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1 DOI: /trd ISSN: (Print)/ (Online) Tuberc Respir Dis 2009;67: CopyrightC2009. The Korean Academy of Tuberculosis and Respiratory Diseases. All rights reserved. 폐섬유모세포에서황사의미세먼지 (Particulate Matter 10) 가활성산소족과 TGF-ß, NF-κB, PDGF-α, Fibronectin 의생성에미치는영향 1 가천의과학대학교의학전문대학원의학과, 2 가천의과학대학교길병원호흡기내과김아현 1, 전수연 2, 윤진영 2, 김유진 2, 경선영 2, 이상표 2, 박정웅 2, 정성환 2 Original Article The Effect of Particulate Matter 10 from Asian Dust on the Production of Reactive Oxygen Species, TGF-ß, NF-κB, PDGF-α and Fibronectin in MRC-5 Fibroblast Cells Ah Hyun Kim, M.D. 1, Suyeon Chon, M.D. 2, Jin Young Yoon 2, Yu Jin Kim, M.D. 2, Sun Young Kyung, M.D. 2, Sang Pyo Lee, M.D. 2, Jeong Woong Park, M.D. 2, Sung Hwan Jeong, M.D., Ph.D. 2 1 Gachon University of Medicine and Science, 2 Division of Pulmonology, Department of Internal Medicine, Gachon University Gil Hospital, Incheon, Korea Background: Dust clouds blown by the wind from the arid deserts of Mongolia and Northeast China are known as Asian dust storms. Ambient particulate matter with a diameter <10 μm (PM 10) is associated with the exacerbation of respiratory diseases and increased mortality of heart and lung disease patients. The fibrotic effects of PM 10 of Asian dust to pulmonary fibroblast cells are unknown. This study examined the production of reactive oxygen species (ROS), TGF-β, NF-κB, PDGF-α and Fibronectin in fibroblasts exposed to Asian dust particles. Methods: Air samples were collected using a high volume air sampler (Sibata model HV500F) with an air flow of 500 L/min for at least 6 hours. The MRC-5 cells were exposed to 0, 50 and 100 μg/ml of PM 10 for 24 hours. ROS was detected by measuring the level of oxidized DCF using FACS. TGF-β, NF-κB, PDGF-α and fibronectin were detected by western blotting. Results: There was no increase in the ROS, TGF-ß and PDGF-α levels in the MRC-5 cells exposed to PM 10. The NF-κB level was higher in the MRC-5 cells exposed to 50 and 100 μg/ml of PM 10 for 24 hours. The fibronectin level in the MRC-5 cells after 24 hours incubation with 50 μg/ml PM 10 was significantly higher than the control group (PM μg/ml ±8.65 of control, p=0.005). Conclusion: PM 10 from Asian dust increases the activation of NF-κB and fibronectin expression in MRC-5 fibroblast cells. Key Words: Particulate Matter; Dust; Reactive Oxygen Species; Pulmonary Fibrosis; Fibroblasts 서 대기오염은호흡기계를비롯하여인체의여러장기에 Address for correspondence: Sung Hwan Jeong, M.D. Division of Pulmonology, Department of Internal Medicine, Gachon University Gil Medical Center, 1198, Kuwol-dong, Namdong-gu, Incheon , Korea Phone: , Fax: jsw@gilhospital.com Received: Sep. 14, 2009 Accepted: Nov. 4, 2009 론 유해하다. 1930년벨기에의 Muese Valley, 1948년 Donora, 1952년런던에서는대기오염으로인해사망률이급증한사건들이있었는데, 이사건들은대기오염의인체유해성에관한연구가시작된계기가되었다 1. 그후급격한산업화로인해대기오염이더욱심해졌으며인체에대한악영향은심각한상태에있다. 대기오염물질중에서도미세먼지 (particulate matter) 는다양한크기, 구성, 발생원에따라다양한독성물질을함유하고있어여러가지건강문제를일으킨다. 10 μm 528

