Counter Clinical Prognoses of Patients With Bloodstream Infections Between Causative Acinetobacter baumannii Clones ST191 and ST451 Belonging to the International Clonal Lineage II

This study was conducted to evaluate the possible clinical and bacteriologic features associated with 30-day mortality from Acinetobacter baumannii (A. baumannii) bloodstream infections (BSIs). We conducted a prospective, multicenter, observational study of 181 entire episodes of A. baumannii BSI from six general hospitals between May 2016 and April 2017 in South Korea. Cox proportional-hazards regression model was used to estimate risks of the primary endpoint, i.e., all-cause mortality within 30 days from the initial blood culture. Most (84.5%) of the A. baumannii blood isolates belonged to the international clonal lineage II (ICLII) and 89.5% of the isolates were either multidrug- or extensively-drug resistant. We identified three risk factors including the old age of patient {hazard ratio, 1.033; [95% Confidential Interval (CI), 1.010-1.056]}, the sequential organ failure assessment score [1.133 (1.041-1.233)], and causative A. baumannii sequence type (ST) 191 belonging to ICLII [1.918 (1.073-3.430)], and three protective factors including causative A. baumannii ST451 belonging to ICLII [0.228 (0.078-0.672)], platelet count [0.996 (0.993-0.999)], and definitive therapy within 72 h [0.255 (0.125-0.519)]. Differing 30-day mortality rate in the dominant ICLII was observed by ST, which was much high in ST191 and low in ST451 and it was likely associated with the molecular traits, rather than the drug resistance.ope

MALDI-TOF Mass Spectrometry Technology as a Tool for the Rapid Diagnosis of Antimicrobial Resistance in Bacteria

Species identification by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a routine diagnostic process for infectious diseases in current clinical settings. The rapid, low-cost, and simple to conduct methodology is expanding its application in clinical microbiology laboratories to diagnose the antimicrobial resistance (AMR) in microorganisms. Primarily, antimicrobial susceptibility testing is able to be carried out either by comparing the area under curve of MALDI spectra of bacteria grown in media with antimicrobial drugs or by identifying the shift peaks of bacteria grown in media including 13C isotope with antimicrobial drugs. Secondly, the antimicrobial resistance is able to be determined through identifying (i) the antimicrobial-resistant clonal groups based on the fingerprints of the clone, (ii) the shift peak of the modified antimicrobial drug, which is inactivated by the resistance determinant, (iii) the shift peak of the modified antimicrobial target, (iv) the peak specific for the antimicrobial determinant, and (v) the biomarkers that are coproduced proteins with AMR determinants. This review aims to present the current usage of the MALDI-TOF MS technique for diagnosing antimicrobial resistance in bacteria, varied approaches for AMR diagnostics using the methodology, and the future applications of the methods for the accurate and rapid identification of AMR in infection-causing bacterial pathogens.ope

Amplification of the Chromosomal blaCTX-M-14 Gene in Escherichia coli Expanding the Spectrum of Resistance under Antimicrobial Pressure

