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1 Journal of Bacteriology and Virology Vol. 46, No. 4 p Communication Eosinophils Regulate Type 2 Immune Responses Following Infection with the Nematode Trichinella spiralis Jayoung Koo Koo and YunJae Jung * Department of Microbiology, School of Medicine, Gachon University, Incheon, Korea Eosinophils are multifunctional leukocytes implicated in protection against helminth infections. Although eosinophils comprise between 1~5% of peripheral blood leukocytes, they primarily reside in the gastrointestinal tract under homeostatic conditions, and rapidly proliferate upon parasitic infection. Intestinal infection with Trichinella spiralis (T. spiralis) induces eosinophilia when the parasite enters the larval stages and larvae finally migrate to the skeletal muscle. Eosinophils are known to mediate parasite death through antibody-dependent cellular cytotoxicity. In this study, we aimed to address the functional significance of eosinophils in the intestinal phase of T. spiralis infection by analysis of immune responses in the Peyer's patch (PP) of infected BALB/c and eosinophil-ablated ΔdblGATA mice. Trafficking of eosinophils to the PP was significantly increased, with upregulation of interleukin-5 at 14 days post infection. Eosinophil deficiency led to a significant augmentation of serum immunoglobulin (Ig) M and IgG1 antibody levels. In accordance with this, IgG1 + B cells in the PP were substantially increased in ΔdblGATA mice compared to that in BALB/c mice. Transforming growth factor-β expression in the PP of infected ΔdblGATA mice was significantly decreased compared to that in BALB/c mice, whereas the number of T. spiralis larvae in the diaphragm was increased. Taken together, these findings indicate that eosinophils contribute to the regulation of Th2 immune responses, and protect the host from T. spiralis attempting to establish larvae in the skeletal muscle. Key Words: Eosinophils, Trichinella spiralis, Intestinal phase, Peyer's patch, Th2 responses INTRODUCTION 호산구는알레르기염증반응을매개하고기생충감염에대한방어작용을나타내는과립구로혈액백혈구의 1~5% 정도로관찰된다 (1, 2). 골수에서호산구의생성은 GATA-1, PU.1, C/EBP 등의전사인자에의해조절되어 GATA-1의결합부위를제거시킨 ΔdblGATA 마우스는전 신적으로호산구가소실되어있다 (3). 일부호산구는골수에서분화가완료되어말초혈액으로이동하는데항상성상태에서상당수의호산구는위장관고유층에분포하며이는호산구에발현된 CCR3가점막조직에높게발현된 eotaxin에반응하기때문이다 (2, 4). 기생충감염과알레르기질환에서호산구증식, 분화를촉진시키는 interleukin-5 (IL-5), IL-3 및 granulocyte-macrophage colony stimulating factor 등의영향으로혈중호산구수가급격히 Received: December 2, 2016/ Revised: December 3, 2016/ Accepted: December 6, 2016 * Corresponding author: YunJae Jung, MD, PhD. Department of Microbiology, School of Medicine, Gachon University, 155 Gaetbeol-ro, Yeonsu-gu, Incheon 21999, Korea. Phone: , Fax: , yjjung@gachon.ac.kr ** This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2016R1D1A1A ). The author thanks Dr. So-Youn Woo (Ewha Womans University, Korea) for supporting works with dblgata mice. T. spiralis was kindly provided by Dr. Hee-Jae Cha (Kosin University, Korea). The author thanks to Eun-Hui Lee (Gachon University, Korea) for technical assistance. CC This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( 295