2 Tuberculosis and Respiratory Diseases Vol. 67. No. 6, Dec 보다큰미세먼지는대개구강과기도에서걸러지지만 10 μm 이하의미세먼지 (particulate matter 10, PM 10) 는흡입시하부기관지및폐의가스-교환부분까지침착하여호흡기계에손상을일으킬수있다고보고되고있다 2. 인공적요인에의한대기오염이외에자연적요인으로는우리나라에매년봄철에생기는황사가있다. 황사는먼지연무 (dust haze) 의일종으로중국, 몽골과같은동북아시아건조지대에서생긴다량의흙먼지가장거리수송되어대기를떠다니며서서히하강하는현상이다. 우리나라에영향을미치는황사입자의크기는대개지름이 10 μm 이하인 PM 10 이다. 국내에서시행된연구에서는황사기간중에 PM 10 이증가하고최대호기유속이감소하여기관지천식환자의호흡기증상악화를유발할수있음이밝혀진바있다 3. 그러나 PM 10 이어떤기전으로호흡기계에악영향을미치는지에대해서는아직정확히밝혀지지않았다. 그동안연구가많이되어있는가설은 PM 10 이세포에산화적스트레스를유발하여 NF-κB 를활성화하여염증을유발하는사이토카인 (cytokine) 들이증가하면서염증이일어난다는가설이다 4. 미세먼지가폐상피세포와대식세포에작용하여산화적스트레스를유발하고활성산소족 (reactive oxygen species, ROS) 을증가시킨다는것은여러연구에서밝혀진바있으며 5, PM 10 이폐상피세포에작용하여 interleukin-1, interleukin-8, GM-CSF 등과같은염증성사이토카인을증가시킨다는것도기존의연구에서증명되었다 6. 최근에는특발성폐섬유화증 (idiopathic pulmonary fibrosis) 의병인에대해세포사멸역설 (apoptosis paradox) 의개념이적용되고있는데그기전에대해제기되는가설은 ROS와 TGF-ß 가섬유화의조절에관여한다는것이다. TGF-ß는세포외기질 (extracellular matrix) 의조합과리모델링에관여하는데과다발현시에는결체조직을비정상적으로축적시켜폐의섬유화를유도한다고알려져있다 7. 세포사멸역설의개념은폐상피세포가외부자극으로인해손상되면세포사멸이발생하고폐의구조를유지하기위해섬유모세포와근섬유아세포 (myofibroblast) 가폐상피세포가세포사멸된부위에몰려들어축적되기때문에 fibroblastic foci를형성한다는것이다 8. 지금까지 PM 10 이폐의상피세포에작용하면여러가지염증성사이토카인의생성을증가시킨다는것이여러연구에서증명되었지만폐섬유모세포에는어떤작용을하는지에대한연구는미미하여, 본연구에서는황사에포함된 PM 10 이폐섬유모세포에작용하여 ROS의생성에어떤 영향을미치는지알아보고 TGF-ß, NF-κB, PDGF-α, fibronectin의생성을변화시키는지관찰하였다. 1. PM 10 의준비및성상 대상및방법 대기시료의포집은 2006년봄철황사기간에인천지역 ( 인천시남동구구월동가천의대길병원응급센터옥상 ) 에서실시하였다. 공기포집기 (HV500F; Sibata, Tokyo, Japan) 를이용하여분당 500 L로하루 6시간씩대기분진을필터 (Pore size 0.25 μm; Millipore, Bedford, MA, USA) 를통해포집하였다. 포집한필터 (filter) 를조각내어 phosphate buffered saline (PBS) 10 ml를넣은튜브 (tube) 에넣어틈틈이소용돌이혼합기 (vortex mixer) 를사용하여 30분간혼합하였다. 필터를건져내고나머지를 10 μm pore의 filter (Mitex TM membrane filters; Millipore) 로여과하였다. 약 1분간초음파로파쇄 (sonication) 하고 121 o C에서 15분간멸균한후사용전까지 20 o C에보관하였다. 국내에유입되는황사의입자크기는대개 10 μm 이하이며본실험에사용된미세먼지입자의크기도평균 6.2 μm 이었다. 실험동안세포에가해줄 PM 10 의농도는각각 0, 50, 100 μg/ml 로정하였다. 2. 세포배양 사람태아폐섬유모세포인 MRC-5 cell (KCLB, Seoul, Korea) 을 10% 우태아혈청이포함된 5.6 mm 포도당함유 Dulbecco s modified Eagle s medium (DMEM) (GiBCO BRL, Goithersburg, MD, USA) 배지에서계대배양하였다. Haematocytometer 를이용하여세포수를세어 6-well plate 의각 well에세포수가 개가되도록접종하고 37 o C, 5% CO 2 배양기에서 24시간배양하였다. 세포가 % 배양되면 0.5% 우태아혈청을포함한 DMEM 배지로교체하여 24시간배양함으로써세포성장을정지시키고 (starvation) 성장주기를동일화하였다. 그후 PM 10 을가해주지않은세포를대조군으로하고, 대조군을제외한각 well에 50, 100 μg/ml 의농도로 PM 10 을가해준후 24시간동안배양하였다. 3. 세포내 ROS 의측정및 confocal microscopy PM 10 을가해주고 24 시간배양한후세포에침투가가능 529

