DBPIA-NURIMEDIA

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1 J Korean Soc Food Sci Nutr 35(1), 1322~1328(26) 한국식품영양과학회지 생약복합조성물 (HemoHIM) 의수지상세포활성화효과 신성해 1 김도순 1 김성호 2 조성기 3 변명우 3 이성태 1 1 순천대학교생물학과 2 전남대학교수의학과 3 한국원자력연구소방사선연구원방사선식품생명공학팀 Effects of a Herbal Composition (HemoHIM) on the Activation of Dendritic Cells Sung Hae Shin 1, Do Soon Kim 1, Sung Ho Kim 2, Sung Kee Jo 3, Mung Woo Byun 3 and Sung Tae Yee 1 1 Dept. of Biology, College of Natural Science, Sunchon National University, Suncheon , Korea 2 College of Veterinary Medicine, Chonnam National Univesity, Gwangju 5-752, Korea 3 Radiation Food and Biotechnology Team, Advanced Radiation Technology Institute, Jeongeup Campus of Korea Atomic Energy Research Institute, Jeonbuk , Korea Abstract In our previous study, a novel herb mixture (HIM I) of Angelica gigas radix, Cnidium officinale rhizoma, and Paeonia japonica radix was developed to protect the intestinal and immune systems and to promote their recovery from radiation damage. A new herbal composition (HemoHIM) with the high immune modulating activity was developed from HIM I. In the present study, we examined the effects of HemoHIM on the maturation process of murine bone marrow (BM) derived dendritic cells (DC). BM cells were cultured in the presence of IL 4 and GM CSF and the generated immature DC were stimulated with HemoHIM for 24 hours. HemoHIM significantly enhanced the expression of co stimulatory molecules, CD8 and CD86, especially. The activation capacity of HemoHIM treated DC was significantly higher than that of immature DC, as analyzed by IL 2 and IFN γ production and proliferation of the responding T cells in the co culture with allogeneic T cells. The antigen presenting capacity of HemoHIM treated DC was also increased by the co culture with OVA specific T cells (HS 1), as analyzed by IL 2 and IFN γ production and the proliferation. These results indicate that HemoHIM causes the maturation and activation of DC, which may be a part of mechanisms of immunomodulation by HemoHIM. Key words: a herbal composition, dendritic cells, IL-2, IFN-γ, co-stimulatory molecules 서론면역계는면역기능에관여하는세포나조직이모여서이루어진것으로서주로림프구와보조세포, 그리고이들이모여서만들어진림프조직으로구성되어있다. 면역반응에서가장중요한역할을하는세포는림프구이지만실제로면역반응은림프구에의해서만나타나는것이아니라, 보조세포라고부르는다른여러가지백혈구들과의상호작용결과로나타난다. 이들보조세포들은항원을림프구에제시하여면역반응을일으키고진행시키기도하며, 항원을제거하는데도중요한역할을담당한다 (1). 보조세포중에서수지상세포 (dendritic cells) 는최근에세포치료제로서응용가능성때문에의학적인주목을받고있으며다양한기능에대해 많은기초적인연구가이루어지고있다 (2,3). 특이적면역반응은 T세포의존적면역반응으로항원제시세포 (antigen presenting cells) 가 MHC 분자에결합한항원펩티드를제시하는것으로시작한다. 특이적면역반응을유도하는과정에서중요한역할을하는항원제시세포를전문적항원제시세포 (professional antigen presenting cells) 라고하며 B세포, 대식세포, 수지상세포를말한다. 그러나불활성화상태의 B세포는탐식작용이활발하지못하고 MHC 분자의발현양도많지않다. 대식세포의경우탐식작용은활발하지만, 면역작용의결과생산되는 IFN-γ와같은사이토카인의도움이없으면 MHC 분자를발현하지못하기때문에항원을제시할수없다. 그러나수지상세포는생체내에널리분포하고있으며성숙하는과정에서다양한 Corresponding author. sungtae@sunchon.ac.kr Phone: , Fax:

2 생약복합조성물 (HemoHIM) 의수지상세포활성화효과 1323 면역반응을조절하는강력한전문항원제시세포로알려져있다 (4). 수지상세포가 T세포에항원을제시하여면역반응을유도하는과정은크게세단계로나눌수있다. 즉수지상세포는말초비림프계기관에서항원을섭취하여처리한다음, MHC 분자와복합체를형성하고, 림프계조직의 T세포영역으로이동하여, 다양한부착 / 공동자극분자와사이토카인으로항원특이적 T세포의증식 / 분화를유도한다 (5). 항원특이적면역반응은 T세포의항원수용체를통해 CD8 + 또는 CD4 + T세포의활성화로일어난다. 이때수지상세포는세포표면의 MHC I과 MHC II 분자와결합한항원펩티드를 T세포에제시할뿐만아니라, 동시에다양한부착 / 공동자극분자 (CD4, CD8, CD86 등 ) 를통해 T세포의 CD154, CD28, CTLA-4 분자와결합하여보조적인자극으로 T세포의활성화를유도한다 (6). 최근수지상세포는 CD4 분자를이용해미경험 B세포의증식과 IgM 항체생산도유도하는것으로알려졌다 (7). 최근에는천연생약재와생리활성물질중에서수지상세포를활성화시킬수있는성분을탐색하는실험이진행되고있다. 즉 candida β-d-glucan(8), Agaricus blazei의물추출성분 (9), Aloe vera에서분리한 acemannan(1) 등이생쥐의골수에서분화한수지상세포의다양한공동자극분자의발현과항원섭취기능등을증가시키며, IL-12 생산량도증가시키는효과가있다는보고가있다. 또한보중익기탕추출물 (11) 과인삼사포닌의성분 (12) 이사람단구세포에서분화한수지상세포의다양한공동자극분자의발현, 항원섭취기능과동종항원반응 T세포의증식반응도증가시키는효과가있다고보고되었다. 본연구팀은동양의학에서사용되고있는다양한생약재및한약처방제의다양한약리작용에대한효과를보고한바있으며 (13-2), 최근에당귀, 천궁, 백작약등 3종의생약재를이용한새로운생약복합물 HIM-I을개발하여면역세포활성화효과, 면역조혈계회복촉진효과, 재생조직및면역조혈계의방호효과, 항산화효과에대해보고하였다 (21). 그리고 HIM-I에서면역및조혈기능활성화효과가더욱강화된새로운생약복합조성물을개발하고자 HIM-I에그조다당분획이첨가된 HemoHIM 을제조하여효능을검증하였다 (22). 그결과생약복합물 HIM-I에조다당분획을첨가하여개발한새로운생약복합조성물 HemoHIM이방사선에의해유발된위장관및면역계조직의손상을감소시켜생존을증가시키는효과가있으며, 특히 HemoHIM 은 HIM-I과비교하여재생조직의산화적손상억제효과는비슷하게유지되면서면역조혈세포방호및회복촉진효과가높은것으로관찰되었다. 본실험은특이적면역반응을유도하는전문항원제시세포인수지상세포의분화와항원제시기능에미치는생약복합조성물 HemoHIM의면역기능증강효과에대해알아보았다. 재료및방법실험동물실험동물은대한실험동물센터 ( 충북음성, 대한민국 ) 에서특정병원체부재 (specific pathogen free) C57BL/6, Balb/c 생쥐를공급받아실험동물사육실에서폴리카보네이트사육상자 (18 2 cm) 당 6개체의밀도를유지하며사육하였다. 이들생쥐는실온에서물과사료를충분히공급하고, 낮과밤의주기를 12시간씩조절하여가능한스트레스를받지않도록사육하면서, 생후 8~12 주사이의생쥐를실험에사용하였다. 사용시약세포배양에필요한 RPMI-164, FBS(fetal bovine serum), antibiotic-antimycotic 은 Gibco BRL(Grand Island, NY, USA) 제품을사용하였으며, 2ME(2-mercaptoethanol), sodium bicarbonate(nahco 3) 는 Sigma Chemical Co.(St. Louis, MO, USA) 제품을사용하였다. 세포표면단백질에대한특이적항체 (anti-cd4, anti-cd86, anti-mhc class II, anti-cd11c mab) 는 Pharmingen(San Diego, CA, USA) 제품을사용하였다. 골수세포에첨가하여수지상세포로분화를유도하기위한 recombinant mouse GM-CSF 와 IL-4는 R&D(Mckinley Place NE, MN, USA) 제품을사용하였다. 생약복합조성물 (HemoHIM) 및분획의제조생약복합조성물 (HemoHIM) 은아래와같은방법으로제조한시료를 ( 주 ) 선바이오텍에서제공받아사용하였다. 서울경동한약재시장에서구입한생약재 3종, 즉당귀 (Danggui, Angelica gigas Nakai) 의뿌리, 천궁 (Chuanxiong, Cnidium officinale Makino) 의근경, 백작약 (Baishaoyao, Paeonia japonica Miyabe) 의뿌리를동일한무게비율로혼합한후, 혼합생약재 1 g당증류수 1, ml을가하고 4시간열탕추출하였다. 추출물의고형분을제거하고감압농축하여생약복합물 HIM-I을얻었다. HIM-I의일부를취하여 4배부피의 1% 에탄올주정을첨가하고 ( 최종에탄올농도약 8%), 25 o C 이하에서 16시간정치한후, 원심분리하여침전된조다당분획 (HIM-I-P) 과상층의에탄올분획 (HIM-I-E) 을수거하였다. 수거한조다당분획의일부를이에해당하는 HIM-I에첨가하여생약복합조성물 HemoHIM을제조하였다. 제조한 HemoHIM은동결건조하여냉동저장하였으며, 실험직전에증류수에녹여사용하였다. 수지상세포분화유도및배양수지상세포는다음과같은방법을이용하여생쥐골수에서분화 유도하여사용하였다 (23). 생쥐 (C57BL/6) 를경추탈골법으로희생시킨후, 1 ml 주사기를이용하여대퇴골 (femur) 과경골 (tibia) 안에있는골수를분리한다음단일세포로만들었다. 적혈구용해완충액 (Tris-buffered ammonium chloride; 9 ml of.16 M NH 4Cl, 1 ml of.17 M

