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1 J Korean Surg Soc 2009;77: DOI: /jkss 원 저 FK506 이 T 림프구사멸에서활성산소생성에미치는영향 전남대학교의과대학외과학교실 이호균ㆍ정상영ㆍ최수진나 The Effect of FK506 to Generate Reactive Oxygen Species on T Lymphocyte Death Ho Kyun Lee, M.D., Sang Young Chung, M.D., Soo Jin Na Choi, M.D. Department of Surgery, Chonnam National University Medical School, Gwangju, Korea Purpose: Tacrolimus (FK506) has been widely used as an immunosuppressant in organ transplanted recipients to suppress organ rejection phenomenon. We investigated the role of oxidative stress and heme oxygense-1 by FK506 on human Jurkat T cells. Methods: The cells viability was examined by DAPI stain, enzyme activity of caspase family proteins, and western blotting for Baks, PUMA, inos, HO-1. Cells were cultured in the absence or presence of CoPPIX or ZnPPIX and the fluorescence intensity was analyzed using a flow cytometry. Results: Treatment with FK506 increased the generation of reactive oxygen species (ROS), including hydrogen peroxide and superoxide anion, and NO in Jurkat cells in a dose-dependent manner. Immunohistochemistry and Western blot analysis data revealed the hemoxygenase-1 (HO-1) was induced by the addition of FK506 in Jurkat cells. Induction of CoPP, HO-1 inducer, resulted in decreased intracellular H 2 O 2 and NO concentrations. Instead ZnPP, an HO-1 competitive inhibitor did it reversely. In addition, ZnPP regulates inos protein synthesis by inhibition of HO-1. Conclusion: Increase of HO-1 expression would induce to decrease the intracellular H 2 O 2 and NO concentrations. Also, HO-1 would regulate inos protein synthesis. Consequently, we can expect the regulation of HO-1 expression with concomitants use of FK506 to suppress organ rejection phenomenon by enhancing apoptosis. (J Korean Surg Soc 2009;77: ) Key Words: FK506, Reactive oxygen species, Heme Oxygenase-1 중심단어 : FK506, 활성산소종, 헴옥시게나아제 -1 서 FK506은토양진균류인 Streptomyces tsukubaensis에서얻어지는 macrolide계항생물질로서 1990년대간이식에최초 책임저자 : 최수진나, 광주시동구학 1 동 8 번지 , 전남대학교병원외과 Tel: , Fax: choisjn@chonnam.ac.kr 접수일 :2009 년 5 월 6 일, 게재승인일 :2009 년 8 월 21 일이연구는 2008 년도전남대학교병원임상의학연구소연구비의지원으로이루어졌음. 론 임상을실시하였다.(1) 특히, FK506의효과는간이식환자에게있어서사이클로스포린을능가하여거의유일한약제로자리매김되어근래는간이식환자과반수가 FK506을복용하고있다. 세포내활성산소종 (reactive oxygen species, ROS) 은활성화된호중구, xanthine oxidase, 미토콘드리아호흡및아라키돈산대사과정을통해서생성된다.(2,3) 세포는환경에서오는다양한스트레스로부터항상성을유지하기위하여즉각적으로반응한다. 대표적인예인헴옥시게나제 (heme oxygenase, HO) 는헴을빌리베르딘, 일산화탄소및유리철로산화분해하여세포내헴을조절하는데중요한역할을 310