3 AH Kim et al: The effect of particulate matter 10 to MRC-5 fibroblast cells 한형광탐촉자인 2,7 -dichlordihydrofluorescin diacetate (DCF-DA) (Molecular Probes Inc., Eugene, OR, USA) 를 5 mm씩가해준후 20분간배양하였다. 그후 PBS 로배지에남아있는 CM-H2DCF-DA 를제거한후세포내의산화를통해생성된 2,7 -dichlordihydrofluorescin (DCFH) 의형광을 fluorescence-activated cell sorting (FACS) (BD Biosciences, San Jose, CA, USA) 를이용하여측정하였다. 측정한세포수는각표본당 20,000개로하였다. 그리고각표본의형광생성정도를 confocal microscope (IX81; Olympus, Tokyo, Japan) 를이용하여사진으로찍었다. 4. NF-κB, TGF-ß, PDGF-α, Fibronectin 에대한 western blot PM 10 을가해주고 24시간배양한후 NF-κB 를관찰할표본은각 well에서배지를 1 ml씩튜브에옮겨담아원심분리하여상층액을얻었다. TGF-ß, PDGF-α, fibronectin 을관찰할표본은세포에 lysis buffer (Pro-PREP protein extraction solution) (Intron Biotechnology Inc., Seoul, Korea) 를각 well당 100 ml를가하여세포를수확한후 20 o C에서 30분간 incubation 한후, 원심분리하여상층액을얻었다. NF-κB 에대한실험은 MRC-5 세포를분쇄한후 ultracentrifuge를시행하여 nuclear protein과 cytosol로나눈후 nuclear protein 에서측정하였으며, 항체로는 p65 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) 를사용하였다. TGF-ß, PDGF-α, fibronectin 을측정할 lysate 는 Bradford assay 를통해단백질이 30 μg 이되도록계산하여준비하였다. 준비된표본을 8% acrylamide gel에각각 loading하여전기영동하였다. 전기영동이끝나면 PVDF membrane (Roche, Indianapolis, IN, USA) 에 1시간동안 100 V로전이하였다. 전이된 membrane을 PBST (Tween 20 1%) 에녹인 5% non fat milk 에담궈 1시간동안 blocking하였다. PBST (Tween20 0.1%) 를이용하여 NF-κB, TGF-ß, PDGF-α, fibronectin 각각에대한일차항체 (Santa Cruz Biotechnology) 을 1:700 1:1,000으로희석하였다. 희석된일차항체용액에 membrane 을담가 4 o C shaker에서 overnight하였다. 그후 PBST (Tween20 0.1%) 로세척하고각각에대한이차항체 (Santa Cruz Biotechnology) 를 PBST (Tween20 0.1%) 에 1:10,000 1:20,000으로희석하여 membrane 을담가 1시간동안 shaking 하였다. PBST (Tween20 0.1%) 로세척한후 WEST-SOL (Intron Inc., Seoul, Korea) 을사용하여 X-ray film에감광시켰다. 감광된 film을 scan하여 density를구하여대조군과비교하였다. 5. 분석방법 결과는평균 ± 표준편차로표시하였다. 분석은 SPSS version 12.0 (SPSS Inc., Chicago, IL, USA) 프로그램을이용하였고 p<0.05인경우에통계학적으로유의하다고판단하였다. 대조군과 PM 10 처리군과의차이는비모수적방법인 Kruskall-Wallis test를통해검정한후 Mann-Whitney test로사후검정하여 p값을표시하였다. 1. 황사의성상 결 황사의주성분은규소계통의모래와황토로서토양기원의원소인알루미늄 (Al), 칼슘 (Ca), 철 (Fe), 마그네슘 (Mg) 등이포함되어있었으며, 특히 2006년봄철인천의대기성분중황사시기에는토양기원의 AlSi, SiO 2 와같은입자들이증가하여있었다 9. 그중가장많은성분은 SiO 2 로서전체중 48% 를차지하였고, 이밖에여러가지중금속물질들이포함되어있었으며, 분석은한국요업기술원 (KICET) 에의뢰하였다 (Table 1). 2. PM 10 이 MRC-5 cell 에서 ROS 의생성에미치는영향 MRC-5 cells에황사기간에채집한 PM 10 을처리하고 24 시간후에세포내 ROS를 FACS로측정한결과를보면각농도에서대조군보다의미있게증가하지않았다 (PM 10 Table 1. Analysis of Asian sand dust Contents Concentration (wt %) SiO AL 2O Na 2O 2.19 Fe 2O CaO 5.18 MgO 2.37 K 2O 3.80 TiO MnO 0.09 Heating loss Korea Institute of Ceramic Engineering and Technology (KICET). 과 530