2 296 J Koo and Y Jung 증가하고관련조직의 eotaxin 발현이증가하면서말초혈액의호산구가조직으로이동하게된다 (2, 5). 염증조직으로이동한호산구는과립단백질을세포바깥으로방출하는데주염기단백질 (major basic protein), 호산구과산화효소 (eosinophil peroxide), 호산구양이온단백질 (eosinophil cationic protein), 호산구유래신경독소 (eosinophil-derived neurotoxin) 등의세포과립은기생충체에손상을입히거나충체제거에관여하는것으로알려져있다 (6~8). 또한호산구는면역반응을조절할수있는다양한종류의사이토카인, 화학주성인자및지질매개물질을분비하여조직의손상과기능부전을유발하게된다 (9). 그러나최근호산구의정상생리조절기능에대한다양한연구성과가발표되고있는데호산구에의해조직재형성과복구가촉진되고 (10), IL-4 분비를통해근육과간재생에필수적인역할을수행하며 (11, 12) 지질대사와인슐린반응성을증가시킨다는것이알려지면서 (13) 호산구의다기능성및면역조절세포로서의기능규명이필요한상황이다. 조직침습기생연충 (tissue-invasive parasitic helminth) 은조직이행기 (larva migration) 를거쳐숙주내선호조직에도착하여숙주의시스템을이용하며기생하게된다 (14). 조직침습기생연충의하나인 Trichinella spiralis (T. spiralis) 감염은감염된고기를익히지않은상태로섭취하여발생하며유충이소장점막에서성숙하여성충이되는장침범기 (intestinal invasion) 로개시된다 (15). 장점막의성충에서배출된유충은조직이행기를거쳐혀, 횡격막등의골격근세포에침입하여 (muscle invasion) 만성감염을일으킨다 (16). T. spiralis와같은조직침습기생충의조직이행기에혈중호산구가급격히증가하고조직내이동이촉진되는데이는감염조직에서 eotaxin 생성이증가하며나타나는현상이다 (17). 이와같이조직에침윤된호산구는충체특이적으로분비된항체와결합하여 antibodydependent cellular cytotoxicity(adcc, 항체의존세포매개세포독성 ) 기전을통해충체를사멸시키는데이는기생충감염시호산구의염증성방어작용에대한주요기전으로제시되어왔다 (18, 19). 그러나호산구세포과립이결핍되거나전신적으로호산구가결핍된마우스에서 T. spiralis 감염에대해정상면역반응이나타나거나오히려충체의생존이억제되고숙주염증반응이증폭된다는연구결과는호산구가기생충감염에대해방어면역기능만을담당하고있지않음을시사하는결과이다 (20~22). 특히장침범기, 조직이행기, 근육침범기의특징적인생 활사를통해감염이성립되는 T. spiralis의경우감염초기의호산구반응에의해숙주의면역반응과충체의만성감염진행의방향이결정될가능성이매우높을것으로예상된다. 본연구에서는정상마우스와 ΔdblGATA 마우스에서 T. spiralis 감염 2주차에비장 (spleen) 과파이어판 (Peyer's patch) 의호산구분획과면역글로불린 (immunoglobulin, Ig) 의생성양상을확인하고파이어판의사이토카인발현을확인하여감염초기에호산구에의해매개될것으로예상되는면역반응의양상을분석하였다. 아울러감염마우스의횡격막내충체분포를관찰하여 T. spiralis 감염에대한초기호산구반응이조직이행기및근육침범기에미치게될영향을분석하고충체와호산구의상호작용기전을제시하고자하였다. MATERIALS AND METHODS 마우스 6~8주령의암컷 BALB/c 마우스 (Orientbio, Gapyeong, Korea) 와 C.129S1(B6)-Gata1 tm6sho /J 마우스 (ΔdblGATA 마우스 ; Jackson Laboratory, Bar Harbor, ME, USA) 를표준실험실온도및습도조건에서유지하였다. 마우스를이용한동물실험은가천대학교동물실험윤리위원회의승인을받아동물실험윤리기준에따라진행하였다 (GIACUC-R ). T. spiralis 분리와감염 T. spiralis는암컷 BALB/c 마우스에서계대감염하여유지하였다. 충체를수거하기위해감염된마우스에서피부, 지방, 내부기관을제거한후근육과뼈를 1% 펩신 (MP Biomedicals, Santa Ana, CA, USA) 과 1% 염산이첨가된소화액에잘라넣은후 37 에서 2시간처리하였다. 용해된조직에서해부현미경 (Olympus, Tokyo, Japan) 관찰하에 T. spiralis 를분리하였다. 마우스에 250개의충체를경구투여하여감염시켰으며마우스는 2주후분석을위해희생시켰다. 유세포분석마우스에서비장과파이어판을적출하여조직을분쇄한후 cell strainer에통과시켜세포를분리하였으며비장세포는 ammonium chloride-potassium lysis buffer로처리하여

Eosinophils Regulate Type 2 Immune Responses Following Infection with the Nematode Trichinella spiralis 297 적혈구를제거하였다. 분리된세포를항마우스 CD16/ CD32 (2.