3 1324 신성해 김도순 김성호 조성기 변명우 이성태 Tris, ph 7.2) 을처리하여적혈구를제거한후, washing용액으로 1,2 rpm에서 5분간 3회원심분리하여충분히세척한다음, 혈구계산반을이용하여세포수를계산하였다. GM- CSF(1, U/mL) 와 IL-4(1, U/mL) 가포함된배지 (RPMI 164, 1% FBS) 에부유된골수세포 (1 1 6 개 /5 ml/well) 를 6-well cell culture plate에서배양하였다. 배양 4일후새배지 1 ml와동량의 GM-CSF(1, U/mL), IL-4(1, U/mL) 를첨가하고 2일간더배양한다음, 세포를 5분간 shaking하여가볍게부착되어있는세포를회수하였다. 회수한세포를 washing용액으로 1,2 rpm에서 5분간원심분리하여충분히세척한다음, 세포 ( 개 /5 ml/well) 배양액에 HemoHIM을농도별 (1, 1, 1 μg/ml) 로첨가한후, 24시간배양하고회수하여실험에사용하였다. 유세포분석기분석수지상세포를 washing 용액 (PBS, 1% FBS,.1% NaN 3) 으로 3회세척한다음 Trypan blue 시약을이용해혈구계산반으로생세포수를계산하였다. 먼저비특이적결합을막기위해세포 (1~5 1 5 개 ) 를 anti-fcγrⅡ/Ⅲ-specific mab (2.4G2) 로 3분간 blocking한다음 washing 하지않고바로 PE-conjugated anti-cd4 그리고 anti-mhc class Ⅱ (M5/114), anti-cd86 mab로각각 4 o C에서 3분간결합시켰다. 그다음 washing 용액으로세척하고, 마지막으로 FITCconjugated anti-cd11c mab로이중결합시킨후유세포분석기 (Epics XL, COULTER, USA) 로분석하였다. 동종항원반응 T세포분리분리한생쥐 (BALB/c) 의비장으로부터단일세포를준비한다음, 1% FBS-RPMI 164 배지에희석하여 nylon wool column 에넣고 37 o C, 5% CO 2 incubator(mco-17a, SANYO Electric Co., Japan) 에 6분간배양한후, Nylon wool column를 37 o C로미리데워진배지로세척하고비부착성인 T 세포만을순수분리하여동종항원반응 (alloreative) T세포로실험에사용하였다. 항원 OVA 특이적 T세포주수립생쥐 (C57BL/6) 를 OVA로 2차면역한후비장을분리하여 OVA에특이적으로증식반응을나타내는 T세포주를수립하였다. 수립된세포주는유세포분석기로분석하여 CD4 + / CD8 - T세포이며항원자극에대해 IL-2와 IFN-γ를생산하는 Type 1 helper T세포인것을확인하였고 HS-1 으로명명하였다. Il-2와 IFN-γ 분비량측정동종항원반응 (alloreative) T세포 (3 1 5 개 /well) 또는 T 세포주 HS-1(5 1 4 개 /well) 과 HemoHIM을농도별 (1 1 μg/ml) 로처리한수지상세포를 well당 1 1 4, 3 1 4, 개씩 96-well flat-bottomed cell culture plate에넣고 24시간배양한후배양상층액을회수하여상층액에포함된 IL-2와 IFN-γ의양을측정하였다. 즉일차항체를 coating buffer(.1 M NaHCO 3) 에희석하여 plate 에적정양을넣고 4 o C에서하룻밤둔다음 washing 용액 (.5% Tween 2/PBS) 으로세척한후, 소혈청 (1% FBS/PBS) 으로 blocking 하였다. 그리고배양상층액을적절하게희석하여넣은다음, biotin이부착된이차항체를첨가하였다. 일정한시간후에 avidin-peroxidase를첨가하고, 기질 (2,2'-azino-bis,.1 M citritic acid, H 2O 2) 을넣어발색시키는효소항체법 (enzymelinked immunsorbent assay: ELISA) 을이용해 Microplate Reader(Sunnyvale, CA, USA) 로 45 nm에서흡광도를측정하였다. T세포증식반응측정 HemoHIM 을농도별로처리한수지상세포를 MMC(mitomycin C) 로처리한다음, T세포와함께 3일또는 4일배양한후세포증식정도를측정하였다. 세포증식정도는 5- Bromo-2'-deoxy-uridine Labeling and Detection KitⅢ (Roche, IN, USA) 를이용하여측정하였다. 항원제시기능측정골수세포에 GM-CSF 와 IL-4를첨가하여 6일간배양한수지상세포를회수하여 HemoHIM 을농도별로처리하고동시에 OVA 항원을처리하여 24시간배양한후에세포를회수하였다. 회수한수지상세포를다시 T세포주 (HS-1) 와 24 시간배양한후상층액을회수하여효소항체법을이용해 IL-2, IFN-γ를측정하고, 일부는 3일또는 4일간배양한후 T세포증식반응을측정하였다. 