Tuberculosis and Respiratory Diseases Vol. 67. No. 6, Dec. 2009 0 μg/ml 113.01±83.4, PM10 50 μg/ml 92.48±70.46, p=0.674, PM10 100 μg/ml 106.80±88.73, p=0.916) (Figure 1).

PM10이 MRC-5 cell에서 TGF-ß의 생성에 미치는 영향 MRC-5 cells에 황사기간에 채집한 PM10을 처리하고 24 시간 후의 TGF-ß 생성 결과를 보면 각 농도에서 대조군보 다 의미 있게 증가하지 않았다 (PM10 50 μg/ml 101.43± 6.99 of control, p=1.000, PM10 100 μg/ml 97.91±26.

PM10이 MRC-5 cell에서 NF-κB의 생성에 미치는 영향 MRC-5 cells에 황사기간에 채집한 PM10을 0, 50, 100 μg/ml의 농도로 처리하고 24시간 후의 NF-κB의 생성 결과를 보면 PM10을 처리했을 때가 처리하지 않은 대조군 에 비해 NF-κB 발현이 증가하는 것을 관찰할 수 있었다 (Figure 4). Figure 1.

4 Tuberculosis and Respiratory Diseases Vol. 67. No. 6, Dec μg/ml ±83.4, PM10 50 μg/ml 92.48±70.46, p=0.674, PM μg/ml ±88.73, p=0.916) (Figure 1). 세포 내의 산화를 통해 생성된 2,7 -dichlordihydrofluorescin (DCFH)를 confocal microscope로 관찰한 결과 에서도 PM10을 처리하고 24시간 후에 ROS의 발현에 큰 차이를 보이지 않았다(Figure 2). 3. PM10이 MRC-5 cell에서 TGF-ß의 생성에 미치는 영향 MRC-5 cells에 황사기간에 채집한 PM10을 처리하고 24 시간 후의 TGF-ß 생성 결과를 보면 각 농도에서 대조군보 다 의미 있게 증가하지 않았다 (PM10 50 μg/ml ± 6.99 of control, p=1.000, PM μg/ml 97.91±26.40 of control, p=1.000) (Figure 3). 4. PM10이 MRC-5 cell에서 NF-κB의 생성에 미치는 영향 MRC-5 cells에 황사기간에 채집한 PM10을 0, 50, 100 μg/ml의 농도로 처리하고 24시간 후의 NF-κB의 생성 결과를 보면 PM10을 처리했을 때가 처리하지 않은 대조군 에 비해 NF-κB 발현이 증가하는 것을 관찰할 수 있었다 (Figure 4). Figure 1. Measured Reactive oxygen species (ROS) by FACS in MRC-5 cells after 24 hours incubation with PM10. MRC-5 cells were exposed to 50 and 100 μg/ml of ambient particulate matter with a diameter of less than 10 μm for 24 hours. Results shown in the box graph are measured ROS values by FACS. The measured ROS in MRC-5 cells after 24 hours incubation with PM10 were not increased (PM10 0 μg/ml ±83.4, PM10 50 μg/ml 92.48±70.46, p=0.674, PM μg/ml ± 88.73, p=0.916). 5. PM10이 MRC-5 cell에서 PDGF-α의 생성에 미치는 영향 MRC-5 cells에 황사기간에 채집한 PM10을 처리하고 24 시간 후의 PDGF-α 생성 결과를 보면 PM10의 농도가 50 μg/ml일 때 대조군에 비해 의미 있는 차이를 보여주지 않았으며, PM10의 농도가 100 μg/ml일 때는 오히려 유의 하게 감소하였다(PM10 50 μg/ml ±8.13 of con- Figure 2. Confocal microscopic findings in MRC-5 cells after 24 hours incubation with PM10. The fluorogenic probe 2 7 -dichlordihydrofluorescin diacetate (DCF-DA) in the cell was oxidized and detected as green color. The oxidized DCF-DA was not increased in MRC-5 cells after 24 hours incubation with PM

