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의학박사학위논문 천식유도된마우스에서예쁜꼬마선충 조항원의기도염증억제효과 Suppressive Effect of Crude Extracts of Caenorhabditis elegans on Airway Inflammation in Mice After Asthma Establishment 2012 년 10 월 서울대학교대학원 의학과박사과정 김성은
Suppressive Effect of Crude Extracts of Caenorhabditis elegans on Airway Inflammation in Mice After Asthma Establishment 천식유도된마우스에서 예쁜꼬마선충조항원의기도염증 억제효과 October, 2012 Department of Parasitology and Tropical Medicine, Seoul National University College of Medicine Sung Eun Kim
천식유도된마우스에서예쁜꼬마선충조항원의기도염증억제효과 지도교수최민호 이논문을의학박사학위논문으로제출함 2012 년 10 월 서울대학교대학원 의학과기생충학전공 김성은김성은의의학박사학위논문을인준함 2012 년 12 월 위원장 ( 인 ) 부위원장 ( 인 ) 위원 ( 인 ) 위원 ( 인 ) 위원 ( 인 )
Suppressive Effect of Crude Extracts of Caenorhabditis elegans on Airway Inflammation in Mice After Asthma Establishment by Sung Eun Kim A thesis submitted in partial fulfillment of the requirements for the Degree of Doctor of Science in Medicine at Seoul National University College of Medicine December 2012 Approved by Thesis Committee: Professor Professor Chairman Vice chairman Professor Professor Professor
학위논문원문제공서비스에대한동의서 본인의학위논문에대하여서울대학교가아래와같이학위논문제공하는것에 동의합니다. 1. 동의사항 1 본인의논문을보존이나인터넷등을통한온라인서비스목적으로복제할 경우저작물의내용을변경하지않는범위내에서의복제를허용합니다. 2 본인의논문을디지털화하여인터넷등정보통신망을통한논문의일부또는 전부의복제 배포및전송시무료로제공하는것에동의합니다. 2. 개인 ( 저작자 ) 의의무 본논문의저작권을타인에게양도하거나또는출판을허락하는등동의 내용을변경하고자할때는소속대학 ( 원 ) 에공개의유보또는해지를즉시 통보하겠습니다. 3. 서울대학교의의무 1 서울대학교는본논문을외부에제공할경우저작권보호장치 (DRM) 를 사용하여야합니다. 2 서울대학교는본논문에대한공개의유보나해지신청시즉시처리해야 합니다. 논문제목 : 천식유도된마우스에서예쁜꼬마선충조항원의기도염증억제효과학위구분 : 석사 박사 학 과 : 기생충학 학번 : 2008-30556 연락처 : 저작자 : 김성은 ( 인 ) 제출일 : 2012 년 12 월 28 일서울대학교총장귀하
초 록 서론 : 최근기생충감염과알레르기질환의유병률이반비례관계에있음이역학적연구나실험연구에서확인되고있다. 이연구는천식에이미이환된마우스에서예쁜꼬마선충의조항원 (crude extract of Caenorhabditis elegans, CEC) 이 ovalbumin (OVA) 으로유도된기도염증반응에어떠한영향을미치는지에대해알아보고자하였다. 방법 : BALB/c 마우스를 OVA로감작및자극시켜천식을유발한후다음네가지방법으로 CEC의천식완화효과를병리학적관찰, 기도과민성, 폐포세척액내염증세포수, 폐포세척액내싸이토카인분비양상, 혈청내총 IgE 항체가를측정하여관찰하였다. OVA로천식을유발할때 CEC를같이감작시킨후가장천식완화효과가좋은 CEC의농도와투여방법을결정하였고천식을유발시킨후 CEC를다양한농도와투여빈도로처치하여천식완화효과를관찰하였다. 또한천식이이미유발된마우스를계속 OVA로자극하면서 CEC를처치하였을때 CEC의천식완화효과를관찰하였다. i