Various forms of adaptive evolution occur in clinical isolates in response to the presence of antimicrobial drugs. Among a total of 171 CTX-M-9 group/family extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli blood isolates recovered between 2016 and 2017 in six general hospitals, 50.3% of the isolates possessed the blaCTX-M-14-like gene in their chromosome rather than in a plasmid. Focusing on this unprecedented way of the blaCTX-M ESBL gene possession, molecular epidemiology of the isolates was assessed and the chromosomal location of the acquired cephalosporinase gene was dissected in an evolutionary point of view. Taking advantage of a complete collection of E. coli blood isolates from a limited period, clonal relatedness of the E. coli isolates carrying the blaCTX-M-14-like gene was clarified and the dominant clone, ST131 H30R, was identified. To control the level of resistance and the resistance spectrum to oxyimino-cephalosporin drugs, transcription level of the blaCTX-M-14-like gene was tuned finely through positioning the gene near the chromosomal initiation dnaA gene and amplifying numbers of the gene in a chromosome using either the copy-and-paste or the tandem amplification methods. Inconspicuous fitness cost by chromosomal location of the gene and free adjustment of the oxyimino-cephalosporin resistance would urge the dominancy of E. coli clinical isolates harboring the blaCTX-M ESBL gene in their chromosome. IMPORTANCE Increasing prevalence of E. coli producing CTX-M ESBL is a major concern in clinical settings because it significantly limits treatment options. Thus, it is important to keep watching current molecular mechanisms of resistance and the scheme for dissemination. Recently, chromosomal locations of the blaCTX-M genes are often documented in clinical settings and the bacterial strategies were needed to be dissected in an evolutionary point of view. Both main mechanisms of fine tuning the chromosomal gene expression, bacterial gene amplification either by copy-and-paste or by tandem amplification and positioning the gene near the chromosomal initiation dnaA gene, were demonstrated in the study, and the fitness cost by the chromosomal location was evaluated.ope

Methicillin-Resistant Staphylococcus aureus Blood Isolates Harboring a Novel Pseudo-staphylococcal Cassette Chromosome mec Element

The aim of this work was to assess a novel pseudo-staphylococcal cassette chromosome mec (ΨSCCmec) element in methicillin-resistant Staphylococcus aureus (MRSA) blood isolates. Community-associated MRSA E16SA093 and healthcare-associated MRSA F17SA003 isolates were recovered from the blood specimens of patients with S. aureus bacteremia in 2016 and in 2017, respectively. Antimicrobial susceptibility was determined via the disk diffusion method, and SCCmec typing was conducted by multiplex polymerase chain reaction. Whole genome sequencing was carried out by single molecule real-time long-read sequencing. Both isolates belonged to sequence type 72 and agr-type I, and they were negative for Panton-Valentine leukocidin and toxic shock syndrome toxin. The spa-types of E16SA093 and F17SA003 were t324 and t2460, respectively. They had a SCCmec IV-like element devoid of the cassette chromosome recombinase (ccr) gene complex, designated as ΨSCCmec E16SA093. The element was manufactured from SCCmec type IV and the deletion of the ccr gene complex and a 7.0- and 31.9-kb portion of each chromosome. The deficiency of the ccr gene complex in the SCCmec unit is likely resulting in mobility loss, which would be an adaptive evolutionary mechanism. The dissemination of this clone should be monitored closely.ope

Reply to Cabrera et al., "Outcomes of Patients with Bloodstream Infections Caused by Ampicillin-Susceptible but Penicillin-Resistant Enterococcus faecalis: Caution in Interpreting the Results"

ope

Performance Evaluation of the Newly Developed BD Phoenix NMIC-500 Panel Using Clinical Isolates of Gram-Negative Bacilli

BACKGROUND: The emergence of carbapenem resistance among gram-negative bacilli (GNB), mediated by carbapenemase production, has necessitated the development of a simple and accurate device for detecting minimum inhibitory concentrations (MICs) and resistance mechanisms, especially carbapenemase production. We evaluated the performance of the BD Phoenix NMIC-500 panel (BD Diagnostic Systems, Sparks, MD, USA) for antimicrobial susceptibility testing (AST) and carbapenemase-producing organism (CPO) detection. METHODS: We used 450 non-duplicate clinical GNB isolates from six general hospitals in Korea (409 Enterobacteriaceae and 41 glucose non-fermenting bacilli [GNFB] isolates). AST for meropenem, imipenem, ertapenem, ceftazidime, and ceftazidime/avibactam, and CPO detection were performed using the Phoenix NMIC-500 panel. Broth microdilution was used as the reference method for AST. The rates of categorical agreement (CA), essential agreement (EA), minor error (mE), major error (ME), and very major error (VME) were calculated in each antimicrobial. In addition, PCR and sequencing were performed to evaluate the accuracy of CPO detection by the BD Phoenix NMIC-500 panel, and the rate of correct identification was calculated. RESULTS: The CA rates were >90% for all antimicrobials tested with the Enterobacteriaceae isolates, except for imipenem (87.2%). The GNFB CA rates ranged from 92.7% to 100% for all antimicrobials. The ME rates were 1.7% for Enterobacteriaceae and 0% for GNFB. The panel identified 97.2% (243/250) of the carbapenemase-producing isolates. CONCLUSIONS: The BD Phoenix NMIC-500 panel shows promise for AST and CPO detection.ope