AH Kim et al: The effect of particulate matter 10 to MRC-5 fibroblast cells Figure 3. TGF-ß in MRC-5 cells after 24 hours incubation with PM 10. TGF-ß was detected by western blotting.

TGF-ß in MRC-5 cells after 24 hours incubation with PM 10 was not increased compared with control group (PM 10 50 μg/ml 101.43±6.99 of control, p= 1.000, PM 10 100 μg/ml 97.91±26.40 of control, p= 1.

5 AH Kim et al: The effect of particulate matter 10 to MRC-5 fibroblast cells Figure 3. TGF-ß in MRC-5 cells after 24 hours incubation with PM 10. TGF-ß was detected by western blotting. The density of band was measured by densitometry. Results shown in the bar graph are the percentage from control values. TGF-ß in MRC-5 cells after 24 hours incubation with PM 10 was not increased compared with control group (PM μg/ml ±6.99 of control, p= 1.000, PM μg/ml 97.91±26.40 of control, p= 1.000). Figure 4. NF-κB in MRC-5 cells after 24 hours incubation with PM 10. MRC-5 cells were exposed to 50 and 100 μg/ ml of ambient particulate matter with a diameter of less than 10 μm for 24 hours. NF-κB was detected by western blotting. trol, p=0.487, PM μg/ml 97.50±1.81 of control, p=0.037) (Figure 5). 6. PM 10 이 MRC-5 cell 에서 Fibronectin 의생성에미치는영향 MRC-5 cells에황사기간에채집한 PM 10 을처리하고 24 시간후의 fibronectin 발현결과를보면 PM 10 의농도가 50 μg/ml 일때대조군에비해유의하게증가하였다 (PM μg/ml ±8.65 of control, p=0.005). 그러나, PM 10 의농도가 100 μg/ml 일때는대조군에비해유의한차이를보이지않았다 (PM μg/ml ± of control, p=0.577) (Figure 6). Figure 5. PDGF-α in MRC-5 cells after 24 hours incubation with PM 10. PDGF-α was detected by western blotting. The density of band was measured by densitometry. Results shown in the bar graph are the percentage from control values. Results shown in the bar graph are the percentage from control values (*p<0.05). PDGFα in MRC-5 cells after 24 hours incubation with 50 μg/ ml PM 10 was not significantly increased compared with control group (PM μg/ml ±8.13 of control, p=0.487). PDGF-α in MRC-5 cells with 100 μg/ml PM 10 was signifi cantly decreased compared with control group (PM μg/ml 97.50±1.81 of control, p=0.037). 고 미세먼지는다양한크기, 구성, 발생원을갖는것으로다양한독성물질을함유하고있어여러가지건강문제를일으킨다. 황사는계절적으로중국북서부나몽골에서발생하여봄철우리나라에자연적으로대기오염을일으키는데입자의크기는대개지름이 10 μm 이하인 PM 10 으로되어있다. 황사의주성분은규소계통의모래와황토로서토양기원의원소인알루미늄 (Al), 칼슘 (Ca), 철 (Fe), 마그네슘 (Mg) 등이포함되어있고최근중국의공업화로인해납 (Pb), 구리 (Cu), 망간 (Mn) 등금속성분이증가하고있다 10. 이들중금속성분의증가는인체로흡입시에산화적스트레스를증가시켜호흡기계를비롯한주요기관에중대한악영향을미칠것으로추정되고있다. 찰 532

