 27 3.14 Protein cargo activity of nanoparticle-treated cells 29 3.15 Globotriaosylceramide degradation 30 3.16 Statistical analysis 31 Chapter 4. Stabilization of cellular mitochondrial enzyme complex activity through supplementation of 30Kc19 protein 32 4.1 Introduction 33 4.2 Effect of 30Kc19 on in vitro mitochondrial enzyme stability 34 4.3 Intracellular and mitochondrial uptake of 30Kc19 35 4.4 Effect of 30Kc19 on intracellular mitochondrial enzyme stability 38 4.5 Effect of 30Kc19 on mitochondrial membrane potential and ATP generation 40 4.6 Conclusions 40 Chapter 5. Synthesis of protein nanoparticles using 30Kc19 protein and human serum albumin 42 5.1 Introduction 43 5.2 Preparation and characterization of 30K-HSA nanoparticles 45 5.3 Cellular toxicity of 30Kc19-HSA nanoparticles 49 5.4 Cellular uptake of 30Kc19-HSA nanoparticles 49 5.5 Conclusions 51 Chapter 6. Protein nanoparticles for protein cargo delivery using 30Kc19 protein and human serum albumin 53 6.1 Introduction 54 6.2 Characterization of β-gal-loaded 30Kc19-HSA nanoparticles 55 6.3 β-Galactosidase loading and in vitro release of 30Kc19-HSA nanoparticles 59 6.4 Cellular toxicity of 30Kc19-HSA nanoparticles loaded with β-Galactosidase 62 6.5 Cellular uptake of 30Kc19-HSA nanoparticles loaded with β-Galactosidase 64 6.6 Intracellular β-galactosidase activity delivered by nanoparticles 66 6.7 Conclusions 69 Chapter 7. Protein nanoparticles for therapeutic protein delivery using 30Kc19 protein and human serum albumin 70 7.1 Introduction 71 7.2 Production and characterization of 30Kc19-HSA nanoparticles 72 7.3 Cellular toxicity of 30Kc19-HSA nanoparticles 76 7.4 Cellular uptake of nanoparticles 76 7.5 Intracellular α-galactosidase activity delivered by nanoparticles 78 7.6 Globotriaosylceramide degradation activity in nanoparticle-treated Fabry disease fibroblasts 80 7.7 Conclusions 84 Chapter 8. Overall discussion and further suggestions 85 8.1 Overall discussion 86 8.2 Conclusion and further suggestions 92 References 96 국 문 초 록 110Docto

Biomechanical characteristics of pelvis and lower limbs in Korean soccer players diagnosed with lumbar facet joint syndrome

의학과/석사[한글] 만성적인 요통의 대표적인 질환으로 후관절 증후군(facet joint syndrome)이 있고, 이것의 원인으로 추정 되는 것은 증가된 요천추 전만(hyper lordosis), 약해진 등/복부 근육, 비정상적인 후관절 형태 그리고 하지 길이차 등에 의한 요추 후관절간 스트레스 증가(increased interfacet stress) 이다. 한편, 장골과 천추을 포함한 골반과 제 5요추는 후관절(제 5요추/제 1천추간)에 의해 밀접하게 연계되어 있어 골반의 비정상적인 움직임이 후관절의 스트레스를 증가시킬 수 있다. 즉, 골반의 편측 회전, 과도한 골반의 전방 기울임, 그리고 골반의 좌우 높이차가 요추 후관절의 스트레스를 증가 시킬 수 있다. 이와 같이 골반의 비정상적인 움직임을 이해하는 것이 요추 후관절 증후군의 유발 원인을 분석하는데 도움이 될 것으로 생각 된다. 본 연구에서는 요추 후관절 증후군을 가진 축구 선수들을 대상으로 골반과 하지의 움직임을 생체 역학적 측정을 통해 얻고, 이것을 요통을 가지지 않은 선수들의 그것과 비교하고자 하였다. 요추 후관절 증후군으로 진단 받은 축구 선수 39명을 연구 대상으로 하였고, 대조군은 요통을 가지지 않은 축구 선수 18명으로 하였다. 