통계처리실험결과는평균값 ±SD로나타내었고, Student's t-test 를이용하여통계처리한후에 p<.5, p<.1 수준에서유의성을검증하였다. 결과및고찰골수세포로부터수지상세포의분화유도및형태학적인특징확인생쥐의골수에서골수세포를분리하여 GM-CSF와 IL-4 를첨가하여 7일간배양하면서수지상세포의증식 / 분화과정을관찰하였다. 먼저배양 2일후부터 GM-CSF와 IL-4에반응하는세포들이분열하여덩어리를형성하며증식하기시작하였으며배양 4일후까지약하게붙어서증식하던세포의덩어리가배양후 6일이지나면서배지내로떨어져나오기시작하였고, 이중일부는세포표면에돌기를가진수지상세포의형태를나타내었다 (Fig. 1). 그리고배양후 7일째는거의모든세포들이배지내로부유하였으며, 전형적인성숙한수지상세포의형태를보였다 (23). 따라서골수세포에고농도의 GM-CSF와 IL-4를공급하면서 7일간배

4 생약복합조성물 (HemoHIM) 의수지상세포활성화효과 1325 Table 1. Effect of HemoHIM on the expression of co stimulatory molecules in CD11c + dendritic cells (DC) (% of double positive staining cells) Surface markers CD11c/MHC class Ⅱ CD11c/CD86 CD11c/CD4 HemoHIM (μg/ml) Bone marrow cells were cultured as described in Fig. 1. On day 6, the cells (Day 6 DC) were washed and stimulated with or without HemoHIM from 6 to 7 days. On day 7, the expression of CD4, CD86, MHC class II and CD11c was analyzed by FACS. Two colour flow cytometry was used to determine the expression level of co-stimulatory molecules in CD11c + DC. The results are representative of three independent experiments. Fig. 1. Phase contrast microscopy of dendritic cell. Bone marrow cells were cultured in complete RPMI 164 medium supplemented with 1, U/mL of GM-CSF and IL-4 for 7 days. Dendritic cells forms loosely adherent proliferating aggregates in the presence of IL-4 and GM-CSF on day 7. :aggregate of dendritic cells. (original magnification 2) 양하여대량의수지상세포를얻을수있다는것을확인하였다. 수지상세포표면단백질분자발현증가효과 수지상세포가 T세포를활성화시키는것은크게두가지신호전달로나눌수있다. 먼저수지상세포가섭취한항원을분해하여자신이가지고있는 MHC 분자에항원펩티드를결합시켜 T세포항원수용체를자극하는신호와수지상세포의세포표면단백질분자로 T세포표면단백질분자를자극하는신호로나눌수있다. 이번실험에서는 HemoHIM 을첨가하여수지상세포를배양하였을때, T세포의세포표면분자인 CD28, CD152, CD154와결합하여 T세포를활성화시킬수있는 MHC Ⅱ (I-A b ), CD4, CD86(B7-2) 과같은공동자극분자를많이발현하는성숙한수지상세포로분화가유도되는지를알아보기위해유세포분석기를이용하여조사하였다. 그결과 HemoHIM을첨가하지않고배양한대조군의 CD11c/CD86 double positive 세포는 36.9% 인데비해 HemoHIM를농도별 (1, 1, 1 μg/ml) 로처리하였을때는 43.1%, 46.4%, 45.8% 로증가하는것을확인할수있었다. 그리고 CD11c/ CD4 double positive 세포는대조군에서 9.4% 인데비해 HemoHIM을처리하였을때 16.8%, 15.3%, 2.2% 로증가하는것을확인할수있었다 (Table 1). 그러나 CD11c/MHC II double positive 세포는대조군에비해 HemoHIM 을처리하였을때유의하게증가하지는않는것으로나타났다. 이러한결과는 Agaricus blazei의물추출성분 (1 μg/ml) 이 CD8, CD86, MHC I, MHC II의발현을유의하게증가시킨결과 (9) 와유사하였고, Aloe vera의추출성분 (1 μg/ml) 이 CD8, CD86, MHC II, CD4, CD54의발현을유의하게증가시킨결과 (1) 와유사하였다. 그리고 candida β-dglucan(25 μg/ml) 이 CD8과 CD86의발현은유의하게증 가시키지만 MHC I과 MHC II 발현은약간증가시키는결과 (8) 와일치하는것으로생각된다. 이상의실험결과, HemoHIM은 T세포를활성화시킬수있는공동자극분자를세포표면에많이발현하여성숙한수지상세포로분화를유도하는효과가있는것으로나타났다. HemoHIM 처리수지상세포의동종항원반응 T세포활성화효과세포표면단백질의발현을분석한결과, HemoHIM에의해수지상세포가성숙한형태로분화하는것을확인할수있었다. 