2 Ho Kyun Lee, et al:the Effect of FK506 to Generate Reactive Oxygen Species on T Lymphocyte Death 311 수행하는효소이고, 이후빌리베르딘은다시세포질내빌리베르딘환원효소에의해항산화제인빌리루빈으로전환된다.(4,5) 세포사멸의과정은크게 2가지로대별되어왔는데, 즉사망수용체 (death receptor, Fas/FasL, TNF-a 등 ) 를통한수용체매개성 ( 외인성 ) 세포사멸경로와항암화학제, 활성산소종 (reactive oxygen species, ROS) 등다양한인자들에의해유발되는미토콘드리아매개성 ( 내인성 ) 세포사멸경로가있다.(6,7) 본연구에서는사람의 T 세포실험대상으로잘알려져있는 Jurkat 세포를이용하여 FK506에의한세포사멸에서세포내활성산소종및 HO-1의역할에대해연구조사하여유의한결과를얻었기에보고하는바이다. 방법 1) 재료 (1) 세포주 : 사람 T 림프구세포주인 Jurkat 세포는한국세포주은행 (KNCC, 서울대학교 ) 으로부터구입, 계대배양하면서실험을실시하였다. (2) 시약및기기 : 실험에필요한 RPMI 1640, 항생제및우태아혈청 (fetal bovine serum, FBS) 은 GIBCO BRL사 (Grand Island, NY, USA) 제품을, 배양용기 (24-well plate와 10 cm dish) 는 Falcon사 (Becton Dickinson, San Jose, CA, USA) 에서구입하여사용하였다. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra zolium bromide (MTT), DAPI, bicinchoninic acid (BCA), dimethyl sulfoxide (DMSO) 는 Sigma사 (St. Louis, Missouri, USA) 에서구입하였다. 2) 방법 (1) Jurkat 세포주배양및시약처리 : Jurkat 세포는 CO 2 세포배양기 (37 o C, 5% CO 2) 에서 10% 우태아혈청이포함된 RPMI 1640 배지로배양하였으며, 24시간간격으로배양액을교체하여 log phase에있는세포에 FK506을처리한후세포고사현상과이에연관된생화학실험을수행하였다. FK506은 10μM 농도로 DMSO에녹여 70 o C에보관하였고, RPMI 1640 배지에희석하여사용하였다. (2) DAPI에의한세포핵염색 : FK506으로처리한세포는포집하여냉 PBS로세척한후 cytospin (Shandon Southern Products Ltd., England) 으로 600 rpm, 3분간회전분사하여슬라이드글라스에부착시켰다. 슬라이드글라스에부착된세 포는 4% formaldehyde 용액으로고정하고 PBS로세척하여, 10μM DAPI로 20분간실온에서반응한후다시 PBS로세척하여형광현미경 (Leica MPS 60, Germany) 으로핵형태를관찰하였다. (3) Western blot analysis: FK506을처리한 Jurkat 세포는포집하여, 냉 PBS로 2회세척하였다. 얻어진세포는파쇄용액 (50 mm HEPES, ph 7.4, 150 mm NaCl, 1% deoxycholate, 1 mm EDTA, 1 mm PMSF, 1μg/ml aprotinin) 과 4 o C에서 30 분반응하였다. 동량의세포파쇄액 (200μg) 과 2X sample buffer를혼합하여 100 o C에서 3분가열하여단백질의변성을유도한후에 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 를시행하였다. 전기영동이끝난 gel의단백질은 electrotransfer system (Ellard Inc, Seattle, WA, USA) 을이용 (0.8 ma/cm) 하여 nitrocellulose membrane으로이동시키고, blocking buffer (5% skim milk) 와상온에서 2시간반응하였다. PERK, phospho- PERK, CHOP, Grp78, Grp94/BiP, Bcl-2, Bak 및 β-actin에대한항체는 0.05% (v/v) 의 tween-20이함유된 tris-buffered saline (TBS-T) 에 1:1,000으로희석하여 nitrocellulose membrane과상온에서 2시간반응하였으며, 각항체에대한이차항체 anti-rabbit IgG conjugated horse radish peroxidase (HRP) 와 anti-mouse IgG conjugated HRP는 TBS-T 로희석 (1:3,000) 하여상온에서 1시간반응한후, enhanced chemilluminescence (ECL) kit (Amersham, England) 를이용하여현상하였다. (4) Caspase계 cystein protease 활성도측정 : FK506처리후포집된 Jurkat 세포 ( ) 를 4 o C에서 15분 lysis buffer (1% TritonX-100, 0.32 M sucrose, 10 mm Tris/HCl, ph 8.0, 5 mm EDTA, 2 mm dithiothreitol [DTT], 1 mm PMSF, 1μg/ ml aprotinin, 1μg/ml leupeptin) 로용해하고 13,000 rpm으로 15분원심분리하였다. 원심분리하여얻은세포파쇄액은 bicinchoninic acid (BCA, Sigma Co. MO, USA) 법으로단백질을정량하고, 세포파쇄액을분석완충용액 (100 mm HEPES, ph 7.5, 10% sucrose, 0.1% Chaps, 2 mm DTT, 1 mm PMSF, 1μg/ml aprotinin, 1μg/ml leupeptin) 에희석된형광기질과 37 o C에서 30분반응한후 Fluorometer (Molecular Devices Co, USA) 로측정하였다. Caspase-3 protease의효소활성의측정은형광기질인 Ac-DEVD-7-amino-4-methylco-umarin (AMC) (Calbiochem Co. CA, USA) 50μM을이용하였으며, 이형광기질의 proteolytic cleavage를측정하여 caspase 활성을결정하였다. 이때의파장은 excitation wavelength (380 nm) 와 emission wavelength (460 nm) 를사용하였다. Caspase-8 pro-

Caspase-9 protease 효소적활성은 Ac-LEHD-AFC (Calbiochem) 50μM을기질로사용하여이들의 proteolytic cleavage를 400 nm (excitation wavelength) 와 505 nm (emission wavelength) 의파장하에서측정하였다.