결과 : 양성대조군인 OVA군에비해서 CEC를처치한실험군에서모두공통적으로기도주위염증세포, 특히호산구의침윤을비롯한병리소견이완화되었으며, methacholine에대한기도과민성이현저하게억제되었고 (P < 0.01), 폐포세척액내총염증세포의수와호산구의수도유의하게감소하였다 (P < 0.01). 또 IL-4와 IL-13 등 Th2 싸이토카인의분비가유의하게감소한반면 Th1 싸이토카인인 IL-12와 IFN-g의분비는증가하였으며 (P < 0.01), 혈청내총 IgE 항체가도유의하게감소하는양상을보였다 (P < 0.01). 천식완화에적합한 CEC 농도를찾고자한실험에서는가장높은투여농도인 50 mg을투여했을때좋은천식완화효과를보였으며, CEC 투여경로에상관없이천식완화효과를보였다. 천식에이미유도된마우스에 CEC 투여시최종투여용량이증가할수록천식완화효과도증가하였으나투여빈도에의한유의한차이는관찰되지않았다. 또, 천식이유도된마우스를 OVA로계속자극하는상태에서 CEC를투여하여도천식완화효과가있음을확인하였다. 결론 : 이연구결과예쁜꼬마선충의조항원이천식모델마우스에서 천식의유발을억제시킬수있을뿐아니라이미천식이유도된 상태에서도천식완화효과가있음을확인하였으며, 이러한효과는 Th1 ii
반응을증가시킴으로써 Th2 반응이억제되어유발되는것으로생각된다. ---------------------------------------------------------------------------------------------- 핵심단어 : 예쁜꼬마선충, 조항원, 기도염증, 기도과민성, 천식모델마우스, Th1 반응, Th2 반응 학번 : 2008-30556 iii
목 차 초록.i 목차..iv 그림목록 v 서론 1 실험재료및방법..10 결과.21 고찰.46 참고문헌 58 초록 ( 영문 ).70 iv
표및그림목록 Fig. 1. The schematic protocols for asthma induction in BALB/c mice and for investigation of the suppressive effect of C. elegans crude extract (CEC) on airway inflammation depending on doses (A) or administration routes (B) of CEC.19 Fig. 2. The schematic protocols for observation of the suppressive effect of C. elegans crude extract (CEC) on airway inflammation after establishment of asthma by OVA sensitization and challenge in BALB/c mice 20 Fig. 3. Histopathological observation of the lungs of mice sensitized with PBS, OVA, or OVA+CEC.24 Fig. 4. Effect of CEC on phenotype of asthma (A) Airway hyperresponsiveness to increasing concentration of methacholine presented as airway resistance. (B) Total cell counts in bronchoalveolar lavage (BAL) fluid collected from mice. (C) Differential cell counts of inflammatory cells in BAL fluid...25 Fig. 5. Effect of CEC treatment on cytokine production in BAL v
fluid.26 Fig. 6. Total IgE titers in sera of mice with OVA-induced airway inflammation 27 Fig. 7. Histopathological observation of the lungs of mice with OVAinduced asthma 30 Fig. 8. Effect of CEC on phenotype of asthma (A) Airway hyperresponsiveness to increasing concentration of methacholine. (B) Total cell counts in BAL fluid. (C) Differential cell counts in BAL fluid 31 Fig. 9. Effect of CEC treatment on