3 Eosinophils Regulate Type 2 Immune Responses Following Infection with the Nematode Trichinella spiralis 297 적혈구를제거하였다. 분리된세포를항마우스 CD16/ CD32 (2.4G2, BD Biosciences, San Jose, CA, USA) 로 4, 15 분처리하여 Fc 수용체를차단시킨후항마우스 CCR3 (83101, R&D Systems, Minneapolis, MN, USA), 항마우스 SiglecF (E , BD Biosciences), 항마우스 CD3 (145-2C11, Biolegend, San Diego, CA, USA) 또는항마우스 B220 (RA3-6B2, BD Biosciences) 를첨가하여 4 에서 30분간반응시켰다. 세포내 IgG1과 IgG2a 발현을확인하기위해 Cytofix/Cytoperm Kit (BD Biosciences) 로고정, 투과시키고항마우스 IgG1 (R6-60.2, BD Biosciences) 또는항마우스 IgG2a (R19-15, BD Biosciences) 로반응시켰다. 파이어판의종자중심 (germinal center) 을확인하기위해파이어판에서분리한세포에 peanut agglutinin (PNA; Vector Laboratories, Burlingame, CA, USA) 을첨가하여 4 에서 30분반응시켰다. 유세포분석은 FACSCalibur (BD Biosciences) 를이용하여실시하였고데이터는 FlowJo software (Tree Star, Ashland, OR, USA) 로분석하였다. T. spiralis 단백질추출감염마우스에서분리한 T. sprialis를 1 mm phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich, Saint Louis, MO, USA) 와 protease inhibitor cocktail (Sigma-Aldrich) 이첨가된 cell extraction buffer (Thermo Fisher Scientific, Waltham, MA, USA) 에부유시킨후얼음에서 30분간반응시켰다. 이후반응액을 20 khz에서 30초간초음파분쇄하였고 T. sprialis 충체용해액은 13,000 rpm에서 10분간원심분리하여상층액을수거하였다. 상층액에포함된단백질은 BCA Kit (Thermo Fisher Scientific) 로정량하여사용하였다. Enzyme-linked immunosorbent assay (ELISA) 마우스혈청의 IgM, IgG1, IgG2a 역가를측정하기위해 microplate에항마우스 IgM (II/41, BD Biosciences), IgG1 (A85-3, BD Biosciences) 또는 IgG2a (R11-89, BD Biosciences) 를부착시켰다. T. sprialis 특이면역글로불린의역가측정을위한 microplate에는 T. spriali의단백추출액 (10 μg/ml) 을부착시켰다. 면역글로불린또는충체단백질로처리된 microplate에계단희석된혈청을첨가한후 biotin 결합된항마우스 IgM (R6-60.2, BD Biosciences), IgG1 (A85-1, BD Biosciences) 또는 IgG2a (R19-15, BD Biosciences) 로반응시켰다. 발색은 streptavidnine-horseradish peroxidase (BD Biosciences) 와 TMB substrate (KPL, Gaithersburg, MD, USA) 를사용하여진행하였으며흡광도 450 nm에서측정하였다. A B Figure 1. Increased eosinophil frequency in the spleen and Peyer's patch (PP) by Trichinella spiralis (T. spiralis) infection. (A and B) Frequency of CCR3 + SiglecF + eosinophils in the spleen (A) and PP (B) of BALB/c and ΔdblGATA mice at 14 days post infection (dpi). Data represent mean ± s.e.m. values. A two-group comparison was performed by a Student's t-test. *p < 0.05, **p < 0.01, ***p <