이들을 대상으로 생체 역학적 분석을 시행하였는데 골반부 측정 항목 선 자세에서의 골반의 좌우 높이차가 있었고 발/발목의 측정 항목은 기립시 종골각 이었다. 연구 결과, 골반 좌우 높이의 비대칭(기립 자세에서 좌측에 비해 우측 골반이 낮음), 기립시 종골각의 비대칭(우측 종골 내반각 증가, 심한 좌/우측 종골각 차이)소견이 발견되었다. 본 연구를 통해, 요추 후관절 증후군을 가진 선수들은 골반과 하지의 생체역학적 이상(부정렬) 소견을 가지고 있음이 밝혀졌고, 향후 이들을 교정하는 것이 요추 후관절 증후군 치료에 도움이 될 것으로 사료된다. [영문]Objective: To investigate and compare biomechanical characteristics in Korean soccer players diagnosed with lumbar facet joint syndrome with those in healthy Korean soccer players./Methods: We recruited thirty-nine Korean soccer players with lumbar facet syndrome that was confirmed by facet block and eighteen players without ailments. We performed biomechanical assessments in pelvis and lower extremities to all subjects. The following parameters were measured; pelvic level (obliquity angle) and resting calcaneal stance position. Results: The followings showed parameters with statistical differences between two groups in which the first set of values identifies facet joint syndrome group and the second set, the control: elevated pelvic level at left side(2.2±1.6; 0.6±0.9) and increased right calcaneal varus angle (2.8±4.0; -0.5±2.6). Conclusion: The asymmetry of pelvis and foot/ankle state in the facet joint syndrome group was identified and could be conclusively considered as one of the major causes of lumbar facet joint syndrome.ope

(A) clinical study of high dose methotrexate, 5-fluorouracil and adriamycin combination chemotherapy in advanced stomach cancer

의학과/석사[한글] 엽산길항제인 methotrexate (MTX)는 이수소화 염산 환원요소를 억제하여 항암효과를 나타내는데 30mg/m**2의 통상 용량의 치료법에서 발전되어 고용량(1-10g/m**2)을 citrovorum factor(CF)와 병용투여하여 골육종, 악성임파종, 두경부종양 등에서 높은 반응율을 나타내고 부작용을 줄일 수 있다고 알려져 있다. 최근 Klein 등은 진행 위암에서 high dose MTX (HDMT7)와 5-FU를 주축으로 하는 복합 화학요법이 높은 반응율(63%)을 얻을수 있다고 보고한바있다. 우리나라에서 가장 흔한 암인 위암에는 5-fluorouracil(5-FU), adriamycin(ADR), mitomycin-C 복합 화학요법(FAM)이 주로 시행되고 있다. 그러나 우리나라에서는 아직 HDMTX 치료성적에 대한 보고 및 약동학적연구가 없어 본 연구자는 진행 위암 환자에서 HDMTX투여 에 따른 혈역동학적 연구와 HDMTX, 5-FU 및 ADR의 복합 화학요법의 항암효과, 독성 정도를 알기 위해 1986년 1월부터 1986년 12월까지 연세대학교 의과대학 부속 연세의료원 및 연세암센터에 입원되고 조직병리학적으로 확진된 진행 위암 환자 18예를 대상으로 관찰하 여 다음과 같은 결과를 얻었다. 1) MTX 1.5 g/m**2 투여후 MTX의 혈중 농도는 투여 종료 30분후 7.17 x 10**-5 ± 3.62. x 10**-5 M(mean±S.D), 6시간후 1.38 x 10**-5 ± 0.75 x 10**-5 M, 12시간후 3.71 x 10**-6 ± 2.23 x 10**-6 M, 24시간후 9.30 x 10**-7 ± 1.12 ㅌ 10**-7 M, 48시간후 1.9 9 x 10**-7 ± 1.70 x 10**-7 M, 그리고 72시간후에는 1.27 x 10**-7 ± 1.25 x 10**-7 M로 시간경과에 따라 감소하였다. 투여 종료 48시간째 1 x 10**-7 M 이하인 경우는 50%였고 72시간째 1 x 10**-7 M 이하인 경우는 66.6%였다. 2) HDMTX, 5-FU 및 ADR 복합 화학요법후의 반응율은 화학요법을 투여받은 기왕력이 있는 환자는 11명 중 1예(9.1%), 받은적이 없는 환자 7명중 1예(14.7%)로 총 18예의 대상환자 중 2예(11.2%)에서 부분관해를 보였다. 3) HDMTX투여에 따른 부작용은 치명적인 백혈구감소증에 의한 패혈증으로 사망한 1예가 있었으며 그외 오심, 구토, 구내염, 발진, 설사등이 동반되었으며 혈중 creatinine의 상승을 동반한 예는 1예뿐이었다. 이상의 결과로 보아 HDMTX투여시 MTX의 혈역동학적 변화는 외국의 보고와 유사하였으며, CF도 최소찬 MTX투여 종료후 72시간 이상 투여해야 안전한 것을 알수 있었다. HDMTX, 5-FU 및 ADR 복합 화학요법의 진행 위암에 대한 반응율은 매우 낮았다. 