이렇게성숙한수지상세포가 T세포를활성화시키는기능을제대로수행하는지를알아보기위해 HemoHIM 을처리한수지상세포의동종항원반응 T세포활성화능력을조사하였다. 시료의농도는앞실험의결과에서세포표면공동자극분자의발현이가장많이증가하는 1 μg/ml 로결정하였다. 수지상세포는성숙하게되면포식된항원을 T세포에제시하고, 항원을제시받은 T세포는분열 증식은물론 IL-2, IFN-γ 와같은사이토카인을분비함으로써면역반응을유도한다. 실험에사용한 allogeneic T세포는수지상세포를분리한 C57BL/6(H-2 b ) 생쥐와 MHC 분자가다른동종이계인 Balb/c(H-2 d ) 생쥐에서유래한세포로서수지상세포자신의 peptide가결합한 MHC 분자를항원으로인식하여반응하는반면, syngeneic T세포는수지상세포와기원이같은동종동계의 C57BL/6(H-2 b ) 생쥐에서분리한세포로서, 수지상세포의 MHC 분자에결합한 OVA 항원 peptide를인식하여반응하는세포주 (HS-1) 를분리배양하여사용하였다. 즉 allogeneic T세포는특정항원과관계없이수지상세포의 MHC 분자를인식하여비특이적으로반응하는데비해다음실험에사용한 syngeneic T세포 clone(hs-1) 은수지상세포의 MHC 분자와결합한 OVA 항원 peptide만을인식하여특이적으로반응한다. 비장세포를 nylon wool colum을통과시켜서얻은 Balb/c

5 1326 신성해 김도순 김성호 조성기 변명우 이성태 Table 2. Effect of HemoHIM stimulated dendritic cells on the IL 2 and IFN γ production of allogenic T cells Conditions T cell (3 1 5 ) DC (1 1 4, 3 1 4, ) DC (1 1 4 )+T cell DC (3 1 4 )+T cell DC (1 1 5 )+T cell IL-2 (pg/ml) IFN-γ (pg/ml) Control HemoHIM Control HemoHIM 62.± ± ± ± ± ± ± ± ± ± ± ±15.9 Day 6 DC were stimulated with or without 1 μg/ml of HemoHIM from 6 to 7 days. Day 7 DC were cultured with allogenic T cells. IL-2 and IFN-γ levels in the supernatant collected after 24 hr were quantified by ELISA. Data are represented as mean±sd of triplicates. p<.5, p<.1: significant difference between untreated control and experimental group. 생쥐의 T세포와다양한수의수지상세포를함께넣고 24시간배양한후, 배양상층액으로분비된 IL-2와 IFN-γ의양을측정하였다. HemoHIM 을처리하지않은수지상세포 (1 1 4 개 ) 와 T세포를넣고배양한대조군의 IL-2 의분비량은 62. pg/ml인데비해 HemoHIM 을처리한실험군은 pg/ml로대조군에비해 IL-2의분비량이약 2배로증가한것을확인할수있었다. 그리고함께배양한수지상세포 (3 1 4, 개 ) 의수가증가할수록모두대조군에비해 IL-2의분비량이증가하는것을확인할수있었다 (Table 2). 또한 IFN-γ 분비량도함께배양한수지상세포의수가증가할수록모두대조군에비해증가하는것을확인할수있었다 (Table 2). 이러한결과는인삼사포닌의성분이사람단구세포에서분화한수지상세포의 IFN-γ, IL-4, IL-5, IL-1 분비량을증가시킨다는보고 (12) 와 Agaricus blazei의물추출성분 (9) 이 IL-2 분비량을증가시키는결과와일치한다. 다음으로수지상세포와 T세포를 3일또는 4일간같이배양한후 T세포증식반응에미치는효과를살펴보았다. 그결과수지상세포의수에비례하여 T세포의증식반응이증가하였고, HemoHIM을첨가하여배양한수지상세포를넣어주었을때 T세포증식반응도수지상세포의수가증가할수록증가하였다 (Fig. 2). HemoHIM 의효과는배양후 3일째수지상세포의수가많을수록확실하게나타났으며배양후 4일째에는가장많은수의수지상세포와함께배양했을때만나타났다. 이러한결과는보중익기탕추출물 (11) 과인삼사포닌의성분 (12) 에의해사람단구세포에서분화한수지상세포가동종항원반응 T세포의증식반응을증가시킨다는보고와일치한다. 이상의실험결과로 HemoHIM 은수지상세포의성숙을유도해동종항원반응 T세포를활성화시켜 IL-2와 IFN-γ 분비량을증가시킬뿐만아니라 T세포증식반응을증가시키는효과도있는것으로나타났다 Control HemoHIM (A) (B) OD at 49nm , 3, 1, 3, 1, 3, 1, 3, Number of DC (per well) Fig. 2. Allogenic T cells were stimulated with dendritic cells derived in the presence of HemoHIM in the MLR assay for 3 (A) or 4 (B) days. C57BL/6 (H-2 b ) bone marrow-derived DC were generated as described in Material and Methods, harvested on day 7, washed extensively and used as stimulators of naive allogeneic Balb/c (H-2 d ) T cells (3 1 5 cells/well) in a one-way MLR. Growth of 72 h (A) and 96 h (B) were measured by BrdU assay kit. : stimulated with DC derived in the presence of HemoHIM. Data are represented as mean±sd for triplicates. p<.5, p<.1: significant difference between untreated control and experimental group.

