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1 패혈증에서 PD-L1 (Programmed Cell Death-ligand 1) 의발현증가기전 서울대학교의과대학내과학교실및폐연구소 이상민 Induction Mechanism of PD-L1 (Programmed Cell Death-ligand 1) in Sepsis Sang-Min Lee, M.D. Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine and Lung Institute, Medical Research Center, Seoul National University College of Medicine, Seoul, Korea PD-L1 is expressed in a variety of antigen-presenting cells and provides T cell tolerance via ligation with its receptor PD-1 and B7-1 on T cells. Stimulation with lipopolysaccharide (LPS) can increase the level of PD-L1 expression in B cells and macrophages, which suggests that this molecule plays a role in the immunosuppression observed in severe sepsis. The aim of this study was to identify which of the downstream pathways of TLR4 are involved in the up-regulation of PD-L1 by LPS in macrophages. Flow cytometry was used to examine the expression of PD-L1 in RAW macrophages stimulated with LPS. The following chemical inhibitors were used to evaluate the role of each pathway: LY for PI3K/Akt, SB for p38 MAPK, and U0126 for MEK. LPS induced the expression of PD-L1 in a time- and dose-dependent manner. Transfection of sirna for TLR4 suppressed the induction of PD-L1. Pretreatment with LY and SB decreased the level of PD-L1 expression but U0126 did not. Overall, the PI3K/Akt and p38 MAPK pathways are involved in the up-regulation of PD-L1 expression in RAW macrophages stimulated with LPS. (Tuberc Respir Dis 2008;65: ) Key Words: PD-L1, LPS, Sepsis, Macrophage 서 패혈증은중환자사망원인의 1위를달리고있는질환으로미국통계에의하면매년 750,000명이상의환자가발생하며그중 210,000 명이상이사망하고있다 1. 최근대증요법이발달하고있지만패혈증의이환율과사망률은감소추세를보이지못하고있는실정으로, 보다근본적인치료법의개발이시급해진상황이다. 그동안많은연구자들은패혈증이세균내독소와같은외부물질에의해유발된염증반응이각종사이토카인의연쇄반응에의하여증폭됨으로써진행한다는사실을발견하고, 이러한염증반응의악순환의고리를끊는치료법개발에몰두하게되었다. 이에염증반응을차단하기위 Address for correspondence: Sang-Min Lee, M.D. Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Seoul National University College of Medicine, 101, Daehangno, Jongno-gu, Seoul , Korea Phone: , Fax: sangmin2@snu.ac.kr 론 하여 steroid 2, antiendotoxin antibody, tumor necrosis factor (TNF) antagonists, interleukin-1-receptor antagonist 등여러약제에대한임상시험이진행되었지만, 안타깝게도그효과가입증된약제는거의없는상황이고, 오히려사망률을증가시킨다는결과도보고되고있는실정이다. 패혈증은조절되지않는염증반응의증폭에의하여발생한다는것이초기의개념이었고, 기존의치료법도염증반응을억제하고자하는데초점을맞추어개발이되었다. 그러나, 위에서살펴본바와같이이러한치료법들이효과가없음이입증이되어패혈증에대한새로운접근방법이필요하게되었다. 1996년도에 Bone 3 은 CARS (compensatory anti-inflammatory response syndrome) 라는개념을제안하여기존의개념과는달리염증반응및면역반응의억제도패혈증의병태생리를이해하는데중요함을주창하였다. 실제로패혈증환자에서 delayed hypersensitivity 가감소되어있고, 병원감염에취약하다는보고들이있어, 이들환자에서면역반응이억제되어있음을시사하고있다. 이를 343