cytokine production in BAL fluid according to the route of CEC administration 32 Fig. 10. Effect of CEC treatment on serum total IgE titers of mice with OVA-induced asthma according to the route of CEC administration 33 Fig. 11. Histopathological observation of the lungs of mice treated with different doses and intervals of CEC administration after establishment of asthma..36 Fig. 12. Effect of CEC on phenotype of asthma (A) Airway vi
hyperresponsiveness to increasing concentration of methacholine in experimental mice. (B) Total cell counts in BAL fluid. (C) Differential cell counts in BAL fluid 37 Fig. 13. Effect of CEC treatment on cytokine production in BAL fluid according to various treatment regimens after establishment of asthma in mice..38 Fig. 14. Serum total IgE titers in mice according to various treatment regimens after establishment of asthma in mice 39 Fig. 15. Histopathological observation of the lungs of mice with established asthma 42 Fig. 16. Effect of CEC on phenotype of asthma (A) Airway hyperresponsiveness to increasing concentration of methacholine. (B) Total cell counts in BAL fluid. (C) Differential cell counts in BAL fluid.43 Fig. 17. Effect of CEC treatment on cytokine production in BAL fluid of mice with established asthma.44 Fig. 18. Serum IgE titers in mice with established asthma 45 vii
서 론 면역반응은병원체감염에대한숙주방어작용에서중요한기전이며건강을유지하는데필수적이다. 그러나면역반응은때때로감염체와상관없이특정항원에의해유도되기도하며, 심각한질병을유발할수있다. 일반적으로알레르기성과민반응은꽃가루, 음식, 약물등과같이본래해롭지않은항원에대해민감하게반응하여일어나며, 이러한알레르기성과민반응의결과로천식, 알레르기비염, 아토피피부염과같은알레르기질환이발생한다. 천식, 알레르기비염, 아토피피부염과같은알레르기질환의유병률이최근수십년사이에기생충감염이드물거나성공적으로관리된지역또는선진국에서현저하게증가하였다 (1, 2). 천식과기생충, 특히연충 (helminth) 감염에서공통적으로관찰되는면역반응은 IgE 항체가의증가, 조직내호산구증가증, 점액분비의증가, IL-4, IL-5, IL-13과같은 Th2 싸이토카인의증가이다 (3). 그러나, 천식은 Th2 싸이토카인에의해활성화되는비만세포, 호산구로인한기도조직의물리적변화, 기도과민성증가, 기도내염증과같은증상이나타나는 - 1 -
반면, 연충에감염된사람에서는천식과같은특징적증상이나타나지않는차이점이있다. 비록알레르기질환과기생충감염이모두 Th2 면역반응을유도한다고알려져있지만최근연구결과에의하면기생충감염은알레르기질환이있는사람이나실험적으로천식이유도된마우스에서항염증반응을유발한다고보고되었다 (1, 3, 4). 기생충이많은후진국에서는알레르기질환의유병률이낮은역학적특징을보이는데, 이러한기생충감염과천식유병률사이의반비례관계를위생가설 (hygiene hypothesis) 로설명하고있다. 위생가설은어릴때위생적인환경에서자라거나항생제, 백신등을접하게될수록자연스럽게각종항원에노출됨으로써면역력이증가될수있는기회를잃게되어성장후항원에노출되면과민반응이유발되어천식및아토피피부염등과같은알레르기성질병들에이환된다는가설이다. 예를들면톡소포자충 (Toxoplasma gondii ) 과 Helicobacter pylori의항원에노출되었을때아토피피부염이유발될위험성이 60% 이상감소했다고한다 (5). 실제로독일과에디오피아에서는도심지에서아토피피부염의유병률이도시와떨어진지역보다월등히높은것으로알려졌다 (6). 이러한위생가설은기생충감염이천식과같은알레르기질환의증상을완화하거나억제할가능성을시사해주고있다 (7-10). - 2 -
