대한진단검사의학회지제 29 권제 4 호 2009 Korean J Lab Med 2009;29:338-44 DOI 10.3343/kjlm.2009.29.4.338 Original Article Diagnostic Immunology 세포주배양에서근적외선조사배지의항염증효과 김상경 1 신임희 2 최창혁 3 최정윤 4 대구가톨릭대학교의과대학진단검사의학교실 1 의학통계학교실 2 정형외과학교실 3 내과학교실 4 Anti-Inflammatory Effect of Near-Infrared Irradiated Cell Culture Media Sang-Gyung Kim, M.D. 1, Im-Hee Shin, Ph.D. 2, Chang-Hyuk Choi, M.D. 3, and Jung-Yoon Choe, M.D. 4 Departments of Laboratory Medicine 1, Medical Statistics 2, Orthopedic Surgery 3, and Internal Medicine 4, Catholic University of Daegu, School of Medicine, Daegu, Korea Background : Near-infrared light (NIR, 0.8-1.5 μm light) has been used in therapeutic devices for various injuries such as infected, ischemic and hypoxic wound. NIR-emitting technology has been developed recently in Korea. We hypothesized that NIR may have an anti-inflammatory effect and investigated the effect of NIR-irradiated media on cell culture. Methods : Three kinds of cell lines, CAPE (vascular endothelial cell), NIH3T3 (fibroblast), and RD (smooth muscle cell) cells were cultured for 4 days in 10% FBS-containing media (1 10 4 cells/ well), which were irradiated or not irradiated (control) by Eco-NFIR Drive (Model #0210, Ecowavetech, Korea). The cells were stimulated by 10 mcg/ml of bacterial lipopolysaccharide (LPS) or sodium nitroprusside (SNP). Cellular proliferation was measured by methylthiazol tetrazolium assay. Expression of interleukin (IL)-1 beta and nitric oxide was measured by ELISA. Expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (inos) was measured by immunofluorescence staining. Results : NIR-irradiated medium was favorable for CAPE cell proliferation (N=8, P=0.000). IL-1 beta secretion from LPS-stimulated NIH3T3 cells incubated in the NIR medium was below that of control medium (N=4, P=0.026). Nitrate production seemed to be low in NIR-irradiated medium although statistically insignificant (N=4, P=0.076). Expression of inos of the LPS-stimulated cells was decreased in NIR medium, however, Cox-2 expression was not different between the two media. Conclusions : NIR-irradiated medium supported vascular endothelial cell proliferation and showed an anti-inflammatory effect on fibroblast culture. These results can be used as basic data for future research on the clinical application of NIR. (Korean J Lab Med 2009;29:338-44) Key Words : Near-infrared (NIR), Anti-inflammatory mediator, IL-1 beta, Nitric oxide (NO), Inducible nitric oxide synthase (inos) Received : January 15, 2009 Manuscript No : KJLM2218 Revision received : May 8, 2009 Accepted : May 13, 2009 Corresponding author : Sang-Gyung Kim, M.D. Department of Laboratory Medicine, Catholic University of Daegu, School of Medicine, 3056-6 Daemyeong 4-dong, Nam-gu, Daegu 705-718, Korea Tel : +82-53-650-4145, Fax : +82-53-653-8672 E-mail : sgkim@cu.ac.kr * 본연구는경북대학교휴먼팩트연구소의지원과 2006 년도대구가톨릭대학교의과학연구소연구비의지원으로이루어졌음. 서론근적외선 (near-infrared radiation, NIR) 은 0.8-1.5 μm 의파장대를가지는가시광선과중적외선사이에존재하는빛이다. 근적외선의이용은 1960년초미국에서농업분야에처음실용화되었고, 이후생화학, 제약, 고분자및의학에도그응용범위가확대되고있다. 의학에서근적외선의이용은주로진단기기 338
Anti-Inflammatory Effect of NIR 339 에사용되었으나, 최근에는다양한손상, 예를들면감염, 허혈성질환, 저산소성창상, 망막및신경손상에치료로사용된다고보고되고있다 [1, 2]. 또한그기전은명확하지않으나국소혈류의개선효과, 자율신경계에대한효과, 항염증효과, 창상치유촉진, 손상된신경조직의치유유도, 통증억제효과등이알려져있다 [1-5]. 국내에서도통증치료에사용한보고가있으며, 근적외선조사후요골동맥의혈류속도개선에유용하게사용될것으로보고하였다 [6, 7]. 최근국내에서근적외선을섬유, 물, 생활용품등에쪼이면지속적으로근적외선이방출되게할수있는기술이개발되어이를이용하여근육의운동강도를증가시켰다는보고가있고, 또한근적외선을쪼인의복을착용하고운동시신체의변화나, 근적외선을쪼인생활용품에서세균의성장억제효과에대한보고가나오고있다 [8-11]. 본연구는근적외선의창상치유효과와근적외선을물, 생활용품에쪼여서지속적으로방출될수있는기술을동시에이용하여, 임상에적용할가능성을알아보기위한기초연구로이미확립되어있는세포주를이용하여세포주의성장과, 염증성자극에대한근적외선조사배지의항염증효과를보고자하였다. 근적외선을직접세포에조사하는것은근적외선조사기등의기구가필요하나, 근적외선이지속적으로방출될수있는기술을이용하는것은미리근적외선을쪼인배지를이용하여방출되는근적외선을이용하므로보다안정적으로근적외선의효과를볼수있을것이다. 류마티스관절염을비롯한관절염들은관절의변형을초래할수있는만성염증성질환으로관절조직, 즉, 활막과주위조직의염증을특징으로하므로염증반응을매개하는여러가지시토카인들을차단하기위한항시토카인항체등생리활성물질들을치료에이용하고자하는노력이활발하나아직해결해야할여러가지문제점들이있다 [12]. 따라서본연구에서는관절염등염증치료에근적외선의적용가능성을알아보고자하였다. 이를위하여근적외선이조사된세포배양배지가체외세포배양에서세포생존력에미치는영향과항염증효과에어떠한영향을미치는지를관찰하고자하였다. 재료및방법 1. 재료 1) 세포주본연구에서는쥐의섬유아세포인 NIH3T3, 소의폐동맥내 피세포인 CAPE, 그리고사람의평활근세포인 RD 세포주를한국세포주은행으로부터구입하여사용하였다. 2) 배지및배양조건세포배양용배지 Dulbecco s modified eagle s medium (DMEM: Gibco BRL Life technologies, Grand Island, NY, USA) 과 RPMI 1640 media (RPMI:Gibco BRL Life technologies) 에근적외선파장조사기 Eco-NFIR Drive (Model #0210, Ecowavetech, Gyeongsan, Korea) 를사용하여근적외선램프와조사물과의거리 10 mm, 조사강도 3.8 W/cm 2 로30분간분말상태배지에조사한후액체배지로제조하여사용하였다. 이기계는삼원색파장혼합방식으로 1,400-1,700 nm 범위의근적외선을발산하는공진환경조성기이다. 대조군으로는근적외선을조사하지않은 DMEM과 RPMI 분말배지를액체로제조하여사용하였다. 우태아혈청을 10% 농도로첨가하여근적외선조사한 DMEM과근적외선조사한 RPMI를조제하여세포주의배양에이용하였으며 37, 5% CO 2 배양기에서 4일간배양하였다. 근적외선조사가세포들의증식과염증반응에어떠한효과가있는지보기위해서염증유발인자인 lipopolysaccharide (LPS: Sigma, St.Louis, MO, USA) 를 10 mcg/ml의농도로배지에첨가하여배양하였다. 또한동시에세포독성에대한근적외선조사의영향을보기위해서세포에직접독성효과가있는 sodium nitroprusside (SNP: Sigma) 를 0.5 mm로배지에첨가하여배양하였다. 2. 방법 1) Methylthiazol tetrazolium assay 배양되는세포 1 10 4 씩을 96 well 배양접시에접종하여 4일간배양하면서매일 methylthiazol tetrazolium (MTT: 3-[4, 5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) assay를시행하였다. MTT assay는살아있는세포의미토콘드리아 succinate dehydrogenase에의해노란색의 MTT가검푸른색으로변하는원리를이용하여살아있는세포수를측정할수있게하는것이다 [13, 14]. 세포증식의정도는배양시작하는날의 MTT 결과를기준으로하여배양일에따른증식의정도를 % 로나타내었다. 2) 염증성시토카인 (Interleukin-1, nitric oxide) 정량세포배양배지에쪼인근적외선이세포독성이나염증유발조건에서어떻게작용하는지를알아보기위해근적외선배지와일