Study of ornament design based on the formative characteristics of spider webs

자연은 오랜 세월 동안 조화로운 질서와 변화를 통해 자연 발생적인 아름다움을 보여주며 그 형상과 형태는 이상적인 미를 제공한다. 다채로운 자연을 보고 느끼는 것은 예술가들이 아름다움을 창조하는 계기가 되며 자연 그대로의 피상적 관찰과 모방이 아닌 세밀한 지적 탐구가 기본이 된다. 또한, 예술가의 미적 원리와 지적 추리력으로 형태를 재구성하고 시대의 흐름에 따른 표현방법을 반영하여 재창조할 수 있도록 지속해서 연구해야 한다. 자연물 중 거미줄은 조형적인 요소가 풍부한 표현의 대상이다. 거미줄은 직선으로 이루어져 원형이나 정형화되지 않은 도형이 모여 거미그물 형태를 이루며 이러한 형상은 자연적이며 원초적인 다양한 조형미를 제공하여 연구자에게 호기심을 자극하는 미적 대상이다. 이에 본 연구는 정주성 거미그물의 생태학적 구조를 살펴 이론적 토대를 다지고 조형적인 특성을 탐색한 후, 연구자의 감성과 시각을 통하여 거미그물을 조형화시킨 장신구작품을 개발하는 데 그 목적이 있다. 연구 내용은 거미와 거미줄의 이론적 고찰을 위해 문헌조사과 선행연구 자료를 토대로 생태 및 특징을 분석하고 거미줄을 이용한 작품 사례를 살펴보았다. 또한, 정주성 거미그물(정상 그물, 바퀴통이 없는 그물, 흰띠 그물, 위장 그물, 설렁줄 그물, 질라형 그물, 말굽형 그물, 삼각 그물, 접시 그물, 깔대기 그물, 줄 그물, 불규칙 그물, 차일 그물)의 형태와 이미지를 고찰하기 위해 자연 현장을 찾아 직접 살피고 전문서적과 사진 자료를 통하여 형태를 분석하였으며 이는 독창적인 조형 작업을 위한 토대가 되었다. 마지막으로 거미줄의 특징과 조형미를 탐구한 이미지를 구성하고 컴퓨터 그래픽프로그램 중 일러스트레이터을 활용하여 디자인한 후 브로치, 목걸이, 반지를 제작하였다. 본 연구의 결론은 다음과 같다. 첫째, 표현 대상으로서 거미그물이 지닌 조형적 가치가 무한함을 인지하였으며 예술가의 지적 탐구, 조형미, 표현 방법에 따라서 독창적으로 재창조할 수 있음을 깨달았다. 둘째, 13 종류의 정주성 거미그물을 관찰하여 균형, 반복, 강조, 탈비례의 조형원리를 살펴볼 수 있었다. 이러한 특징을 연구자의 조형과정에서 미적 원리와 연결해 과장, 삭제, 변화, 재구성하여 거미그물의 조형미를 표현할 수 있었다. 셋째, 거미그물의 특징 중 다공성과 투시성을 표현하고자 금속의 기법 중 양면부식의 사용은 효과적이었으며 일러스트레이터 프로그램을 활용한 정밀하고 정확한 디자인 작업에 따라 부식의 섬세한 결과가 결정되고 투각은 금속의 무게감을 최소화시켜 장신구의 역할로서 장점을 가지며 연구자에게도 거미줄을 조형화하는데 크기에 제한을 두지 않고 표현할 수 있었다. 넷째, 금속의 양면부식기법은 황동의 두꺼운 정도에 따라 섬세하고 예민한 거미그물을 표현할 때 제한적이었다. 판이 얇을수록 선과 선이 만나는 각이 섬세하고 두꺼워질수록 둥글게 표현되었다. 본 연구의 결과를 통하여 거미그물의 생태학적 특징과 조형적 아름다움을 토대로 독창적인 장신구 디자인을 하는 데 의의가 있다. 거미줄(그물)의 조형성은 디자인적 모티브로써 무궁무진하며 앞으로 연구자는 장신구에 제한을 두지 않고 다양한 분야의 영역과 소재의 확장을 통하여 거미줄의 미적, 감성적, 그리고 형태적 시각을 넓히고 급변하는 디자인의 흐름 속에서 새로운 감성을 전달할 것이다.;Nature manifests its autogenic beauty throughout the harmonious order and transformation over the centuries and the form and shape provide an ideal beauty. Viewing and feeling a variety of nature provide artists with the opportunities of creating the beauty, and the base must be built on minute intellectual quest and not on superficial observation and imitation of the nature as it is. Moreover, nature is a field requiring steady inquiry in order for artists to reconstruct its form based on aesthetic principle and intellectual reasoning ability, as well as to recreate it by reflecting the expressive method in the stream of time. Spider webs, among many other natural objects, are an object of expression abundant in formative elements. Spider webs are composed of straight lines and are formed as a pattern of net of spiders gathered by circular figures or non-structured figures. This form is an aesthetic object stimulating my curiosity as a researcher by providing various natural and elemental formative beauties. Hereupon, this study aims to develop ornament works inspired from the shaped form of spider webs through my own emotion and perspective, after inquiring into the ecological structure of sedentary spider webs for theoretical fundament and examining their formative characteristics. As for the research content, literature review and preceding studies were carried