Tuberculosis and Respiratory Diseases Vol. 67. No. 6, Dec. 2009 Figure 6. Fibronectin and β-actin in MRC-5 cells after 24 hours incubation with PM 10.

The density of band was measured by densitometry. Results shown in the bar graph are the percentage from control values (*p<0.05).

6 Tuberculosis and Respiratory Diseases Vol. 67. No. 6, Dec Figure 6. Fibronectin and β-actin in MRC-5 cells after 24 hours incubation with PM 10. MRC-5 cells were exposed to 50 and 100 μg/ml of ambient particulate matter with a diameter of less than 10 μm for 24 hours. Fibronectin was detected by western blotting. The density of band was measured by densitometry. Results shown in the bar graph are the percentage from control values (*p<0.05). Fibronectin in MRC-5 cells after 24 hours incubation with 50 μg/ml PM 10 was significantly increased compared with control group (PM μg/ml ±8.65 of control, p=0.005). Fibronectin in MRC-5 cells with 100 μg/ml PM 10 was not increased compared with control group (PM μg/ml ±11.91 of control, p=0.577). 실제로미세먼지를인체세포에가하면산화적스트레스가발생하면서 ROS가증가하거나, 염증성사이토카인들이증가하여세포손상을발생시킬수있다는것이여러연구에서밝혀진바있다 5,6,11. 또신장세포에서는인위적으로과산화수소를투여하여 ROS를증가시키면 fibronectin 생성이증가하고 plasminogen activator inhibitor-1 도증가되면서세포외기질 (extracellular matrix) 의축적을초래하게됨이밝혀졌다 12. 그러나본연구에서는황사의미세먼지에의해폐섬유모세포인 MRC-5 세포에서 ROS가의미있게증가하지않았다. 이는저자들이폐상피세포가황사의 PM 10 에의해서자극받았을시에 ROS 의생성이의미있게증가하는것을 보고한것과는다른결과인데, 이는상피세포와섬유모세포간에미세먼지에대한반응이차이가있기때문이라고추측된다 12. 기존의연구에서보면폐의손상시에가장먼저민감하게손상받는세포는제1형폐상피세포이다. 폐포손상이발생하면그자리를채우기위해제2형폐상피세포가이동하여제1형폐포세포의자리를대신한다. 폐포벽의표면에있는상피세포와는달리폐섬유모세포는간질에있으며일반적으로외부자극에의한손상에저항성이있다. 폐섬유모세포는폐상피세포가손상을받으면증식하면서간질에세포외기질을구성하는성분들을생산하여폐섬유화를발생시키는데, 본연구의결과에서두세포사이에황사미세먼지에대한반응의차이를보이는것이이런기능상의차이가작용하여나타난결과로유추해본다 13. 또한이전의연구에서폐섬유모세포에과산화수소 (hydrogen peroxide) 를직접가해서활성산소를발생시키면 TGF-ß1 이증가한다는연구결과가있었으나 14, 본연구에서는황사의미세먼지 (PM 10 ) 를 MRC-5 세포에가했을때 ROS와함께 TGF-ß 도증가시키지않는것으로나타났는데, 본연구에서이들이증가하지않은것은, 폐섬유모세포는 PM 10 에대하여직접노출시에도저항성이크기때문으로사료되고, 이로인해 ROS의형성에크게변화가없으면서 TGF-ß 나 PDGF-α의발현도증가시키지않은것으로추측되나, 이과정에서 CTGF같은다른사이토카인이나성장인자 (growth factor) 등의경로가존재할수있을가능성이있기때문에, 이의증명을위해서는 TGFß이외의다른성장인자들에대한추가적인연구가필요할것이다. 본연구의주요한결과는황사기간에포집한미세먼지 (PM 10 ) 는 50 μg/ml 의농도에서폐섬유모세포인 MRC-5 세포에가해주면 24시간 incubation 후에 fibronectin 의양이유의하게증가했다는것이다. 다만 100 μg/ml의농도에서는의미있는증가가발생하지않았는데이는고농도의미세먼지하에는세포의활성도 (viability) 가감소되면서나타난소견으로생각된다. 세포외기질의구성성분인 fibronectin 은 collagen과같은물질에대한접착력이강하여증가시세포외기질이축적되어기도의재형성 (remodeling) 을유발한다. 그러므로이결과는황사의미세먼지가폐로흡인되었을때폐에세포외기질을축적할수있고나아가섬유화를유발할가능성이있음을시사한다. 미세먼지가폐에손상을일으켜 533