2 SM Lee: Induction mechanism of PD-L1 바탕으로패혈증발생기전에대한개념이변화되었는데, 초기에는염증반응물질의과도한생성이일어나지만, 패혈증이진행할수록염증반응및면역반응이억제되는상황으로전이되어간다는것이다 4. 이로써, 패혈증에서면역반응이억제되는기전에대한관심이높아지고이에대한연구가진행되어지게되었다. 한편면역반응에중요한역할을담당하고있는 T lymphocyte 가증식하고분화하는데있어 APC (antigen presenting cell) 에서제공되는여러가지신호전달이중요하다고알려져있으며이러한신호전달에각종 costimulator 가중요한역할을담당하고있다. APC 표면에발현되는단백질중 PD-L1 (programmed death receptor ligand 1) 은 B7 family 에속하는표면단백질로서 B7-H1 이라고도불린다. PD-L1 은기존의 B7-1 이나 B7-2 등의다른 B7 family와는달리 T lymphocyte 표면의 CD28 이나 CTLA-4 등과는결합을하지않고, PD-1 (programmed death receptor 1) 이라는특정 receptor 와결합을한다고알려져있다. PD-L1 과 PD-1 이결합함으로써 T-cell receptor (TCR) 을통한신호전달체계를억제하여 T lymphocyte 의증식이나싸이토카인분비력을억제한다고보고되고있다 5. 이러한 PD-L1 은 T lymphocyte, B lymphocyte, macrophage 및 dendritic cell 표면에발현되며 LPS를포함한몇몇자극에의하여발현이유도된다. 또한심장의내피세포, 췌장의베타세포, 근육의 glial cell 등에서도발현됨이발견되어림프계기관뿐만아니라비림프계기관의면역조절기전에도관여함이시사되고있다 6-8. 최근 PD-L1 은 macrophage 와 B cell에서는 LPS 자극에의하여발현이증가한다는보고가있어, 패혈증모델에서 LPS 에의해 PD-L1 발현이증가된다면 PD-L1 과 PD-1의상호작용이패혈증에서나타나는면역반응억제의한기전일가능성을고려해볼수있다. 그렇지만, 이제까지의 PD-1/PD-L1 관련연구는주로자가면역질환이나종양분야에집중되어있는경향을보이고있고, 패혈증에서의면역억제반응과 PD-1/PD-L1 이관여하는지, 관여한다면어떤기전에의하여발현이조절되는지에대한연구는거의이루어지지않고있는현실이다. 본연구에서는 mouse macrophage cell line을이용하여 LPS 자극에의하여 PD-L1 발현이증가하는지를확인하고, 발현증가에어떤기전이관여하는지를알아보고자하였다. 대상및방법 1. 세포주본연구에는 mouse macrophage cell line 인 RAW 을사용하였다. RAW 세포주는 10% FBS (fetal bovine serum), 페니실린 30 mg/ml, 스트렙토마이신 50 mg/ml 이첨가된 RPMI-1640 배지를이용하여 37 o C, 5% CO 2 incubator 에서배양하였다. 2. Western 분석법 Whole lysis buffer (0.1% Nonidet P-40, 5 mm EDTA, 50 mm Tris (ph ), 250 mm NaCl, 50 mf) 를이용하여총세포단백을추출하였다. 30 g의세포단백을 10% SDS-polyacrylamide gel에서전기영동시켰다. 4시간동안 400 ma의일정한전류로단백질들을 nitrocellulose membrane으로 transfer 시키고, 이 membrane을 blocking solution (5% skim milk in 1 X PBS/Tween 20) 으로 1시간동안 block 시킨후 anti-pd-l1 항체, anti-tlr4 항체, 혹은 anti-actin 항체를 1:1,000 으로첨가하여 12시간동안반응시켰다. 세척후이차항체를 1:2,000으로첨가하여반응시킨후면역신호의검출은 ECL Western blotting detection system을이용하였다. 3. Flow cytometry 세포표면의 PD-L1 발현증가를확인하기위하여 Flow cytometry 기법을이용하였다. RAW 세포에 LPS를필요한시간동안처리한후 PE conjugated anti-mouse B7-H1 (CD274, B7H1, PD-L1) 를넣고 4 o C에서 30분간배양하였다. Cytometry analyzer는 Epics XL (Beckman Coulter, Fullerton, CA, USA) 를이용하였고, mean fluorescence intensity (MFI) 를측정하여비교하였다. 4. MTT assay 3-(4,5-dimethyl thiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay를이용하여세포생존율을측정하였다. 96 well plate에 well 당 개의세포를분주하고 4 5시간뒤약물을처리하여정해진시간동안배양하였다. 배양된세포에 5 mg/ml 의 MTT 용액 (Sigma, St. Louis, MO, USA) 을 15 μl 첨가하고, CO 2 incubator에 37 o C에서 5시간동안배양하였다. 배양액을제거한후 50 μl 의 DMSO를첨가하여녹이고분광광도계를이용하여 590 nm 파장에서흡광도를측정하였다. 각각 4개 well 의 344