6 생약복합조성물 (HemoHIM) 의수지상세포활성화효과 1327 Table 3. Effect of HemoHIM stimulated dendritic cells on IL 2 production of OVA specific T cell clone Cell number DC (1 1 4 )+T cell DC (3 1 4 )+T cell HemoHIM (μg/ml) 1,911.8±42.4 5,2.7± ,169.8±18.1 3,61.2±36.3 5,613.4± ,44.2± ,839.5±54.5 7,85.5±2. (pg/ml) Day 6 DC generated from C57BL/6 were stimulated OVA with or without HemoHIM from 6 to 7 days. Day 7 DC were cultured with OVA-specific T cell line (5 1 4 cells/well). IL-2 levels in the supernatant collected after 24 hours were quantified by ELISA. Data are presented as mean±sd of triplicates. p<.5, p<.1: significant difference between untreated control and experimental group. Table 4. Effect of HemoHIM stimulated dendritic cells on IFN γ production of OVA specific T cell clone Conditions DC (1 1 4 )+T cell DC (3 1 4 )+T cell HemoHIM (μg/ml) 113.3± ± ± ± ± ± ± ±3.5 (pg/ml) Day 6 DC generated from C57BL/6 were stimulated OVA with or without HemoHIM from 6 to 7 days. Day 7 DC were cultured with OVA-specific T cell line (5 1 4 cell/well). IFN-γ levels in the supernatant collected after 24 hours were quantified by ELISA. Data are presented as mean±sd of triplicates. p<.5, p<.1: significant difference between untreated control and experimental group. HemoHIM 처리수지상세포의 OVA-specific T세포활성화효과다음은수지상세포의항원전달기능에미치는 HemoHIM 의영향을알아보기위하여 OVA 단백질항원에특이적으로반응하는 T세포주 (HS-1) 를수립하여실험에사용하였다. GM-CSF 와 IL-4를첨가하여 6일간분화를유도한수지상세포에 OVA 항원 (1 mg/ml) 과 HemoHIM을농도별로첨가하여다시 24시간배양한수지상세포를 T세포주 (HS-1) 와함께배양하였다. HemoHIM 을처리하지않은수지상세포 (1 1 4 개 ) 와 HS-1을함께넣어배양한대조군의 IL-2 분비량 1,911 pg/ml 에비해 HemoHIM(1, 1, 1 μg/ ml) 을처리한실험군은 5,2 pg/ml, 5,169 pg/ml, 3,61 pg/ml 로모두대조군에비해유의하게증가한것을확인할수있었다 (Table 3). 그리고 개의수지상세포와함께배양한경우에도대조군에비해유의하게증가하였다. 이때고농도보다저농도의 HemoHIM 을처리하였을때 IL-2 분비량이더많은것으로나타났다. 그리고 IFN-γ 분비량도대조군에비해 HemoHIM 처리실험군에서증가하였다 (Table 4). 다음으로같은조건에서 3일또는 4일간배양하여 HS-1 의증식반응을알아본결과, HemoHIM 처리시험군에서 HS-1의증식반응이대조군에비해증가하였고, 수지상세포의수가많을수록증식반응도크게증가하는것으로나타났다 (Fig. 3). 그리고수지상세포의수가동일한경우에는 HemoHIM 1 μg/ml을처리하였을때가장높은증식반응이나타났고최대증식반응은배양후 4일째관찰되었다. 이상의실험결과, HemoHIM 을첨가하여배양한수지상세포는대조군에비해 T세포를충분히활성화시켜 IL-2 와 IFNγ 분비량과증식반응을증가시키는것을확인할수있었다. 