out for theoretical considerations of spiders and spider webs, this way, their ecology and characteristics were analyzed and some real cases of works done based on spider webs were inquired into. Moreover, so as to examine the forms and images of sedentary spider webs (normal web, web without the hub of wheel, white-band web, camouflage web, bell rope web, zilla-type web, horseshoe-type web, triangle web, sheet web, hopper web, string web, irregular web, awning web), on-spot field study was carried out and specialized publications and photographic materials were equally used for the analysis of forms. All these elements served for building the basement of formative works full of originality. In the last place, the images exploring the features and formative beauty of spider webs were composed and the Illustrator among many computer graphic programs was used for design. Finally, brooches, necklaces and rings were made. The findings of this study are as follows: Firstly, the formative values of spider webs as an object of expression are infinite and it was discovered that there is a possibility of original recreation depending on intellectual quest as a researcher, formative beauty and expressive method according to the stream of time and trends. Secondly, it was possible to inquire into the formative principle of balance, repetition, transformation and non-proportion, by observing 13 types of sedentary spider webs. These characteristics were linked with aesthetic principle in my own shaping process as a researcher, and they went through exaggeration, elimination, transformation and reconstruction processes for expressing the formative beauty of spider webs. Thirdly, the use of corrosion among metal techniques was effective to express porosity and seeing-though (penetration) traits, and precise design work based on Illustrator Program helped to determine detailed results of corrosion. The bratticing techniques helped to minimize the sense of weight and give advantages to the role as an ornament. For this reason, it was possible to express without the limit of size in modeling the spider webs. Fourthly, the metal corrosion technique of both sides encountered restriction when delicate and sensitive spider web was supposed to be expressed according to the thickness degree of brass. While the plate was thinner and the angle encountering two lines was more delicate and thicker, the expression was closer to be rounder. The formativeness of spider webs (net) can be infinite in terms of design motives, therefore, in quality of researcher, I will not place a limit upon the ornaments but expand the area towards a wide range of field and materials, I will extend the perspective of spider webs in term of aesthetic, emotional and morphological dimension and will convey new emotions in the stream of rapidly changing design.Ⅰ.서론 1 A.연구 목적 1 B.연구 내용 및 방법 2 Ⅱ.본론 4 A.거미의 생태와 거미줄 4 1.거미의 생태 4 2.거미줄의 종류 및 생태적 특징 5 B.거미줄의 조형미 연구 9 1.조형원리를 통한 거미줄 분석 9 2.거미줄과 직조의 구조적 연관성 14 Ⅲ.작품 제작 및 분석 21 A.작품 제작 의도 21 B.작품 제작 방법 23 C.작품 및 작품 분석 29 Ⅵ.결론 53 참고문헌 55 ABSTRACT 5