3 Tuberculosis and Respiratory Diseases Vol. 65. No. 4, Oct 성적을분석하였다. 5. Chemical inhibitor 처리 각 signaling pathway 를 blocking 하기위해서 chemical inhibitor 를처리하였다. PI3K/Akt pathway 를억제하기위해서 LY μm 을, ERK/MEK pathway를억제하기위해서 U μl 를, 그리고 p38 MAPK pathway를억제하기위해서 SB μl 를전처치하였다. 각각의 chemical inhibitor 는 2시간동안처리되었다. 결 1. PD-L1 is up-regulated by LPS in time-dependent & concentration-dependent manner in mouse macrophage cell line RAW 세포주에서 LPS 자극에의하여 PD-L1 의발현이증가하는지평가하기위하여 LPS 1.0 μg/ml 의농도로 24시간동안전처치후 RAW 세포주표면의 PD-L1 발현정도를 flow cytometry 를통해분석하였다. Control 군에서도 PD-L1 발현이관찰되어 macrophage 에서의 constitutional expression 을반영하고있었고, LPS 자극후에 PD-L1 발현이증가함을알수있었다. Control 군에서 mean fluorescence intensity (MFI) 가 30.16이고, LPS 처리군에서 MFI가 로통계적으로유의하게 (p=0.001) LPS 처리한경우 PD-L1 발현이증가함을관찰할수있었다 (Figure 1). LPS 자극에의한 PD-L1 발현의증가는 LPS 처리후시간이증가함에따라 time-dependent manner 로나타남을알수있었고, LPS 처리농도를변화시켰을때도농도에비례하여 dose-dependent manner 로 PD-L1 발현의증가가관찰되었다 (Figure 2). 과 2. Blocking of TLR4 with sirna suppress LPS-induced PD-L1 expression LPS 자극에의한 PD-L1 발현의증가가 toll-like receptor 4 (TLR4) 를통해서일어나는지알아보기위하여 small interfering RNA (sirna) method를이용하여실험을진행하였다. RAW 세포주에 TLR4 에대한 sirna 를 transfection하였을때 control sirna를 transfection하였을때보다 TLR 발현이효과적으로감소하는것을 western blot을통하여확인하였다 (Figure 3A). Control sirna 를 transfection 한뒤 LPS 자극후의 MFI를 100% 로하였을때, TLR4 sirna를 transfection한후의 MFI가감소됨을 flow cytometry 를통하여확인할수있었다 (Figure 3B). 이를통해 LPS 자극에의한 PD-L1 발현의증가는 TLR4 를통하여이루어짐을추정할수있었다. 3. PI3K/Akt pathway is involved in up-regulation of PD-L1 by LPS 다음으로 TLR4의 downstream pathway 중하나인 PI3K/Akt pathway가 LPS 자극에의한 PD-L1 발현의증가에관여하는지알기위하여 PI3K/Akt pathway 에대한 chemical inhibitor인 LY294002를전처치한뒤실험을진행하였다. LY 전처치시 RAW 세포주에서 PD-L1 의 constitutional expression 에는영향을미치지않음을알수있었으나, LPS 자극후의 PD-L1 발현의증가는 LY 전처치시감소함을확인할수있었다 (Figure 4). MTT assay로 cell viability를평가하였을때 vehicle control 과차이가없어이러한효과는 cell death 에의한것은아님을알수있었다. 이러한결과를통해 LPS 자극후의 PD-L1 발현의증가에는 PI3K/Akt pathway 가관여 Figure 1. PD-L1 is up-regulated by LPS in mouse macrophage cell line. RAW cells were treated with LPS (1.0 μg/ml) for 24 hrs. Surface expressions of PD-L1 were measured by flow cytometry and expressed via MFI (mean fluorescence intensity). Number: mean fluorscence intensity (MFI). Gray: isotype control, Black: PD-L1. 345