340 김상경 신임희 최창혁외 1 인 반배지에 LPS와 SNP로처리한조건하에서세포를배양하면서배양상층액을배양후 4일간매일모아 -70 에냉동보관하였다. Interleukein-1 beta (IL-1 beta) 정량은상용화된 ELISA kit (R&D, Minneapolis, MN, USA) 를사용하였으며, nitric oxide (NO) 농도는 nitrate reductase에의해 nitrate가 nitrite 로되는효소반응에서생성되는 NO를측정하는 griess reaction 을근거로한상용화된 kit (Nitric Oxide parameter assay, R & D, Minneapolis, MN, USA) 를이용하였다 [15]. 3) inducible nitric oxide synthase와 cyclooxygenase-2 발현측정배양된 1 10 4 세포들을 chamber slide에서 4일간근적외선조사배지와일반배지에서 LPS, SNP 처리하여배양하면서매일 paraformaldehyde로고정후 FITC가 conjugated 된 inducible nitric oxide synthase (inos) 항체 (BD biosciences, San Jose, CA, USA) 를 1:100으로희석하여염색한후음성대조로는 isotype 항체를이용하여형광현미경 (NiKon, micro- FXA, Tokyo, Japan) 으로판독하였다. Cyclooxygenase-2 (COX-2) 염색은일차항체는 1:100, 2차항체는 1:50으로희석하여사용하였다. 두사람의숙련된판독자가각각독립적으로판독하여녹색의형광을띠면양성으로판독하였다. 3. 통계분석실험의결과는평균 ± 평균의표준오차로표현하였으며 SPSS 통계프로그램 (Win version 14.0, Chicago, IL, USA) 을이용하여, 두요인반복측정으로분석하였다. P 값은 0.05 이하일때를통계적으로유의하다고분석하였다. 결과 1. 정상조건에서각세포주의증식정상조건에서근적외선조사배지의효과는세포종류에따라다르게나타났다. CAPE 세포는소의혈관내피세포로근적외선조사배지에서 3일째 200% 이상증식하여일반배지보다증식이잘되었다 (N=8, P=0.000). 그러나 NIH3T3 세포와 (N=16, P=0.170) RD 세포는 (N=8, P=0.295) 근적외선조사한배지와일반배지에서세포증식에유의한차이가없었다 (N=8, P=0.285) (Fig. 1). 2. 염증유발물질 LPS와세포독성물질 SNP 자극후세포증식염증유발물질인 LPS로자극하였을때 CAPE, NIH3T3, RD 세포주들의세포증식은일반배지와근적외선조사배지에서통계학적으로유의한차이를보이지는않았다. 1) CAPE cell CAPE 세포는 LPS를 24 시간처리후일반배지와근적외선조사배지에서모두증식억제가나타났다. SNP를처리한 CAPE 세포는근적외선조사배지에서 3일째 170% 이상으로증식하여일반배지의 150% 증식보다더증식하였으나통계적으로유의한차이는아니었다 (data not shown). 2) NIH3T3 cell NIH3T3 세포는 LPS를 24 시간처리한후배양 2일째근적 2.4 CAPE cell NIH3T3 cell RD cell 2.2 0.8 2.0 2.0 1.6 1.2 1.8 1.6 1.4 0.6 0.4 1 2 3 4 1 2 3 4 1 2 3 4 Fig. 1. Methylthiazol tetrazolium test result of the cells. NIR-irradiated medium provided favorable condition for CAPE cells in terms of cellular mitochondrial enzyme (succinate dehydrogenase) actvity (mean±se, N=8, P=0.000). However, there was no significant difference of cellular proliferation of NIH3T3 cell (mean±se, N=16, P=0.170) and RD cell (mean±se, N=8, P=0.295) between NIR-irradiated medium (N, ) and control medium (C, ). Abbreviations: C, control medium; N, NIR-irradiated medium; MTT, methylthiazol tetrazolium; NIR, near-infrared irradiated.