온도에 민감한 수용성 젤로써 폴리(에틸렌 글라이콜)-폴리(알라닌)수용액을 이용한 사람의 진피 세포의 3차원 배양과 상처 치료

As a new application of a thermogel, a poly(ethylene glycol)-b- -poly(L-alanine) (PEG-L-PA) gel encapsulating fibroblasts was investigated for wound healing. The fibroblasts were encapsulated by the temperature sensitive sol-to-gel transition of the polymer aqueous solution. Under the in vitro three-dimensional (3D) cell culture condition, the PEG-L-PA thermogel was comparable with MatrigelTM for cell proliferation and significantly better than MatrigelTM for collagen type I formation. After confirming the excellent 3D microenvironment of the PEG-L-PA thermogel for fibroblasts, in vivo wound healing was investigated by injecting the cell-suspended polymer aqueous solution on incisions of rat skin, where the gel encapsulating cells was formed in situ. Compared with the untreated system and the PEG-L-PA thermogel, the PEG-L-PA thermogel encapsulating cells not only accelerated the wound closure but also improved epitheialization and the formation of skin appendages such as keratinocyte layer (epidermis), hair follicles, and sebaceous glands. This paper proved the potential of thermogels for cell therapy as an injectable tissue engineering scaffold.;본 연구에서는 사람의 피부 진피세포를 넣은 폴리(에틸렌 글라이콜)-b-폴리(L-알라닌) (PEG-L-PA) 젤이 상처 치료를 위한 새로운 온도 민감성 젤로 사용되었다. PEG-L-PA 고분자 수용액은 온도에 민감하게 졸에서 젤로 변하는 과정에서 진피세포를 지지해줄 수 있는 3차원적인 매트릭스가 될 수 있다. PEG-L-PA 온도 민감성 젤과 비교군으로 매트리젤TM을 사용하여 3차원으로 세포를 키우는 생체 외 배양 세포 증식 실험 결과, PEG-L-PA에서 배양한 진피세포가 세포 증식에서 매트리젤TM만큼 좋은 결과를 보여주었고 콜라겐 타입 I 의 생성이 매트리젤TM보다 더 잘 된 것을 관찰할 수 있었다. PEG-L-PA 온도 민감성 젤이 진피세포의 3차원 배양에 적절하다는 것을 확인한 후, 외부에서 세포를 함유한 젤을 랫의 피부상처에 주입하여 상처 치료 과정을 관찰하는 동물실험이 진행되었다. 피부 상처에 Phosphate buffered saline만 처리한 군과 PEG-L-PA 온도 민감성 젤을 처리한 군에 비하여 진피세포를 함유한 PEG-L-PA 온도 민감성 젤을 처리한 군은 상처 닫힘이 가장 빠르게 진행됨을 확인할 수 있었다. 뿐만 아니라, 세포를 함유한 PEG-L-PA 온도 민감성 젤을 처리한 군은 다른 비교군보다 가장 빠르게 효과적으로 피부 진피층의 표피조직, 모낭, 피지샘과 같은 피부 구성물들을 재생시킨 것을 조직 연구에서 확인할 수 있었다. 따라서 본 연구는 온도 민감성 젤이 조직공학적으로 세포를 3차원적 배양시킬 수 있는 스캐폴드로써, 피부 상처에 대해 세포 치료 기법이 가능함을 보여주고 있다.CHAPTER 1. INTRODUCTION 1 1.1 Tissue engineering and biomaterials 2 1.2 Skin regeneration 5 1.3 Thermosensitive hydrogel 10 1.4 Three-Dimensional (3D) Culture 14 1.5 Aims for study 16 1.6 References 17 CHAPTER 2. Human dermal fibroblast 3D culture in PEG-L-PA hydrogel 19 2.1 Introduction 20 2.2 Experimental Section 23 2.2.1 Materials 23 2.2.2 Synthesis 24 2.2.3 Methods 25 2.3 Results and Discussion 32 2.3.1 Synthesis and Characterization 32 2.3.2 Gelation study of PEG-L-PA 37 2.3.3 Cell 3D culture 40 2.3.4 Wound Healing and Histology 44 2.4 Conclusions 48 2.5 References 49 국문 요약 52 감사의 글 5