4 SM Lee: Induction mechanism of PD-L1 Figure 2. PD-L1 is up-regulated by LPS in time-dependent & concentration-dependent manner. RAW cells were treated with LPS (1.0 μg/ml) for the indicated times (A) and with the indicated concentrations of LPS for 24 hrs (B). Surface expressions of PD-L1 were measured by flow cytometry and expressed via MFI. 함을추정할수있었다. 4. MEK/ERK pathway has no role in up-regulation of PD-L1 by LPS 다음으로 TLR4의 downstream pathway 중하나인 MEK/ERK pathway 가 LPS 자극에의한 PD-L1 발현의증가에관여하는지알기위하여 MEK/ERK pathway 에대한 chemical inhibitor인 U0126를전처치한뒤실험을진행하였다. U0126 전처치시 PD-L1의 constitutional expression 에는차이가없었으나, PI3K/Akt pathway 의경우와는다르게 LPS 자극후오히려 MFI가증가해 PD-L1 발현이다소늘어남을확인할수있었다 (Figure 5). 이는반복실험을통해서도확인할수있어 MEK/ERK pathway 는 LPS 자극후의 PD-L1 발현증가에영향을미치지않음을알수있었다. 5. p38 MAPK pathway is involved in up-regulation of PD-L1 by LPS 마지막으로 TLR4의 downstream pathway 중하나인 p38 MAPK pathway 가 LPS 자극에의한 PD-L1 발현의증가에관여하는지알기위하여 p38 MAPK pathway 에대한 chemical inhibitor인 SB202190를전처치한뒤 LPS 자극후의 PD-L1 발현을평가하였다. 앞에실험에서의같이 SB 전처치는 PD-L1의 constitutional expression 에는영향을미치지않았으나, LPS 자극후의 PD-L1 증가정도는 MFI로평가하였을때줄어듦을알수있었다 (Figure 6). 이를통해 p38 MAPK pathway 는 LPS 자극후의 PD-L1 발현증가에관여함을확인할수있었다. 고 찰 PD-L1 은 inhibitory function 을가지는 costimulatory 346

Tuberculosis and Respiratory Diseases Vol. 65. No. 4, Oct. 2008 Figure 3. Blocking of TLR4 with sirna suppress LPS-induced PD-L1 expression. RAW 264.

0 μg/ml) for 24 hrs, surface expressions of PD-L1 were measured by flow cytometry and expressed via MFI (B). molecule 의하나로알려져있다 9.

5 Tuberculosis and Respiratory Diseases Vol. 65. No. 4, Oct Figure 3. Blocking of TLR4 with sirna suppress LPS-induced PD-L1 expression. RAW cells were transfected with control sirna and sirna for TLR4. The levels of TLR4 in cellular extracts were detected by Western blot analysis (A). After cells were treated with LPS (1.0 μg/ml) for 24 hrs, surface expressions of PD-L1 were measured by flow cytometry and expressed via MFI (B). molecule 의하나로알려져있다 9. Naive T cell은 MHC와 TCR의작용과함께, costimulatory molecule 의작용에의하여 activated T cell 이된다. 이때 chronic infection이나 persistent antigen stimulation 과같은상황에서, APC에서발현되는 PD-L1은 T세포에있는 inhibitory costimulatory molecule인 PD-1과상호작용하여 inhibitory signal을만들고, T cell은 exhausted T cell로변하게되어 immune tolerance 를일으키게된다. PD-1 이 T lymphocyte 에대한 negative stimulatory function을갖는다는사실은 PD-1 Figure 4. PI3K/Akt pathway is involved in up-regulation of PD-L1 by LPS. RAW cells were pretreated with LY (50 μm) for 2 hrs and then stimulated with LPS for 24 hrs. Surface expressions of PD-L1 were measured by flow cytometry and expressed via MFI. Cell viability by MTT assay:>90% of vehicle control. knockout mice에서자연적으로자가면역질환이발생한다는실험결과에서밝혀지기시작했다 10,11. 이러한 PD-1- PD-L1 interaction은 HBV, HCV, HIV, H. pylori와같은만성감염과종양면역에서 immune tolerance 를일으키는기전의하나로잘알려져있다 9. 한편 PD-1의 ligand 로알려진 PD-L1 에관해서는몇몇질병 model에서 immune tolerance 기전에관여함이알려지고있다. 우선 NOD (non-obese diabetes) mice의췌장베타세포에서 PD-L1 발현이증가되어있으며, PD-1 knockout NOD mice 에서제1형당뇨병이더잘발생한다는사실이보고되었다 12. 또한, 대표적인 immune tolerance 현상인 347