7 AH Kim et al: The effect of particulate matter 10 to MRC-5 fibroblast cells 소기도의섬유화와재형성을유발하는데는 NF-κB 의활성화가중요하다는것이여러연구에서밝혀졌는데 15, 본연구에서도황사의미세먼지를 MRC-5에가하면 NF-κB 의발현이증가하는것을확인할수있었다. 이결과는황사이외의다른종류미세먼지를폐상피세포에가했을때 NF-κB 의발현이증가되었다는기존의연구와비슷한결과이다 16. NF-κB 는전염증성분자들을조절하는 DNA 에대한전사인자로서미세먼지를폐상피세포에가하면 NF-κB 를매개로하여염증성사이토카인인 interleukin (IL)-6, IL-8, tumor necrosis factor 등이증가한다는연구가있다 17. 그증가기전은아직밝혀지지않았지만미세먼지의내독소, 또는미세먼지에포함된철과같은중금속성분이관여된다고주장하는연구들도있다 18,19. 결론적으로황사의미세먼지는폐섬유모세포에서 NF-κB 의발현을통해 fibronectin 의증가를유발할것이라고예측된다. 다만폐섬유모세포가황사의미세먼지 (PM 10 ) 에의하여자극을받았을시에 NF-κB 가활성화되면서어떤신호전달과정에의하여 cytokine 이나 growth factor 의발현이증가되어 fibronectin 의발현이증가하는지는향후추가적인연구가필요할것으로사료된다. 참고문헌 1. Bell ML, Davis DL. Reassessment of the lethal London fog of 1952: novel indicators of acute and chronic consequences of acute exposure to air pollution. Environ Health Perspect 2001;109 Suppl 3: Donaldson K, Stone V, Clouter A, Renwick L, MacNee W. Ultrafine particles. Occup Environ Med 2001;58: Park JW, Lim YH, Kyung SY, An CH, Lee SP, Jeong SH, et al. Effects of ambient particulate matter (PM 10) on peak expiratory flow and respiratory symptoms in subjects with bronchial asthma during yellow sand period. Tuberc Respir Dis 2003;55: Donaldson K, Stone V. Current hypotheses on the mechanisms of toxicity of ultrafine particles. Ann Ist Super Sanita 2003;39: Becker S, Soukup JM, Gilmour MI, Devlin RB. Stimulation of human and rat alveolar macrophages by urban air particulates: effects on oxidant radical generation and cytokine production. Toxicol Appl Pharmacol 1996;141: Kim JH, Jeon HK, Kim MK, Kyung SY, An CH, Lee SP, et al. Particulate matter from Asian dust storms induces the expression of proinflammatory cytokine in A549 epithelial cells. Tuberc Respir Dis 2006;60: Sheppard D. Transforming growth factor beta: a central modulator of pulmonary and airway inflammation and fibrosis. Proc Am Thorac Soc 2006;3: Thannickal VJ, Horowitz JC. Evolving concepts of apoptosis in idiopathic pulmonary fibrosis. Proc Am Thorac Soc 2006;3: Hwang HJ, Kang SJ, Kang SE, Park YM, Kim HK, Roh CU. Single particle characterization of aerosol samples collected during Asian dust storm events and non-asian dust period in Incheon. Proceedings of the 2007 Environmental Societies Joint Conference; 2007 May 2-4; Busan, Korea. Seoul: Korean Society for Atmospheric Environment; p Baek KW, Chung JD. Study on the yellow sandy