4 298 J Koo and Y Jung Real-time PCR 분석파이어판에서 QIAzol lysis reagent (Qiagen, Hilden, Germany) 와 RNeasy Mini Kit (Qiagen) 를통해 RNA를추출하였다. RNA는 DNase I (New England Biolabs, Ipswich, MA, USA) 으로처리한후 iscript cdna synthesis kit (Bio-Rad, Hercules, CA, USA) 로 cdna를합성했다. Real-time PCR 은 iq SYBR Green supermix (Bio-Rad) 를사용하여 CFX Connect real-time System (Bio-Rad) 에서진행되었다. 사용한 primer set는다음과같다. Il5: forward, 5'-ACAAGCAATG- AGACGATGAG-3'; reverse, 5'-CCAGCGGACAGTTTGAT- TCTT-3'; Il6: forward, 5'- GAGGATACCACTCCCAACAG-3'; reverse, 5'-AAGTGCATCATCGTTGTTCA-3'; Tgfb1: forward, 5'-CTCCCGTGGCTTCTAGTGC-3'; reverse, 5'-GCCTTAGTT- TGGACAGGATCTG-3'; Gapdh: forward, 5'-CTGGTATGAC- AATGAATACGG-3'; reverse, 5'-GCAGCGAACTTTATTGA- TGG-3'. 조직학적관찰마우스에서분리한횡격막을 10% 포르말린에고정하여파라핀에포매하고 4 μm 두께로잘라슬라이드에부착시켰다. 조직내충체를확인하기위해헤마톡실린- 에오신염색을실시한후광학현미경 (Olympus) 으로관찰하였다. RESULTS AND DISCUSSION 호산구증가양상 T. spiralis 구강감염시약 2주간의장침범기가개시되어충체가증식하며성충에서배출된유충이혈행성으로골격근에전파되며장관내충체가사라지게된다 (23). 따라서감염 2주이내에장관내호산구매개반응이매우강력하게나타날것으로예상되며전신과장관특이적반응을분석하기위해각각비장과파이어판의호산구분획을확인하였다. T. spiralis 감염 2주후 BALB/c 마우스 A B Figure 2. Increased total and Trichinella-specific immunoglobulin (Ig) levels after T. spiralis infection. (A and B) Total (A) and Trichinellaspecific (B) IgM, IgG1, and IgG2a titers in sera of BALB/c and ΔdblGATA mice at 14 dpi. Data represent mean ± s.e.m. values. A two-group comparison was performed by a Student's t-test. *p < 0.05, **p < 0.01.

5 Eosinophils Regulate Type 2 Immune Responses Following Infection with the Nematode Trichinella spiralis 299 비장에서 CCR3와 SiglecF를발현하는호산구분획은 1.5 ± 0.3% 에서 3.2 ± 0.6% 로유의하게증가하여장내기생충감염에서전형적으로나타나는호산구증가가확인되었다 (Fig. 1A). 파이어판은소장점막고유층에존재하는림프소절로장관으로유입되는항원에대한면역반응이개시되고항체의동형전환 (isotype switching) 이활발하게발생하여특이항체의생성이촉진되는조직이다 (24). 파이어판에호산구는정상적으로거의존재하지않으며 Th2형면역반응항진시증가하는것으로알려져있다 (25). T. spiralis 감염 2주동안파이어판의 CCR3 + SiglecF + 분획이 0.04 ± 0.02% 에서 0.5 ± 0.2% 로 10배이상증가하는것이관찰되었다 (Fig. 1B). 따라서 T. spiralis 감염 2주후장관특이적, 전신적으로호산구증가가유발됨을확인할수있었다. 항체생성양상기생충감염시증가하는호산구는충체에대한방어항체와결합하여 ADCC 기전을통해충체를사멸시킨다 (18, 19). 또한 T. spiralis 충체에대한방어항체는체내에존재하는충체의수에의존적으로생성되어항체의역가가높으면감염충체의수가감소하여감염의조절및재감염예방에도움이된다 (26). 