3 312 J Korean Surg Soc. Vol. 77, No. 5 tease 효소적활성은 Ac-IETD-7-amino-4-trifluoromethylcoumarin (AFC) (Calbiochem Co. Germany) 50μM를기질로사용하였으며이들의 proteolytic cleavage를 400 nm (excitation wavelength) 와 505 nm (emission wavelength) 의파장하에서측정하였다. Caspase-9 protease 효소적활성은 Ac-LEHD-AFC (Calbiochem) 50μM을기질로사용하여이들의 proteolytic cleavage를 400 nm (excitation wavelength) 와 505 nm (emission wavelength) 의파장하에서측정하였다. (5) 세포내 ROS 생성의측정 : FK506에의한세포내활성산소종의생성을측정하기위하여형광 probe 2, 7 -dichlorofluorescein diacetate (DCF-DA; Sigma) 를이용하였다. 비형광물질인 DCF-DA는세포내 hydrogen peroxide와관련된 peroxides 존재시형광의 DCF로변환되어녹색의형광을발한다. Jurkat 세포에 FK506을처리한후세포를수확하기전에 5μM DCF-DA를처리하여 37 o C에서 30분배양하였다. 배양한세포는 PBS (ph 7.4) 로세척하여 flow cytometry (FACSCalibur, BD Biosciences) 로형광을측정하고 CellQuest software (Becton Dickinson) 를이용하여분석하였다. (6) 세포내 NO 생성의측정 : FK506에의한세포내산화질소 (Nitric Oxide, NO) 의생성을측정하기위하여형광 probe 2', 7 -diaminofluorescein diacetate (DAF-DA; Sigma) 를이용하였다. 비형광물질인 DAF-DA는세포내산화질소존재시형광의 DAF로변환되어녹색의형광을발한다. Jurkat 세포에 FK506을처리한후세포를수확하기전에 5μM DAF-DA를처리하여 37 o C에서 30분배양하였다. 배양한세포는 PBS (ph 7.4) 로세척하여 Flow cytometry (FACSCalibur, BD Biosciences) 로형광을측정하고 CellQuest software (Becton Dickinson) 를이용하여분석하였다. (7) 미토콘드리아막전위측정 : 미토콘드리아막전위의변화를조사하기위하여 JC-1 염색을시행하였다. FK506을처리한세포는포집하여냉 PBS로세척한후 cytospin (Shandon Southern Products Ltd., England) 으로 600 rpm, 3분간회전분사하여슬라이드글라스에부착시켰다. 슬라이드글라스에부착된세포는 PBS로세척하여, 10μg/ml JC-1과 20분간실온에서반응한후다시 PBS로세척하여형광현미경 (Leica MPS 60, Germany) 으로형광의변화를관찰하였다. (8) 통계처리 : 표시된결과는 3번이상의독립적인실험결과이며, 실험결과의통계처리는 student's t-test에준하여처리하였고, P-value가최대치 0.05 (P<0.05) 이하인경우를유의한것으로판정하였다. 결과 1) FK506이 T 림프구, Jurkat 세포의세포사멸에미치는영향 세포사멸의특징적인현상을초래하는세포내신호전달기전에서 caspase의중요성이이미잘알려져있다. FK506 에의한 Jurkat 세포의고사현상이 caspase 활성화와관계가있는지를확인하기위하여 caspase-3 proteases의효소적활성을이효소의형광기질 (fluorogenic substrate) 을이용하여 12시간간격으로측정하였다. 그결과 Caspase-6, -8, 및 -9 protease의활성은대조군에비교하여유의한변화가없었으나 ( 미발표성적 ), caspase-3 protease는시간의존적으로증가하여 FK506 처리 72시간후대조군에비하여최대 4.2배의증가된활성을보였다 (Fig. 1A). 또한, FK506에의한세포사멸에서형태학적특징을관찰하기위하여핵산의 Fig. 1. FK506 induced cytotocixity and nuclear fragment on Jurkat cells. (A) Cells were treated with 10μM FK506 for 12 to 72 hr and lysed to measure the activity of caspase proteases by using fluorogenic biosubstrates. Data represent the mean±standard deviation (S.D.) of quadruplicates. (B) Cells were treated with FK506 (10μM) for 72 hr. Then, cells were stained with DAPI and observed under fluorescence microscopy.