6 SM Lee: Induction mechanism of PD-L1 Figure 5. MEK/ERK pathway has no role in up-regulation of PD-L1 by LPS. RAW cells were pretreated with U0126 (10 μm) for 2 hrs and then stimulated with LPS for 24 hrs. Surface expressions of PD-L1 were measured by flow cytometry and expressed via MFI. Cell viability by MTT assay:>90% of vehicle control. Figure 6. p38 MAPK pathway is involved in up-regulation of PD-L1 by LPS. RAW cells were pretreated with SB (10 μm) for 2 hrs and then stimulated with LPS for 24 hrs. Surface expressions of PD-L1 were measured by flow cytometry and expressed via MFI. Cell viability by MTT assay:>90% of vehicle control. 임신과관련된연구결과도발표되었다. Guleria 등 13 은 murine abortion model을이용하여태반세포에 PD-L1 발현이증가되어있으며, PD-L1 을억제하는 blocking Ab 를처리하였을때유산 (abortion) 이늘어남을밝혀임신에서의 immune tolerance 에도 PD-1/PD-L1 이관여함을보여주고있다. 이외에 PD-L1 에대한연구는악성종양분야에서도진행되고있는데, Thompson 등 14 은신장암환자를대상으로한연구를통해 tumor cell 및 lymphocyte 에서 PD-L1 발현이증가되어있는환자가더빨리사망에이른다는사실을밝혔다. 또한, 식도암환자에서도 PD-L1 발현이증가된환자의예후가더나쁘다는보고도있어 tumor cell 이면역체계를피해서증식하는기전에도 PD-L1 발현이중요함을시사하고있다 15. PD-L1은 Activated macrophages 및 B lymphocytes, dendritic cells, activated T cells 등여러 APC에서발현되며, 특히 IFN-γ stimulation 은 endothelial cell이나 tumor cell 같은 non-lymphoid tissue에서도 PD-L1의발현을증가시키는것이잘알려져있다. LPS stimulation 에의해서는 non-lymphoid tissue에서발현이증가된경우는없었고 dendritic cell에서도 LPS자극이 PD-L1의발현을증가시키지않는다. 그러나 macrophage 에서는 LPS에의하여발현이증가한다는몇몇보고가알려져있다. Yamazaki 등 16 은 PD-L1이 macrophage 와 B cell에서 348

7 Tuberculosis and Respiratory Diseases Vol. 65. No. 4, Oct LPS 자극에의하여발현이증가한다는발표를하였으며, 이후 mouse peritoneal macrophage 에서 LPS 자극에의해 PD-L1 의발현이증가하는것이 TLR4의 downstream effect 라는것을주장한실험결과가보고되기도하였다 17. 따라서 PD-L1 과 PD-1의상호작용이패혈증에서나타나는 immune suppression 의한기전일가능성을유추해볼수있다. 그러나실제 macrophage 에서 LPS 자극시에 PD-L1 의발현이증가하는것에 TLR4 의어떤 downstream pathway 에의한것인지는알려져있지않아본연구를진행하였다. 패혈증에대한개념이바뀌어, 면역억제반응이패혈증후기에는더중요하다는사실이밝혀지게되었고, PD-L1 이이런면역억제반응에관여할수있음을보여주고있어, PD-L1 이패혈증이지속되는현상을이해하는데중요한열쇠가될수있음을시사하고있다. 패혈증에서 LPS 로시작된염증신호전달체계는 TLR4 를거쳐서이하 downstream pathway 에전달이된다. 이러한 downstream pathway 에는 PI3K/Akt, ERK, p38 MAPK 등여러신호경로가관여함이알려져있고 18, 이러한신호경로사이에는서로 crosstalk 이일어난다는보고도있는실정이다. 이에본연구에서는 PI3K/Akt, ERK 및 p38 MAPK 경로가 LPS 자극에의한 PD-L1 증가에관여하는지에초점을맞추어연구를시행하였다. PI3K/Akt 경로가 PD-L1 증가에직접적으로관여하는지에대한연구결과는많지않은실정이다. Dermal fibroblast cell을이용하여 IFN-gamma 를처리한경우 PD-L1 발현의증가를관찰하였다는보고가있었고 19, 이때 PI3K 의일시적인인산화가관찰되어서이러한 PI3K/Akt pathway 의활성화가 PD-L1 발현증가에관여할가능성을시사하였다. 