Anti-Inflammatory Effect of NIR 341 외선조사배지에서더많이증식한데비해일반배지에서는증식이둔화되었다. 그러나배양 4일간의차이는통계적으로유의하지않았다 (N=16, P=0.285). 한편 NIH3T3 세포에세포독성물질인 SNP를처리한후배양하였을때는일반배지와는달리근적외선조사배지에서 2일째까지계속증식을보였다 (N=16, P=0.000) (Fig. 2). 3) RD cell RD 세포는 LPS를 24 시간처리후일반배지와근적외선조사배지에서모두증식억제가나타났다. SNP 처리후 RD 세포는정상조건보다는증식이억제되었으나, 일반배지보다근적외선조사배지에서는더증식하여정상조건에서와통계학적으로유의한차이가없었다 (data not shown). 3. IL-1 beta 측정 NIH3T3 세포에 10 mcg의 LPS를배지에첨가하였을때배양액의 IL-1 농도는배양 1일째근적외선조사배지에서 24±0.36 pg/ml로일반배지의 29±0.56 pg/ml보다낮았으며, 배양 2 일째도근적외선조사배지에서 31±0.51 pg/ml로일반배지에서35±0.60 pg/ml보다낮았다 (N=4, P=0.026) (Fig. 3). 혈관내피세포인 CAPE 세포와근육세포인 RD 세포는 LPS 자극에대해세포증식억제를보였으며 IL-1 beta가측정되지않았다. 4. 세포배양액의 nitric oxide 정량 LPS 처리한 NIH3T3 세포배양액의 NO는 5.48±0.45 μmol/ L로일반배지의 7.25±0.83 μmol/l보다낮았다. 하지만통계적으로유의하지는않았다 (N=4, P=0.076) (Fig. 4). 혈관내피세포인 CAPE 세포와근육세포인 RD 세포는 LPS 자극에대해 NO 생성의차이를보이지않았다. IL1 beta (pg/ml) 5. Inducible nitric oxide synthase 와 cyclooxygenase-2 발현 NIH3T3 세포는 LPS 24 시간처리한후근적외선조사배지 35.0 30.0 25.0 20.0 1 2 3 Fig. 3. IL-1 beta concentration of the supernatant of NIH3T3 cells which were stimulated with LPS (10 mcg/ml). The IL-1 beta secretion of NIH3T3 cells incubated in NIR-irradiated medium ( ) was below that in the control medium ( ) (mean±se, N=4, P= 0.026). Abbreviations: See Fig. 1. 0.50 9.0 0.45 0.40 NO (μmol/l) 8.0 7.0 6.0 0.35 5.0 0 1 2 3 Fig. 2. Methylthiazol tetrazolium test result of SNP-stimulated NIH3T3 cells. The SNP-stimulated NIH3T3 cells incubated in the NIR-irradiated medium ( ) proliferated more than the cells incubated in the control medium ( ) at day 2 (mean±se, N=16, P=0.000). Abbreviations: See Fig. 1. Control NIR Fig. 4. Nitric oxide production of NIH3T3 cells which were stimulated with LPS (10 mcg/ml). The NO level of NIH3T3 cells incubated in NIR-irradiated medium (NIR) was lower than that in the control medium (control), although statistically insignificant (mean ±SE, N=4, P=0.076). Abbreviations: See Fig. 1.