4 Ho Kyun Lee, et al:the Effect of FK506 to Generate Reactive Oxygen Species on T Lymphocyte Death 313 DAPI염색을수행하였다. Jurkat 세포에 FK506 (10μM) 을 72시간처리한후 DAPI 염색을시행한결과, 정상대조군의핵은타원형의온전한형태의균질한형광염색을나타냈으나, FK506 72시간처리군의핵은여러조각으로분절된형광절편이증가되어관찰되었다 (Fig. 1B). 2) FK506에의한 Jurkat 세포사멸에서세포내활성산소 H 2 O 2 의생성에미치는영향 FK506에의한세포내활성산소인 H 2O 2 생성변화를관찰하기위하여 DCF-DA로염색하고 flow cytometry로분석하였다. DCF-DA는세포내 H 2O 2 와반응시형광을띠는 DCF 로전환된다. 그결과, FK506 농도의존적인증가를보였다 (Fig. 2). 이상의결과는 FK506에의한세포내활성산소인 H 2O 2 생성이세포사멸을유도할가능성을시사하였다. 3) FK506이 Jurkat 세포사멸에서 NO의생성및 inos 단백질의발현에미치는영향 FK506에의한세포내활성산소종의생성변화를확인하 기위하여 DAF-DA 염색을통해세포내 NO의생성변화를 flow cytometry로분석하였다. 그결과 FK506 처리농도에의존적으로 NO의양이증가하였다 (Fig. 3A). 또한, 세포내 NO 합성효소인 NOS의발현변화를 Western blot 방법으로조사한결과 inos 단백질의발현이 FK506 처리 12시간부터증가하여 24시간에최대의발현량을보였으며 48시간이후부터점차감소하는양상을보였다 (Fig. 3B). 따라서 FK506 처리에의한 Jurkat 세포의세포사멸은 inos 단백질의발현증가에따른 NO의생성증가에의해초래됨을시사하였다. 4) FK506에의한 Jurkat 세포사멸에서미토콘드리아막전위변화와 PUMA 단백질발현에미치는영향 FK506에의한 Jurkat 세포의미토콘드리아의막전위차변화를확인하기위하여 JC-1 형광염색을시행하였다. 10 μm의 FK506을 Jurkat 세포주에 36시간처리후 JC-1으로염색하여형광현미경으로관찰하였다. 그결과 Jurkat 세포는 FK506을처리하지않은대조군점상형 (punctuated-pat- Fig. 2. Production of H 2O 2 in FK506 treated Jurkat cells. Cells were treated with indicated dose of FK506 for 72 hrs. Then, cells were incubated with the dye 2', 7'-dichlorofluorescin diacetate (5μM) and the fluorescence intensity of more than 10,000 cells was analyzed using a flow cytometry.