그러나 대상 예가 적어 추가적인 연구 및 평가가 필요하다고 사료된다. [영문] The folic acid antagonists, methotrexate (MTX), were shown to have antitumor activity due to inhibition of the enzyme, dihydrofolate reductase. In the last two decades, the drug has been administered in conventional doses of 30mg/m**2; however during the last 10 years, very high doses of methotrexate in the range of 1-10g/m**2, coupled with citrovorum factor rescue, has been found to be strikingly more effective for chemotherapy of osteogenic sarcoma, malignant lymphoma, and squamous carcinoma of the head and neck. Klein(1983) reported a high response rate(63%) by MTX and 5-FU combination chemotherapy in cases with advanced stomach cancer. In Korea, the incidence of stomach cancer is higher than other tumors, and the usual chemotherapy program for advanced stomach cancer is a 5-fluorouracil, adriamycin and mitomycin-C combination regimen(FAM) ; but there is no pharmacokinetic study or clinical application of MTX. This paper presents a pharmacokinetic and clinical study of 18 cases with advanced stomach cancer, admitted from Jan. 1986 to Dec. 1986. The resells were as follows: 1) After intravenous infusion of MTX 1.5gm/m**2, the plasma MTX level were recorded as follows: At 30 min after discontinuation of MTX infusion, plasma MTX level was 7.17 x 10**-5 ± 3.63 x 10**-5 M. At 6 hours, it was 1.38 x 10**-5 ± 0.75 x 10**-5 M; at 12 hours, 3.71 x 10**-6 ± 2.23 x 10**-6 M ; at 24 hours, 9.30 x 10**-7 ± 1.02 x 10**-7 M ; at 48 hours, 1.99 x 10**-7 ± 1.70 x 10**-7 M; and at 72 hours, 1.27 x 10**-7 ± 1.25 x 10**-7 M. Forty-eight hours after discontining the drug, the plasma level of MTX fell below 1 x 10**-7 M in half of the cases. After 72 hours, 66.6% of the caes fell below the 1 x 10**-7 M level. 2) The response rate for the HDMTX, 5-FU and ADR combination chemotherapy was 11.2% in all cases. One case(9.1%) out of 11 cases with previous chemotherapy, showed a partial response; also, out of 7 case without previous chemotherapy, one case(14.2%) showed a partial response. 3) Toxicities with high dose MTX were : pancytopenia(1 case), nausea and vomiting(14 cases), stomatitis(8 cases), skin rash(2 cases) and diarrhea(2 cases). In our study the patient with pancytopenia died due to sepsis. There was only one case with an elevated creatinine level. In summary, the results of this study for pharmacokinetic changes of that MTX were similar to other reports. However, over 72 hours after discontinuation of high dose MTX and 5-FU combination chemotherapy, the risk of toxicity is reduced by the administration of CF. The response rates of HDMTX, 5-FU and ADR combination chemotherapy were low; therefore further studies should be done to elucidate a more exact response rate.restrictio