호산구에의한감염초기의방어항체생성조절능력을확인하기위해혈청에서 IgM, IgG1, IgG2a 역가를분석한결과 BALB/c 마우스에서감염 2주후에 total IgM과 IgG1 뿐만아니라 anti- Trichinella IgM과 anti-trichinella IgG1이유의하게증가하여항체생성및항원특이적 Th2 항체의동형전환증가를확인할수있었다 (Fig. 2). 특이한것은이들항체역가 A B C Figure 3. Increased isotype responses in the PP after T. spiralis infection. (A and B) Frequency of CD3 +, B220 +, IgG1 +, and IgG2a + cells in the spleen (A) and PP (B) of BALB/c and ΔdblGATA mice at 14 dpi. The frequency of IgG1 + and IgG2a + cells was analyzed with B220-gated cells. (C) Frequency of PNA high cells in the PP of BALB/c and ΔdblGATA mice at 14 dpi. Data are mean ± s.e.m. values. A two-group comparison was performed by a Student's t-test. *p < 0.05, **p < 0.01, ***p <

6 300 J Koo and Y Jung 의증가가 ΔdblGATA 마우스에서 BALB/c에비해더욱증폭되어나타난것으로 T. spiralis 감염초기호산구에의해 Th2 면역반응이억제되며이는항원특이적으로발생한다는것을시사하는결과이다 (Fig. 2). Total IgG2a의경우감염에관계없이 BALB/c에비해 ΔdblGATA 마우스에서높게관찰되었지만 anti-trichinella IgG2a는실험그룹간유의한차이가나타나지않았다 (Fig. 2). 따라서 T. spiralis 장침범기와근육침범기전환기에전신적으로 Th2 반응이증폭되며해당시기에발생하는호산구증가가과다한 Th2 반응의조절에관여할가능성이매우높은것으로사료된다. 항체생성세포분획의변화양상전신과장관특이적항체생성세포의변화를분석하기위해비장과파이어판의세포를분리하여유세포분석을진행하였다. BALB/c와 ΔdblGATA 마우스모두감염전후비장과파이어판의 CD3 및 B220 양성세포의분획은차이가없어 T 세포와 B 세포의양적차이는발생하지않 는것으로확인되었다 (Fig. 3). 그러나파이어판의 B220 발현세포분획내에존재하는 IgG1과 IgG2a 양성세포의수는감염 2주후유의하게증가하였으며이는 BALB/c 에비해 ΔdblGATA 마우스에서더욱현저하게관찰되었다 (Fig. 3B). IgG1과 IgG2a 양성세포의증가가비장에서는관찰되지않아감염 2주차의항체생성반응은장관면역계에서더욱강력하게나타나는현상임을시사하였다. 파이어판에서항체의동형전환이가장활발하게발생하는부위는종자중심으로 glycan 발현이높기때문에 PNA의결합이높게나타난다 (27). 파이어판에서분리한세포를 B220와 PNA로염색하고분석한결과감염 2주후 B220 + PNA + 분획이유의하게증가하였으며 ΔdblGATA 에서증가의정도가더욱높아항체생성을위한종자중심반응이 BALB/c 마우스에비해증가한것을확인할수있었다 (Fig. 3C). 파이어판면역반응정도와근육내충체분포파이어판의면역반응양상을분석하기위해사이토 A B Figure 4. Immune responses in the PP and muscle larvae after T. spiralis infection. (A) mrna expression of cytokine in the PP of BALB/c and ΔdblGATA mice at 14 dpi. (B) Hematoxylin and eosin stained sections of diaphragm collected from BALB/c and ΔdblGATA mice at 14 dpi. Arrows indicate representative T. spiralis larvae in the diaphragm. Numbers of larvae per five fields were counted with three sections of each group of mice. Original magnification 40. Data are mean ± s.e.m. values. A two-group comparison was performed by a Student's t-test. *p < 0.05, **p < 0.01.