5 314 J Korean Surg Soc. Vol. 77, No. 5 Fig. 3. Effects of NO production and inos protein expression in Jurkat cells. (A) Cells were treated with indicated dose of FK506 for 72 hrs. Then, cells were incubated with the dye DAF-DA (5 μm) and the fluorescence intensity of more than 10,000 cells was analyzed using a flow cytometry. (B) Histogram status of A Cells were treated with 10μM FK506 for various periods. The equal amounts of protein from cell lysate were subjected on 10% SDS-PAGE, transferred onto nitrocellulose membrane and immunoblotted with anti-inos and anti-β-actin antibodies. tern) 의오렌지형광 (Fig. 4A, a) 이녹색형광으로시간의존적으로변화하면서미만형 (diffused-pattern) 으로세포질전체에균질하게분포하였다 (Fig. 4). 또한이때 Bcl-2 family 단백질중 Bak과 PUMA 단백질의발현변화를조사하기위하여 Jurkat 세포에 10μM의 FK506을처리한후 Western blot을실시하였다. 그결과, 6시간이후부터 pro-apoptotic 기능을갖는 Bak 단백질의발현을증가를보이다점차감소하는양상을보였고 72시간이후대조군수준으로감소하였고, anti-apoptotic 기능을갖는 PUMA 단백질은 FK506 처리시간에의존적으로감소되는발현양상을보였다 (Fig. 4B). 이때단백질양은 β-actin을통하여동일한양임을확인하였다. 이상의결과는 FK506 처리가 Bak의발현증가와 PUMA의발현감소에따른 Jurkat 세포의미토콘드리아막 전위차감소를초래하였음을시사하였다. 5) FK506이 Jurkat 세포사멸에서 CoPP 및 ZnPP가 H 2O 2 의생성및 HO-1 단백질의발현에미치는영향 FK506에의한세포내활성산소종생성에대한 HO-1의역할을조사하기위하여 HO-1 유도제인 CoPP와 HO-1 효소활성저해제인 ZnPP를각각 30분전처리한후 10μM FK506을처리 48시간후에 H 2O 2 의생성변화및 HO-1 단백질의발현을조사하였다. 먼저세포내 H 2O 2 의생성변화는 DCF-DA 염색을통해 flow cytometry로분석하였다. 그결과 10μM 농도의 FK506 처리 48시간후에대조군에비하여증가하였던 H 2O 2 의양이 CoPP 전처리에의해현저히감소하였으며, 이때 CoPP 자체영향은없었다. 그러나 ZnPP 전

FK506 treated cells were stained with 10μg/ml of JC-1 visualized under a fluorescent microscope. (a) Control cells, and (b) FK506 treated cells for 36 hrs.

6 Ho Kyun Lee, et al:the Effect of FK506 to Generate Reactive Oxygen Species on T Lymphocyte Death 315 Fig. 4. Change of mitochondrial membrane potential transition and differential expression of Bak and PUMA in FK506 treated Jurkat cells. (A) Cells were treated with 10μM FK506 for 36 hr. FK506 treated cells were stained with 10μg/ml of JC-1 visualized under a fluorescent microscope. (a) Control cells, and (b) FK506 treated cells for 36 hrs. (B) Cells were treated with 10μM FK506 for various periods. The equal amounts of protein from cell lysate were subjected on 15% SDS-PAGE, transferred onto nitrocellulose membrane and immunoblotted with PUMA, anti-bak and anti-β-actin antibodies. 처리시 FK506에의해생성증가된 H 2O 2 의양은감소하지않았으며특히 ZnPP 단독처리군에서증가된양상을보였다 (Fig. 5A, B). 이때 CoPP 처리에의하여 Jurkat 세포의 HO-1 단백질의발현은증가되었으며, ZnPP 처리시 HO-1의발현은감소되는양상을보였다 (Fig. 5C). 이상의결과는 HO-1의 inducer 및 inhibitor 처리를통해 HO-1의발현에따른세포내 H 2O 2 생성의연관관계를확인하였다. 6) FK506이 Jurkat 세포사멸에서 CoPP 및 ZnPP가 NO 의생성및 inos 단백질의발현에미치는영향 FK506에의한세포내 NO 생성에대한 HO-1의역할을조사하기위하여 HO-1 유도제인 CoPP와 HO-1 효소활성저해제인 ZnPP를각각 30분전처리한후 10μM FK506을처리 48시간후에 NO의생성변화및 inos 단백질의발현을조사하였다. 