dust phenomena in Korean peninsula and chemical compositions in fine particles at background sites of Korea. Korean J Sanit 2004;19: Park KS, Kim YJ, Yoon JY, Kyung SY, An CH, Lee SP, et al. Particulate matter 10 from Asian dust storms induces the expression of reactive oxygen species, NF-kappaB, TGF-beta and fibronectin in WI-26 VA4 epithelial cells. Tuberc Respir Dis 2008;65: Jiang Z, Seo JY, Ha H, Lee EA, Kim YS, Han DC, et al. Reactive oxygen species mediate TGF-beta1-induced plasminogen activator inhibitor-1 upregulation in mesangial cells. Biochem Biophys Res Commun 2003;309: Rennard SI, Bitterman PB, Crystal RG. Response of the lower respiratory tract to injury. Mechanisms of repair of the parenchymal cells of the alveolar wall. Chest 1983;84: Junn E, Lee KN, Ju HR, Han SH, Im JY, Kang HS, et al. Requirement of hydrogen peroxide generation in TGF-beta 1 signal transduction in human lung fibroblast cells: involvement of hydrogen peroxide and Ca 2+ in TGF-beta 1-induced IL-6 expression. J Immunol 2000; 165: Churg A, Brauer M, del Carmen Avila-Casado M, Fortoul TI, Wright JL. Chronic exposure to high levels of particulate air pollution and small airway remodeling. Environ Health Perspect 2003;111: Shukla A, Timblin C, BeruBe K, Gordon T, McKinney W, Driscoll K, et al. Inhaled particulate matter causes expression of nuclear factor (NF)-kappaB-related genes and oxidant-dependent NF-kappaB activation in vitro. Am J Respir Cell Mol Biol 2000;23: Quay JL, Reed W, Samet J, Devlin RB. Air pollution 534

8 Tuberculosis and Respiratory Diseases Vol. 67. No. 6, Dec particles induce IL-6 gene expression in human airway epithelial cells via NF-kappaB activation. Am J Respir Cell Mol Biol 1998;19: Chandel NS, Trzyna WC, McClintock DS, Schumacker PT. Role of oxidants in NF-kappa B activation and TNF-alpha gene transcription induced by hypoxia and endotoxin. J Immunol 2000;165: Jiménez LA, Thompson J, Brown DA, Rahman I, Antonicelli F, Duffin R, et al. Activation of NF-kappaB by PM 10 occurs via an iron-mediated mechanism in the absence of IkappaB degradation. Toxicol Appl Pharmacol 2000;166:

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386-390.hwp 386 HANYANG MEDICAL REVIEWS Vol. 29 No. 4, 2009 우리나라 미숙아의 통계와 의료비용 Statistics and Medical Cost of Preterm in Korea 윤혜선 을지대학교 노원을지병원 소아청소년과학교실 Hye Sun Yoon, M.D., Ph.D., Department of Pediatrics, Nowon