342 김상경 신임희 최창혁외 1 인 Table 1. Result of immunofluorescence staining of inducible nitric oxide synthase expression of the NIH3T3 cells Medium Treatment Findings Day 1 Day 3 Control None Positive Weakly positive Control LPS Positive Weakly positive Control SNP Positive Positive NIR None Positive Negative NIR LPS Positive Negative NIR SNP Positive Weakly positive Abbreviations: LPS, lipopolysaccharide; SNP, sodium nitroprusside; NIR, near-infrared irradiated. 에서배양 3일째일반배지에서보다 inos 발현이감소되었다 (Table 1, Fig. 5). 그러나이때동시에시행한 COX-2 expression은근적외선조사여부에관계없이모두음성이었다. 혈관내피세포인 CAPE 세포와근육세포인 RD 세포는 LPS 자극에대해세포증식억제를보였으며 inos와 COX-2 발현에차이가없었다. 고찰근적외선은특정파장대를가지는빛으로인체에무해하며, 적혈구에조사하였을때헤모글로빈에의해흡수되어적혈구의모양을변화시키고, 적혈구막의음전하 (electric zeta potential) 를변화시키며, 용혈을일으킨다고보고되었다 [16]. Ells 등 [2] 은근적외선이백혈병환자의구강점막증치료에유용하였으며 cytochrome oxidase가주된광수용체 (photoacceptor) 라고보고하였다. 이와같이근적외선은최근의료에도활발히적용되어특히창상치유를촉진시키며, 허혈성상처에도효과가있는것으로보고되었으나아직그기전은명확히밝혀지지않았다 [17, 18]. 본연구는근적외선조사배지의세포증식에미치는영향과항염증효과를알아보기위해여러가지의세포주를이용하여배양하면서배지에근적외선을조사하여대조군과비교하였다. 연구결과세포증식에근적외선이조사된배지는세포의종류에따라다른결과를보여주었다. 즉, 소의혈관내피세포주인 CAPE 세포는증식이증가되어통계적으로유의한차이를보여주었다. 그러나섬유아세포주인 NIH3T3와근육세포인 RD 세포에는근적외선조사배지가세포증식에통계적으로유의한차이를보여주지않았다. 즉, 근적외선은혈관내피세포의증식에유리한것으로생각되었으며, 이는근적외선이혈관재생및국소혈류를개선시킬수있다는보고와연관될것으로생각되 Fig. 5. LPS (10 mcg/ml) stimulated NIH3T3 cells show green fluorescence after Immunofluorescence staining of inos-fitc antibody (epi-fluoresence microscopy, NiKon, micro-fxa 400). Abbreviations: See Table 1. 었다 [7]. 섬유아세포인 NIH3T3 세포는염증유발물질로알려진 LPS 를처리하였을때와세포독성물질인 SNP를처리하였을때배양 2일째까지는근적외선을조사한배지에서계속증식한데비해일반배지에서는증식이둔화되었다. 그러나배양 4일간의차이는통계적으로유의하지않았다 (N=16, P=0.285). 또한염증유발물질인 LPS를배지에첨가하였을때 NIH3T3 세포는근적외선조사배지에서일반배지보다 IL-1 beta 농도가낮아근적외선이 LPS에의해유도되는 IL-1 beta의분비를억제하는효과가있는것으로생각되었다. 한편세포독성물질인 SNP를첨가하였을때근적외선조사한배지에서는 NIH3T3 세포의증식억제가어느정도방지되는것으로보아근적외선은세포독성물질에대해세포를어느정도보호할수있을것으로생각되었으며 SNP는 IL-1 beta 분비와무관한것으로나타났다. 혈관내피세포인 CAPE 세포와근육세포인 RD 세포는 LPS 자극에대해세포증식억제를보였으며 IL-1 beta를생성하지는않는것으로나타났다. 배양상층액의 NO 농도와동시에이들세포에서 inos 표현을조사한결과통계적으로유의하지는않았으나일반배지에서보다근적외선조사배지에서의 NO 양이낮아근적외선은 NIH- 3T3 세포의 NO 분비를억제할것으로기대할수있었다. 이때동시에측정한 inos 발현이억제되었고, COX-2 발현은차이가없었던것으로보아이는 inos 발현억제에기인한것으로생각할수있었으나, 이에관해서는앞으로좀더많은연구가필요할것으로생각되었다. 혈관내피세포인 CAPE 세포와근육세포인 RD 세포는 LPS 자극에대해 inos나 COX-2 발현에차이를
Anti-Inflammatory Effect of NIR 343 보이지않아염증반응과는무관해보였다. 이와같은연구결과로미루어보아근적외선조사배지는혈관내피세포증식을도우며, 염증성자극물질인 LPS에의해섬유아세포에서분비되는염증성시토카인 IL-1 beta의분비를억제하고, inos 발현억제를통하여염증매개물질인 NO 생성을억제하는것으로생각되었다. 본연구는 1,400-1,700 nm 범위의근적외선을배지에쪼여지속적으로방출되는근적외선이세포의염증성자극에미치는영향을보고자하는기초연구로, 앞으로근적외선을실제임상에적용하기위하여보다다양한세포와더많은수의검체에대한연구가필요할것이다. 요약배경 : 근적외선은 0.8-1.5 μm 파장대의빛으로다양한손상, 감염, 허혈성질환에치료로사용된다고보고되고있다. 최근근적외선을물, 섬유등에쪼이면지속적으로근적외선이방출되게하는기술이개발되었다. 본연구는근적외선을적절히조사한배지에서근적외선이지속적으로방출되어항염증효과를가질것으로가정하고, 우선세포배양에서근적외선조사배지가세포생존력과염증성자극에대한반응에미치는영향을알아보고자하였다. 