먼저세포내 NO의생성변화는 DAF-DA 염색을통해 flow cytometry로분석하였다. 그결과 10μM 농도의 FK506 처리 48시간후에대조군에비하여증가하였던 NO의양이 CoPP 전처리에의해현저히감소하였으며, 이때 CoPP 자체영향은없었다. 그러나 ZnPP 전처리시 FK506 에의해생성증가된 NO의양은증가하는양상을보였으며특히 ZnPP 단독처리군에서현저히증가하였다 (Fig. 6A, B). 이때 CoPP 처리에의하여 Jurkat 세포의 inos 단백질의발현은 FK506 단독처리군에비하여감소되었으며, ZnPP 처리시 inos의발현은뚜렷한증가를보였다 (Fig. 6C). 이상의결과는 HO-1의 inducer 및 inhibitor 처리를통해 HO-1의발현에따라세포내 NO 생성및 inos의발현의조절의연관관계를확인하였다. 고찰 FK506은실험적장기이식술에서강력한면역억제제로작용하며독성이훨씬적고스테로이드사용을줄인다. 특히 1980년대부터임상에사용된사이클로스포린 A (cyclosporine A) 는각종장기이식의성공률을향상시켜이식분야발전에지대한공헌을한것으로평가받고있는데, 1990 년대에새로개발된강력한면역억제제인 FK506는급성거부반응의예방뿐만아니라난치성거부반응의구조요법으로효과적이며, 각종장기이식에사용되어사이클로스포린 A을대신할수있는우수한면역억제제로보고되었다.(7) 그러나, 사이클로스포린 A에비교하여 FK506에의한 T 림프구의증식억제및세포사멸에대한신호전달과정및그작용기전에대한연구가미흡한실정이다. 따라서본연구에서는 FK506에의한사람 T 림프구 Jurkat 세포사멸에서활성산소에의한스트레스와관련된신호전달기전을조사하였다. 그결과 FK506은농도의존적으로 Jurkat 세포의생존율의감소를초래하였으며, 세포사멸의형태학적특징인핵의분절현상과 caspase-3 protease의활성을유도하였다. FK506은세포내활성산소종인 H 2O 2 와 NO의생성을증가시켰으며, 이는세포내산화질소합성효소인 inos의발현증가에기인하였다. 또한 FK506은사람 T 림프구세포에서 Bak 단백질의발현증가, PUMA 단백질의감소등미토콘드리아기능이상을통한세포사멸의신호전달기전을활성화시켰다. 활성산소종 (reactive oxygen species, ROS) 은활성화된호중구, xanthine oxidase, 미토콘드리아호흡및아라키돈산

7 316 J Korean Surg Soc. Vol. 77, No. 5 Fig. 5. Effects of H 2O 2 production and HO-1 protein expression in Jurkat cells by CoPP or ZnPP. (A) Cells were cultured in the absence or presence of CoPP or ZnPP for 48 hrs. Cells were treated with 10μM FK506 for various periods. Then, cells were incubated with the dye DCF-DA (5μM) and the fluorescence intensity of more than 10,000 cells was analyzed using a flow cytometry. (B) Histogram status of A. (C) The equal amounts of protein from cell lysate were subjected on 10% SDS-PAGE, transferred onto nitrocellulose membrane and immunoblotted with anti-ho-1 and anti-β-actin antibodies. The immunoreactive signals were visualized by ECL detection kit.

8 Ho Kyun Lee, et al:the Effect of FK506 to Generate Reactive Oxygen Species on T Lymphocyte Death 317 Fig. 6. Effects of NO production and inos protein expression in Jurkat cells by CoPP or ZnPP. (A) Cells were cultured in the absence or presence of CoPP or ZnPP for 48 hrs. Cells were treated with 10μM FK506 for various periods. Then, cells were incubated with the dye DAF-DA (5μM) and the fluorescence intensity of more than 10,000 cells was analyzed using a flow cytometry. (B) Histogram status of A. (C) The equal amounts of protein from cell lysate were subjected on 10% SDS-PAGE, transferred onto nitrocellulose membrane and immunoblotted with anti-inos and anti-β-actin antibodies. The immunoreactive signals were visualized by ECL detection kit.