Study of MMP-9/2 activities on breast cancer metastasis inhibited by novel ALK5 inhibitor

Extracellular matrix는 소극적인 형태의 생물학적 장벽으로 인식되어 왔다. 그러나 Steven Paget의 “seed and soil” 이론이 대두되면서, 암세포의 고착이 일어나는 장소인 tumor microenvironment에 주목하게 되었다. ALK5 억제제인 EW-7197과 EW-7273은 TGF-βR1에 선택적으로 작용하는 항암제로 개발되었다. 종양은 끊임없이 생체 내의 다른 세포, 다른 조직들과 상호작용하고 있기 때문에 ALK5 억제제인 EW-7197, EW-7273이 tumor microenvironment를 이루는 개별 요소들에 어떤 영향을 주는지 연구하였다. MMPs는 ECM의 분해에 있어서 필수적인 단백분해효소인데, 이 중에서도 gelatinases는 collagenases에 의해 분해된 fibral collagen을 최종적으로 분해하는 역할을 함으로써 전이에서 중요한 역할을 하고 있다. EW-7273은 MDA-MB-231, 4T1 세포에서 TGF-β에 의해 유도되는 MMP-2, MMP-9의 mRNA의 생성을 억제하며, MDA-MB-231, 4T1, MCF-10A에서는 MMPs 효소 활성 또한 기저 수준으로 저하시키는데, 이로써 EW-7273은 유전자 발현 및 효소로서의 활성 발현을 억제하여 암세포의 침투, 전이, 혈관신생에 필요한 과정인 ECM 분해 및 tissue remodelling을 억제한다. 또한, EW-7197은 MCF-10A에서 TGF-β에 의해 유도되는 효소 활성 및 단백량을 조절하며, MMP-2/MMP-9 mRNA 역시 감소시키는 것으로 확인되었다. 이러한 gelatinases의 생체 내 활성을 확인하기 위해서 in vivo gelatin zymography를 실시하였다. 그 결과, 1차 고형암에서의 MMP-2 및 MMP-9의 활성은 농도 의존적으로 높아지는 경향을 나타내었으나, 폐에서는 투여 농도 의존적으로 활성이 낮게 나타나 유방암이 1차적으로 전이되는 폐에서의 gelatinase의 활성이 낮았다. 이것은 EW-7197이 유방암의 대표적 전이 장소인 폐의 ECM의 분해를 억제하여, 전이성 암세포가 정착하기 어렵게 함으로써 암의 전이를 억제하는 것으로 생각된다. 또한, 폐에서의 MMP-9/MMP-2 효소활성비는 암의 전이정도와 상관관계를 보인다는 것을 확인하였다. 그리고 in vitro에서는 확인되지 않았던 MMP-2의 존재가 in vivo에서 확인됨으로써, MMP-2의 활성에 영향을 미치는 면역세포의 역할을 주목하게 되었다. Tumor-associated macrophages는 암조직에서 가장 많이 존재하고 있는 면역세포의 형태이다. 암의 전이에서 TAMs이 암세포와 맺고 있는 상호관계는 매우 중요하다. 따라서 면역세포와 암세포간의 상호작용을 살펴보기 위해 RAW264.7 및 4T1 세포를 이용하여 in vitro co-cultivation을 실시한 결과, MMP-2의 발현은 살펴볼 수 없었으나 MMP-9 외에 55 kDa에서 또 다른 gelatinolytic band가 관찰됨으로써, 그들 간의 상호작용이 다른 종류의 MMPs를 발현시키는데 관여한다는 사실을 확인하였다. 전이성 암의 치료에 있어서 세포 외 기질 분해 억제 효과 이외에 혈관신생억제 효과도 중요한 기전이다. 혈관신생에 미치는 ALK5 억제제의 효능을 알아보기 위해 in vitro HUVECs tube formation assay를 실시하였다. 그 결과, VEGF의 존재 하에서 ALK5 inhibitor인 EW-7197이 혈관 신생을 억제한다는 것을 확인하였다. 따라서, EW-7197은 TGF-β에 의한 MMPs 효소 활성의 억제에 기인한 ECM 분해 억제 효과를 가짐과 동시에 VEGF에 의한 혈관신생도 함께 억제함으로써, EW-7197이 효과적인 항암작용을 가진다는 것도 확인할 수 있었다. 이번 연구를 통해, EW-7273과 EW-7197의 ECM에 대한 in vitro, in vivo에서의 작용, 혈관신생에 대한 작용, 암세포 주변 세포들과의 상호작용에 대해서 개괄적으로 살펴보았다. 