지금까지의실험결과를종합하면, 특이적면역반응을유 OD at 49 nm. OD at 49 nm DC DC(1 13) DC DC(3 13) DC DC(1 14) 4 ) DC DC(1 13) DC DC(3 13) DC DC(1 14) 4 ) (A) Concentration of HemoHIM (µg/ml) (B) Fig. 3. Effect of HemoHIM stimulated dendritic cells on the proliferation of OVA specific T cell clone. Day 6 DC generated from C57BL/6 were stimulated by OVA with or without HemoHIM from 6 to 7 days. Day 7 DC were cultured with OVA-specific T cells (5 1 4 cells/well). Cell growth at 72 h (A) and 96 h (B) were measured by BrdU assay kit. Data are presented as mean±sd of triplicates. p<.5, p<.1: significant difference between untreated control and experimental group. 도하는전문항원제시세포인수지상세포가 T세포의활성화를유도하는과정에서, 생약복합조성물인 HemoHIM 이수지상세포의분화를유도하여 T세포의 IL-2와 IFN-γ 생산량을

7 1328 신성해 김도순 김성호 조성기 변명우 이성태 유의하게증가시키고, 증식반응역시증가시키는것을확인하였다. 따라서 HemoHIM 은생체내의특이적면역반응을강화하는면역기능조절제로서사용이가능할것으로생각된다. 요 특이적면역반응을유도하는전문항원제시세포인수지상세포의분화와항원제시기능에미치는 HemoHIM의면역기능증강효과에대해알아보았다. HemoHIM은수지상세포의분화 / 증식과정에첨가하였을경우에공동자극분자인 CD4, CD86 분자의세포표면발현량을증가시키는것으로나타났다. 이수지상세포는동종항원반응 T세포를활성화시켜 IL-2, IFN-γ 분비량과증식반응을유의하게증가시켰다. 그리고 HemoHIM을처리한수지상세포는대조군에비해 OVA 항원을 T세포주에충분히전달하여 IL-2와 IFNγ의분비량을유의하게증가시켰을뿐만아니라 T세포주의증식반응도유의하게증가시켰다. 따라서 HemoHIM 은생체내의특이적면역반응을유도하는수지상세포의기능을강화하는면역기능조절제로서사용이가능할것으로생각된다. 약 감사의글 본연구는과학기술부의원자력연구개발중장기계획사업 (M A92-212) 의지원을받아수행하였기에감사드립니다. 문 1. Hart DN Dendritic cells: Unique leukocyte populations which control the primary immune response. Blood 9: Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, Pulendran B, Palucka K. 2. Immunobiology of dendritic cells. Annu Rev Immunol 18: Schuler G, Schuler-Thurner B, Steinman RM. 23. The use of dendritic cells in cancer immunotherapy. Current Opinion in Immunology 15: Banchereau J, Steinman RM Dendritic cells and the control of immunity. Nature 392: Inaba K, Witmer-Pack M, Inaba M, Hathcock KS, Sakuta H, Azuma M, Yagita H, Okumura K, Linsley PS, Ikehara S, Muramatsu S, Hodes RJ, Steinman RM The tissue distribution of the B7-2 costimulator in mice: Abundant expression on dendritic cells in situ and during maturation in vivo. J Exp Med 18: Rattis FM, Peguet-Navarro J, Staquet MJ, Dezutter- Dambuyant C, Courtellemont P, Redziniac G, Schmitt D Expression and function of B7-1 (CD8) and B7-2 (CD86) on human epidermal Langerhans cells. Eur J Immunol 26: Liu YJ, Arpin C Germinal center development. Immunol Rev 156: Kikuchi T, Ohno N, Ohno T. 22. Maturation of dendritic 헌 cells induced by Candida beta-d-glucan. Int Immunopharmacol 2: Kim GY, Lee MY, Lee HJ, Moon DO, Lee CM, Jin CY, Choi YH, Jeong YK, Chung KT, Lee JY, Choi IH, Park YM. 25. Effect of water-soluble proteoglycan isolated from Agaricus blazei on the maturation of murine bone marrow-derived dendritic cells. Int Immunopharmacol 5: Lee JK, Lee MK, Yun YP, Kim YS, Kim JS, Kim YS, Kim KJ, Han SS, Lee CK. 21. Acemannan purified from Aloe vera induces phenotypic and functional maturation of immature