위와같은위생가설을설명하기위해기생충감염과알레르기질환발병률의반비례관계에대하여연구한결과에의하면회충 (Ascaris lumbricoides) 에감염된사람들은회충에감염되지않은사람보다천명 (wheezing) 의생김이적고 (11) 만손주혈흡충 (Schistosoma mansoni) 유행지역에거주하는사람들이비유행지역에사는사람들에비해천식과같은폐질환의발병률이낮은것으로보고되었다 (7, 8). 가봉에서는방광주혈흡충 (Schistosoma haematobium) 에감염된아이들에서집먼지진드기에의한아토피성피부질환이감염되지않은아이들보다적게발병되는것이확인되었으며, 장기간장내기생충을치료하면집먼지진드기에대한아토피성반응이증가하는것으로확인되었다 (10). 숙주의면역반응을피하여숙주체내에오랜기간서식하는기생충의특성상기생충이숙주체내에서성공적인정착, 이동, 물질대사를위하여숙주의면역체계를조절한다고생각되며, 기생충의구성물질중숙주면역반응을조절하여스스로를보호하는기능을가진물질이있을것으로생각된다 (12). 또기생충에감염된사람이나동물에서천식증상이완화되는현상은기생충감염에의해면역관용 (immune tolerance) 이유도됨을시사하며 (11, 13), 면역관용은기생충감염시 - 3 -
증가하는 regulatory T cell( 조절 T 세포 ) 에의한면역조절효과때문일것으로도추측된다 (14). 많은역학연구와동물모델을이용한실험적인연구결과에의하면사람과동물모델에서기생충감염이천식을억제할수있는것으로보고되고있는데, 특히구충 (hookworms) 과같은장내선충류와주혈흡충감염이알레르기질환의위험도를유의하게감소시켜준다 (2, 15). 돼지편충의충란을사용하면궤양성대장염과크론병과같은염증성장질환을치료하는데효율적인것으로알려져있으며 (16, 17), 구충을천식환자에실험적으로감염시키면통계적으로유의하지는않지만대조군환자와비교하여천식환자의기도과민성을개선시키는효과가있음이보고된바있다 (18). 기생충항원외에도세균에서분비되는단백물질을천식유도마우스모델에처리했을시천식이억제된다는보고도있다 (19). 기생충감염시유도되는조절 T 세포는숙주의면역반응을억제함으로써기생충이숙주체내에서성공적으로생존할수있도록도와주는것으로추측된다 (20). 이렇게유도된조절 T 세포는기생충뿐만아니라숙주에도이로운결과를유도하는데, 주혈흡충증의병변진행을억제하거나알레르기증상을완화시킬수있다. 일본주혈흡충 (Schistosoma japonicum) 의충란항원에의해증가한 - 4 -
CD4+CD25+FoxP3+ T 세포는천식마우스모델에서천식증상을억제하는효과를보이며 (21), 장내선충인 Heligosomoides polygyrus를실험동물에감염시켰을때에도 CD4+CD25+FoxP3+ T 세포의증가와천식억제가확인된바있다 (22). 또, 기생충감염자에서비감염자에비해 IL-10의증가가관찰되는데, 이러한현상은 IL-10- secreting Treg이천식증상억제와관련이있음을시사한다 (23). 쥐의장내선충인브라질구충 (Nippostrongylus brasiliensis) 또한감염시 IL-10의분비를유도하여염증을완화시킨다고보고되었다 (13). 최근기생충의천식에대한영향을밝히기위한역학연구나실험연구에서브라질구충 (Nippostrongylus brasiliensis) 감염시분비되는항원이알레르기성기도염증반응을악화시킨다는결과도보고되고되었고이와는반대로돼지회충 (Ascaris suum) 에서분비되는항원은 IL-10 의존적으로수지상세포의기능을억제하여오히려점막성알레르기염증반응을완화시킨다는상반된연구결과도보고되고있다 (24, 25). 이와비슷한예로개회충 (Toxocara canis) 역시오히려 ovalbumin(ova) 으로유도된알레르기면역반응을악화시키는것으로알려져있다 (26). 동일기생충이라하더라도단백질의종류에따라상반된효과를보이기도하는데, 돼지회충 (Ascaris suum) 의조항원중 - 5 -