9 318 J Korean Surg Soc. Vol. 77, No. 5 대사과정을통해서생성된다.(8) 비록 superoxide dismutase, catalase, glutathione peroxidase 효소등이세포내에서생성된활성산소종을제거하기위한생리적방어기전으로작용하지만, 과도한활성산소종은이들방어체계를초과하여세포손상을초래한다.(9) FK506 처리시세포내유의한 H 2O 2 생성증가가관찰되었다. 이러한세포내활성산소는미토콘드리아기능이상에의해초래되거나, 반대로미토콘드리아기능이상을초래한다고알려져있다.(9) 본연구결과에서는 FK506에의한미토콘드리아의막전위차변화를 JC-1 형광염색으로확인하였으며, Bak 단백질의발현증가와 PUMA 단백질의발현감소를통해다시한번확인하였다. Heme Oxygenase는 1968년 Tenhunen 등 (10) 에의해처음발견되었으며지금까지 HO-1, HO-2, HO-3 세가지종류의아형이알려져있다. 이중 HO-1은세포가다양한종류의산화적스트레스에노출되면유도되어헴을빌리베르딘일산화탄소유리철로분해하는효소이다. Stocker 등 (11) 은과거에는단순한생물학적대사과정의노폐물로만여겼던빌리루빈과빌리베르딘이강력한항산화작용이있다는것을규명하였고 Verma 등 (12) 도헴분해후생성되는일산화탄소가혈관확장이나신경전달등의생리적기능을중재하는역할에관여함을보고함으로써 HO-1의헴분해가불필요한헴을제거하는과정일뿐아니라빌리루빈이나일산화탄소등생체에이로운기능을할수있는물질을새로생성해내는동화과정임을밝혔다. 저산소증산화질소등다양한물질에의해유도될수있는데 Applegate 등은활성산소종 (reactive oxygen species, ROS) 에대한반응, 즉세포내환원제의양이감소하거나혹은활성산소종이증가하는상태에서는원인에상관없이공통적인과정으로서이유도될수있고이러한결과를근거로 HO-1이세포를보호하는역할을수행할가능성이있다고언급하였다. 최근에는 Tanaka 등 (13) 이실험적종양모델에서 HO 억제제인 Zinc protoporphyrin IX (ZnPP IX) 을투여시광범위한세포사멸이초래됨을보고하였다. 본연구에서도 FK506 처리시세포내 H 2O 2 와 NO의양이증가하였으며, HO-1의발현은 FK처리초기에증가하다가감소하는양상을보였다. 그러나 CoPP 처리에의해 HO-1의발현이증가하면세포내활성산소가감소하였으며, ZnPP을처리시세포내 H 2O 2 및 NO의양이증가하여세포사멸을촉진하는결과를보였다. 과량의산화질소생성은패혈성쇼크, 자가면역질환, 뇌경색과당뇨병같은질환들과연관되어있으며, 이들질환 에서는산화질소매개세포사멸이자주관찰된다.(14) 산화질소는단백질과핵산반응을포함한몇가지세포독성을가지며, DNA 손상을유도하고 p53 유도와 poly (ADP-ribose) polymerase (PARP) 의활성화를통하여세포사멸을유발한다.(15) 산화질소는 inos로부터생성된다. inos는주로심각한감염을포함한염증질환에서유도된다.(16) enos에의해생성된작은양의산화질소는동맥경화증, 허혈성질환, 고혈압을방지한다. 그러나 inos에의해생산되는과량의산화질소는동맥경화증, 허혈 / 재관류, 심부전을포함한다양한혈관질환의병리기전에도관련이있으며,(16,17) ER stress 경로는심혈관질환의발병과관련이있다.(18) 이러한질환병변에있는염증세포와내피세포에서 inos가유도되는것이확인되었다. Zhou 등 (19) 은 CHOP 발현을포함한 ER stress 경로가동맥경화증에서혈관벽을침범하는대식세포에서활성화되어있음을보고하였다. 본연구에서는 FK506 처리시 inos 단백질의발현이처리 12 시간후부터시간의존적으로증가하였으며, 세포내 NO의생성도증가하였다. 그러나 HO-1의발현증가시 NO 생성이감소하였으며, HO-1의활성억제시 inos의발현증가에따른 NO의생성이증가되는것을확인하였다. 이상의결과를종합하면, FK506은사람 T 림프구인 Jurkat 세포에세포사멸을유도하였고, 또한 inos의발현증가를유도하여 NO의생성을증가시켰고, Bak 단백질의발현증가에따른미토콘드리아의기능이상과세포내활성산소인 H 2O 2 의생성증가를초래하였다. 따라서, HO-1의발현조절에대한추가적인분자생물학적연구가요구되며면역억제제의작용기전에대한임상적용의과학적기반을제공할수있으리라사료되며, HO-1의조절을통해 FK506 와병용처리시세포사멸항진효과에따른용량조절과거부반응억제효과가기대된다. REFERENCES 1) Pirsch JD, Miller J, Deierhoi MH, Vincenti F, Filo RS. A comparison of tacrolimus (FK506) and cyclosporine for immunosuppression after cadaveric renal transplantation. FK506 Kidney Transplant Study Group. Transplantation 1997;63: ) Margreiter R; European Tacrolimus vs Ciclosporin Microemulsion Renal Transplantation Study Group. Efficacy and safety of tacrolimus compared with ciclosporin microemulsion in renal transplantation: a randomised multicentre study.

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경락경혈학회지 Vol.27, No.1, pp.87 106, 2010 Journal of Meridian & Acupoint Dept. of 1 Meridian & Acupoint, 3 Acupuncture & Moxibustion, College of Oriental Medicine, Daejeon University 2 Division of Clinical