따라서, EW-7197 및 EW-7273은 암의 전이 및 혈관 신생을 모두 억제할 수 있는 효과적인 항암제로서의 귀추가 주목된다.;Extracellular matrix has been recognized as a biological barrier of the passive form. However, emerged Steven Paget’s "seed and soil" theory, the importance of tumor microenvironment where cancer cell adhesion may occur was emphasized. EW-7197 and EW-7273, ALK5 inhibitors, had been developed as anti-cancer agents that act selectively on TGF-βR1. Because the tumor cells always interact with other cells and tissues, this study was dealt with how ALK5 inhibitors work on these different elements of the tumor microenvironment. MMPs are proteolytic enzymes that are necessary for degradation of ECM. Above all, gelatinases play an important role in metastasis by finally degrading fibral collagen primarily degraded by collagenases. EW-7273 reduces TGF-induced MMP2/9 mRNAs and MMP2/9 enzyme activities in MDA-MB-231 and 4T1, and EW-7197 reduces MMP2/9 mRNA and enzyme activities in MCF-10A. Therefore, this study confirmed that EW-7197 and EW-7273 suppressed ECM degradation and tissue remodelling. To determine the in vivo activity as a gelatinases, in vivo gelatin zymography was performed in tissues of 4T1 xenograft BALB/c mice. As a result, in the lung where primarily metastatic sites of breast cancer, MMP-2 and MMP-9 activities were inversely proportional to EW-7197 dose. Tumor-associated macrophages are most abundant immune cells in the tumors. Therefore, it is important how TAMs interact with cancer cells. Under the cultivation of combination with RAW264.7 and 4T1, gelatinolytic band by MMP-2 was not identified, but at 55 kDa, other gelatinolytic band was observed, which would be identified whether other types of MMPs was expressed or not. In the treatment of metastatic cancer, inhibition of angiogenesis is as important as inhibitory effect for the degradation of the extracellular matrix. In vitro HUVECs tube formation assay was performed for ALK5 inhibitors’ effect on angiogenesis. As a result, in the presence of VEGF, EW-7197 was found to inhibit angiogenesis. Thus, EW-7197 has a suppressive effect on ECM degradation caused by inhibition of the