방법 : CAPE ( 혈관내피세포 ), NIH3T3 ( 섬유아세포 ), RD ( 평활근세포 ) 등세가지종류의세포주를 10% 우태아혈청이포함되고, 근적외선이조사된배지와일반배지에서 37, 5% CO 2 배양기에서 4일간배양하였다 (1 10 4 cells/well). 근적외선의항염증효과를보기위해배지에 10 mcg/ml의 lipopolysaccharide (LPS) 와 sodium nitroprusside (SNP) 를첨가하였다. 세포증식은 methylthiazol tetrazolium (MTT: 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) assay로측정하였고, interleukin (IL)-1 beta와 nitric oxide는 ELISA 법, inducible nitric oxide synthase (inos) 와 cyclooxygenase-2 (COX-2) 는간접면역형광법을이용하였다. 결과 : 근적외선조사배지는 CAPE 세포증식에유리하였으며 (N=8, P=0.000), LPS 처리한 NIH3T3 세포에서 IL-1 beta 분비를감소시켰고 (N=4, P=0.026), nitrate 생성을억제시키는경향이있었으나통계적으로유의하지않았다 (N=4, P=0.076). 근적외선조사배지는 NIH3T3 세포에서 inos 발현을억제하였으나, COX-2 발현은두가지배지에서차이가없었다. 결론 : 근적외선조사배지는일반배지에비하여세포배양시혈관내피세포의증식을촉진시키고, 섬유아세포에는항염증효 과를나타내었다. 이러한결과는임상적으로염증성질환에근적외선을적용할기초자료로이용될수있을것이다. 참고문헌 1. Wong-Riley MT, Liang HL, Eells JT, Chance B, Henry MM, Buchmann E, et al. Photobiomodulation directly benefits primary neurons functionally inactivated by toxins: role of cytochrome c oxidase. J Biol Chem 2005;280:4761-71. 2. Eells JT, Wong-Riley MT, VerHoeve J, Henry M, Buchman EV, Kane MP, et al. Mitochondrial signal transduction in accelerated wound and retinal healing by near-infrared light therapy. Mitochondrion 2004;4:559-67. 3. Danno K, Mori N, Toda K, Kobayashi T, Utani A. Near-infrared irradiation stimulates cutaneous wound repair: laboratory experiments on possible mechanisms. Photodermatol Photoimmunol Photomed 2001;17:261-5. 4. Kandolf-Sekulovic L, Kataranovski M, Pavlovic MD. Immunomodulatory effects of low-intensity near-infrared laser irradiation on contact hypersensitivity reaction. Photodermatol Photoimmunol Photomed 2003;19:203-12. 5. Mochizuki-Oda N, Kataoka Y, Cui Y, Yamada H, Heya M, Awazu K. Effects of near-infra-red laser irradiation on adenosine triphosphate and adenosine diphosphate contents of rat brain tissue. Neurosci Lett 2002;323:207-10. 6. Oh WS, In JY, Ha KH, Hong KH. Application of polarized light irradiation to meralgia paresthetica. Korean J Anesthesiol 2000;38:183-6. ( 오완수, 인준용, 하경호, 홍기혁. 직선편광근적외선조사에의한지각이상성대퇴신경통의치료경험. 대한마취과학회지 2000;38:183-6.) 7. Han SM and Lee SC. The change of blood flow velocity of radial artery after linear polarized infrared light radiation near the stellate ganglion: comparing with the stellate ganglion block. Korean J Pain 2001;14:37-40. ( 한승문및이상철. 성상신경절부위의직선편광근적외선조사후요골동맥에서의혈류속도의변화 : 성상신경절차단술과의비교. 대한통증학회지 2001;14:37-40.) 8. Kim J, Otzel D, Kim W, Janelle CM. Near-infrared light and expectancy effects on maximal isokinetic strength performance: a randomized, double-blind, placebo-controlled study. J Strength Cond Res 2006;20:378-82. 9. Choi CH, Lee HS, Seo HS, Kim SG, Shin IH. Biochemical changes and recovery after half-course marathon. J Korean Orthop Soc Sports
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