dendritic cells. Int Immunopharmacol 1: Nabeshima S, Murata M, Hamada M, Chong Y, Yamaji K, Hayashi J. 24. Maturation of monocyte-derived dendritic cells by Hochu-ekki-to, a traditional Japanese herbal medicine. Int Immunopharmacol 4: Takei M, Tachikawa E, Hasegawa H, Lee JJ. 24. Dendritic cells maturation promoted by M1 and M4, end products of steroidal ginseng saponins metabolized in digestive tracts, drive a potent Th1 polarization. Biochem Pharmacol 68: Kim SH, Lee SE, Oh H, Kim SR, Yee ST, Yu YB, Byun MW, Jo SK. 22. The radioprotective effects of Bu- Zhong-Yi-Qi-Tang: A prescription of traditional Chinese medicine. Am J Chin Med 3: Jo SK, Yu YB, Oh H, Kim SR, Kim SH. 2. The effects of Shi-Quan-Dai-Bu-Tang and its ingredients on the survival of jejunal crypt cells and hematopoietic cells in irradiated mice. J Korean Soc Food Sci Nutr 29: Kim SH, Lee SE, Oh H, Yang JA, Chung CY, Jang JS, Yu YB, Jo SK The radioprotective effect of Kuei-Pi- Tang as a prescription of traditional Chinese medicine in mice. J Korean Soc Food Sci Nutr 28: Oh H, Park HR, Jeong IY, Kim SH, Jo SK. 22. Protective effects of Paeonia japonica against radiation-induced damage. J Korea Asso Radiat Prot 27: Kim SH, An MR, Nah SY, Lee JH, Kim JH, Jo SK, Jang SJ, Shin DH. 21. The effects of herbs on the radiationinduced apoptosis in intestinal crypt cells. J Korea Asso Radiat Prot 26: Kim SH, Oh H, Kim SR, Jo SK, Byun MW, Kim KS, Lee JH, Shin DH. 21. The radioprotective effects of radices herbs. Korea J Vet Res 41: Lee SE, Oh H, Yang JA, Jo SK, Byun MW, Yee ST, Kim SH Radioprotective effects of two traditional Chinese medicine prescriptions: Si-Wu-Tang and Si-Jun-Zi-Tang. Am J Chin Med 27: Kim SH, Oh H, Lee SE, Jo SK, Byun MW Effect of Si-Wu-Tang and Si-Jun-Zi-Tang on the survival of jejunal crypt cells and hematopoietic cells in irradiated mice. Korean J Food Sci Technol 3: Park HR, Kim SH, Yee ST, Byun MW, Jo SK. 25. The effects of a herb mixture (HIM-I) on the protection of the hematopoietic-immune system and self-renewal tissues against radiation damage. J Korean Soc Food Sci Nutr 34: Jo SK, Park HR, Jung UH, Oh H, Kim SH, Lee SE. 25. Protective effect of a herbal preparation (HemoHIM) on the self-renewal tissues and immune system against γ- irradiation. J Korean Soc Food Sci Nutr 34: Son YI, Egawa S, Tatsumi T, Richard RE Jr, Kalimski P, Kanto T. 22. A novel bulk-culture method for generating mature dendritic cells from mouse bone marrow cells. J Immunol Methods 262: (26 년 9 월 6 일접수 ; 26 년 9 월 15 일채택 )

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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