enzymatic activity of MMPs and an inhibitory effect on VEGF-induced angiogenesis. This study provided overview for EW-7273 and EW-7197 about the effects in vivo and in vitro, the effects on angiogenesis and the interaction of cells surrounding the cancer cells. Thus, EW-7197 and EW-7273 are noted as effective anti-cancer drugs to inhibit cancer metastasis and angiogenesis.I. 서론 1 A. TGF-β에 의한 Smad/non-Smad 신호전달 3 B. TGF-βR1에 작용하는 유기합성 억제제 (small molecule inhibitor) 5 C. Tumor invasion에 요구되는 단백분해효소계 (protease system) 5 1. MMPs (matrix metalloproteinases)와 TIMPs (Tissue Inhibitor of matrix metalloproteinases) 6 2. Plaminogen activator system 8 D. Angiogenesis 9 E. TAMs (Tumor-associated macrophages) 11 II. 실험 재료 및 방법 12 A. 실험 재료 및 기구 12 B. 세포 배양 13 C. RNA extraction and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis 14 D. Western Blot 17 E. Zymography 18 F. In vitro Tube formation assay 19 G. 동물실험 19 H. Statistics 20 III. 결과 21 A. In vivo 4T1 BALB/c xenograft mice에서 EW-7197에 의한 MMPs 활성 조절 21 1. EW-7197 경구투여에 의한 약효 평가 21 2. In vivo 4T1 xenograft mice의 조직에서의 MMPs 발현 24 3. MMPs와 metastatic nodule 간의 상관관계 28 B. In vitro에서의 EW-7273 및 EW-7197의 Matrix metalloproteinases에 대한 활성 평가 31 1. MMP-9에 대한 EW-7273 및 EW-7197의 영향 31 a. 생쥐유방암세포주에서의 MMP-9 효소 활성 및 관련 유전자 발현에 미치는 영향 31 b. 사람유방암세포주에서의 MMP-9 효소 활성 및 관련 유전자 발현에 미치는 영향 38 2. MMP-2에 대한 EW-7197 및 EW-7273의 영향 43 a. 생쥐유방암세포주에서의 MMP-2 효소 활성 및 MMP-2 mRNA의 변화 43 b. 생쥐유방암세포주에서의 MMP-2 효소 활성화에 기여하는 MT1-MMP 및 TIMP-2의 유전자 발현 조절 47 c. 사람유방암세포주에서의 MMP-2 효소 활성 및 MMP-2 mRNA의 변화 51 d. 사람유방암세포주에서의 MMP-2 효소 활성화에 기여하는 MT1-MMP 및 TIMP-2의 유전자 발현 조절 54 3. MMPs의 negative regulator인 RECK 유전자 발현의 in vitro에서의 조절 57 C. MCF-10A에서의 EW-7197 및 EW-7273에 의한 MMPs 효소활성, 유전자의 발현과 단백발현간 상관관계 61 D. In vitro co-culture system에서의 EW-7197 및 TGF-β의 작용 68 E. Plasminogen activation system 70 1. In vitro 생쥐유방암세포주에서 EW-7273에 의한 plasminogen activation system의 조절 70 2. In vitro 사람유방암세포주에서 EW-7273에 의한 plasminogen activation system의 조절 75 F. Angiogenesis에 미치는 EW-7197의 영향 80 III. 고찰 82 참고문헌 93 ABSTRACT 10

군보건의료체계에 대한 군인의 태도와 관련 요인 연구

학위논문(석사)--서울대학교 보건대학원 :보건학과 보건정책관리 전공,2001.Maste