그렇지만, 실제 PI3K pathway 를 block하는방법등을이용하여직접적으로 PI3K/Akt pathway 가관여하는지를입증하지는않았다. 본연구에서는 PI3K/Akt pathway 의 chemical inhibitor 를전처치하였을때 LPS에의한 PD-L1 발현증가가억제됨을보여, PI3K/Akt pathway가직접적으로관여함을보여주었다. 한편기존에 ERK 경로와 PD-L1 과의연관성에대한연구들은주로 PD-L1 을억제하였을때나타나는현상중의하나로서 ERK pathway 에변화가나타난다는데초점이맞추어져있었고 20,21, 반대로 ERK pathway 에의해 PD-L1 발현이어떤영향을받는지에대한연구결과는거의없는실정이다. 본연구에서는 ERK pathway 에대한 chemical inhibitor 인 U0126을처리한뒤 PD-L1 expression 을평가 하여, ERK pathway 는 LPS에의한 PD-L1 발현증가에직접적인영향을미치지는않음을보여주었다. 그렇지만, p38 MAPK에대해서는 chemical inhibitor 인 SB 전처치에의하여 LPS에의한 PD-L1 발현증가가억제됨을보여, PI3K/Akt pathway 와마찬가지로 p38 MAPK pathway가직접적으로관여할수있음을밝혔다. 결론적으로 mouse macrophage cell line을이용하여 LPS 자극시세포표면의 PD-L1 발현증가가일어나고, 이러한기전에는 TLR4 가관여하며 downstream pathway 중 PI3K/Akt 경로및 p38 MAPK 경로가관여함을확인할수있었다. 참고문헌 1. Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR. Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med 2001;29: Bone RC, Fisher CJ Jr, Clemmer TP, Slotman GJ, Metz CA, Balk RA. A controlled clinical trial of high-dose methylprednisolone in the treatment of severe sepsis and septic shock. N Engl J Med 1987;317: Bone RC. Sir Isaac Newton, sepsis, SIRS, and CARS. Crit Care Med 1996;24: Hotchkiss RS, Karl IE. The pathophysiology and treatment of sepsis. N Engl J Med 2003;348: Okazaki T, Honjo T. The PD-1-PD-L pathway in immunological tolerance. Trends Immunol 2006;27: Iwai Y, Terawaki S, Ikegawa M, Okazaki T, Honjo T. PD-1 inhibits antiviral immunity at the effector phase in the liver. J Exp Med 2003;198: Liang SC, Latchman YE, Buhlmann JE, Tomczak MF, Horwitz BH, Freeman GJ, et al. Regulation of PD-1, PD-L1, and PD-L2 expression during normal and autoimmune responses. Eur J Immunol 2003;33: Wiendl H, Mitsdoerffer M, Schneider D, Chen L, Lochmuller H, Melms A, et al. Human muscle cells express a B7-related molecule, B7-H1, with strong negative immune regulatory potential: a novel mechanism of counterbalancing the immune attack in idiopathic inflammatory myopathies. FASEB J 2003;17: Sharpe AH, Wherry EJ, Ahmed R, Freeman GJ. The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection. Nat Immunol 2007;8: Nishimura H, Minato N, Nakano T, Honjo T. Immunological studies on PD-1 deficient mice: implication of 349

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Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee University College of Medicine & Hospital E mail : ycell2@yahoo.co.kr Abstract