7 Eosinophils Regulate Type 2 Immune Responses Following Infection with the Nematode Trichinella spiralis 301 카인의발현을분석한결과감염 2주후 BALB/c와 ΔdblGATA 모두 IL-5 발현이증가하여 Th2 반응이증폭되고있음을확인할수있었다 (Fig. 4A). ΔdblGATA 에서 BALB/c 대비 IL-5 발현이증가한경향성이나타났지만통계적유의성은없었다 (p = ). IL-6의경우 T. spiralis 감염에의해변화가발생하지는않았지만감염에관계없이 ΔdblGATA 에서 BALB/c보다높게발현되어호산구에의해 IL-6 생성에관여하는염증세포의활성이조절될가능성이높을것으로사료된다 (Fig. 4A). 호산구결핍마우스의장관에염증을유발하였을때대조군에비해호중구의침윤이현저하며 IL-6 등염증성사이토카인의발현이증가하는것으로알려져있다 (28). 또한호산구에의해증폭되는 IL-4/IL-13 신호전달에의해염증반응이억제되어대식세포와호중구의침윤및염증성사이토카인발현이억제된다는연구결과가최근에발표된바있다 (29). 따라서호산구결핍마우스에서관찰되는 IL-6의증가기전을확인하기위해 T. spiralis 감염시호산구와면역세포의상호작용을분석하는추가연구가필요할것으로사료된다. T. spiralis 근육침범기에서 transforming growth factor (TGF)-β 발현은염증반응과근육내의유충수를조절하는것으로알려져있다 (30). 즉유충분포와 TGF-β 발현은역의상관관계가있을것으로예상되는데감염 2주후 BALB/c 마우스파이어판에서 TGF-β 발현은감소하는경향이관찰되었고 ΔdblGATA 마우스의경우유의한수준의발현감소가확인되었다 (Fig. 4A). 감염 2주차는 T. spiralis 감염의장침범기에서근육침범기로의전환이일어나는시기이므로파이어판의면역반응양상과근육내유충의분포정도를확인하기위해유충의침범이쉽게발생하는횡격막의충체분포를분석하였다. 근육내의 T. spiralis 유충은염색체의구조가치밀해지며주변에투명대가형성되어근육과쉽게구분이되며현미경관찰을통해유충의수를관찰한결과 ΔdblGATA 에서유의한수준의유충수증가를확인할수있었다 (Fig. 4B). 즉호산구가존재하지않을경우 T. spiralis 장침범기에서파이어판의 Th2 면역반응이증폭되고혈청의 Th2형방어항체의역가가증가함에도불구하고근육침범기의충체수가적절히조절되지않는것으로사료된다. 이는호산구가단순히 Th2 염증반응을증폭시키기보다는조직침습기생연충의효율적인제거와이과정에서발성하는과도한 Th2 반응을조절시키는다양한기능을발휘하고있음을 시사하는결과이다. 숙주의면역반응과기생충의면역회피기전에의해숙주조직의염증성손상이최소화하는만성감염이성립된다 (14). 따라서 BALB/c와 ΔdblGATA 을이용하여감염초기면역반응을분석한본연구결과는감염시기에따라조직내분포가특징적으로변화하는 T. spiralis 감염증에서기생충과숙주상호작용을구체적으로분석하는후속연구에활용될수있을것이다. REFERENCES 1) Jung Y. Eosinophils are required for immune responses induced by oral immunization. J Bacteriol Virol 2015;45: ) Rothenberg ME, Hogan SP. The eosinophil. Annu Rev Immunol 2006;24: ) Yu C, Cantor AB, Yang H, Browne C, Wells RA, Fujiwara Y, et al. Targeted deletion of a high-affinity GATA-binding site in the GATA-1 promoter leads to selective loss of the eosinophil lineage in vivo. J Exp Med 2002;195: ) Jung Y, Rothenberg ME. Roles and regulation of gastrointestinal eosinophils in immunity and disease. J Immunol 2014;193: ) Rosenberg HF, Phipps S, Foster PS. Eosinophil trafficking in allergy and asthma. J Allergy Clin Immunol 2007;119: ) Lee TD. Helminthotoxic responses of intestinal eosinophils to Trichinella spiralis newborn larvae. Infect Immun 1991; 59: ) Kazura JW, Aikawa M. Host defense mechanisms against Trichinella spiralis infection in the mouse: eosinophilmediated destruction of newborn larvae in vitro. J Immunol 1980;124: ) Specht S, Saeftel M, Arndt M, Endl E, Dubben B, Lee NA, et al. Lack of eosinophil peroxidase or major basic protein impairs defense against murine filarial infection. Infect Immun 2006;74: ) Kita H. The eosinophil: a cytokine-producing cell? J Allergy Clin Immunol 1996;97: ) Jacobsen EA, Helmers RA, Lee JJ, Lee NA. The expanding role(s) of eosinophils in health and disease. Blood 2012; 120: ) Goh YP, Henderson NC, Heredia JE, Red Eagle A,

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α α α α α α α α 太陰調胃湯加減方 dbdb 마우스 肝에 대한 아디포사이토카인 및 발현에 미치는 영향 SREBPs 와 섞어서 하고 마커 또한 하여 전기 영동을 하였다 전기영동을 한 후에 에 를 쪼여서 각 를 확인하였다 이 를 프로그램을 이용해 수치화하 여 분석하였다 肝 조직 동결 절편 분리한 조직은 로 시간 동안 고정 시킨 후 에 세척한 후 물기를