APAS-3(allergenic protein of A. suum) 는 Th2 면역반응을유도하여호산구성기도염증과기도과민성을유발하는반면, PAS- 1(suppressive protein of A. suum) 은오히려염증반응을억제한다고보고되었다 (27). 비슷한예로만손주혈흡충에감염된쥐의면역반응은충란의유무에따라서다르게나타난다. 즉, 숙주에게암컷과수컷주혈흡충을동시에감염시켰을때는 OVA로유도되는기도염증반응이더악화되는반면, 수컷충체만감염시켰을경우에는오히려기도과민성이억제되었다 (23). 이와같이기생충감염과알레르기질환유병률의관계는기생충의종 (species), 기생충에서분비되는물질, 감염빈도또는감염량, 감염시기등여러요인에따라다양한결과로관찰된다 (26, 28). 최근알레르기질환또는염증성질환치료에기생충감염을적용하고자하는시도가다양하게진행되고있다 (16-18, 29). 실제로돼지편충 (Trichuris suis) 의충란을염증성장질환 (Inflammatory Bowel Diseases, IBD) 환자에인위적으로감염시켜 IBD 증상을완화시킬수있음이보고된바있다 (16, 17). 그러나기생충을이용한치료요법 ( 연충치료요법, helminth therapy) 은기대하는치료효과외에면역억제, 과민반응, 알레르기항원과의상호작용등여러가지부작용이발생할수있다 (1). 한예로, 돼지편충의충란을사용하여치료받은크론병 - 6 -
환자에서돼지편충감염이확인되기도하였다 (30). 또 SPF(specific pathogen free) 인치료용기생충충란이나유충을만들기위한제작과정이복잡하고, 제작비용이비싸치료비용이증가할수있으며, 환자들이기생충자체를치료방법으로사용하는사실에대해심리적거부감을가질수있다. 이러한연충치료요법의제한점을극복하기위해기생충유래물질중항알레르기성또는항염증성기능을가진치료후보물질을선정하여작용기전과효과를관찰한후치료법으로사용하는방안을모색할수있다 (29). 이미동물모델이나임상실험에서몇몇의기생충에서유래된항염증물질이숙주의면역반응을조절하고염증완화에효과가있음을보고한연구결과가발표되고있다 (1, 29, 31). 사상충에서분비되는단백분해효소억제제인 cystatin은 OVA로유도된 Th2 반응을억제하는기능이있는데, 이러한기능은조절 T 세포가아닌대식세포와 IL-10에의존적인것으로알려져있다 (32). Cystain의면역억제기능은알레르기성질환뿐아니라황산염으로유도되는대장염의증상도감소시킨다 (33). 쥐의사상충 (Acanthocheilonema viteae) 에서분비되는 ES-62라는물질역시알레르기성염증반응을감소시키는항염증기능을가지는대표적인기생충유래물질중하나로알려져 - 7 -
있다. ES-62는피부와폐에서발생하는대식세포의존적인과민반응을조절하고 (34) 관절염역시억제시키는것으로확인되었다 (35). 이외에도개심장사상충 (Dirofilaria immitis) 에서유래된항원 (DiAg)(36) 과간질에서생성되는 thioredoxin peroxidase (37) 등과같이알레르기성질환을완화시킬것으로기대되는기생충유래물질들이보고되고있으나아직실제모델에적용을못한경우가많다. 천식은알레르기항원에의해직접유발되는 Th2 면역반응을동반하는복잡한질환으로서기도과민성과기도염증을유발한다 (38-40). 천식의주요치료법으로흡입코르티코스테로이드제를사용하거나함께지속성 b2-항진제등을사용하는방법이있지만약을규칙적으로투여하는등지속관리가필요하며중증천식에서사용할약제가많지않는등아직도해결되지못한부분이많이있다 (38). 천식이유발되는초기에 Th2 면역반응을유도하는알레르기항원을감소시키면증상이완화되는사실은 Th2 반응을억제또는조절하는물질을치료후보물질로사용할수있음을시사한다 (38). 최근 OVA 로천식을유발시킨마우스에서 예쁜꼬마선충조항원 (Crude extract of Caenorhabditis elegans, CEC) 이 - 8 -
천식완화효과를보임이보고된바있다 (41). 즉, 마우스에천식을유도하기위해 OVA로감작할때 CEC를같이처리하였을경우 Th2 싸이토카인의분비가억제되고 Th1 싸이토카인인 IL-12, IFN-g의분비가증가하였으며, 기도과민성과호산구의증가가억제되는등천식유발이억제됨이확인되었다 (41). 이러한결과는 CEC가천식의병리기전을억제함으로써천식의유발을억제하거나증상을완화할수있음을시사해준다. 그러나임상에서접하는대부분의천식환자들은이미질병이진행된상태에서치료를받기위해내원하는환자들임을감안할때 CEC가천식치료제로활용될수있기위해서는천식이이미확립된모델동물에서 CEC의천식억제효과를관찰하는것이중요하다. 따라서본연구에서는마우스에 OVA로천식을유도한후 CEC를처리하여기도염증발생을억제하고이미생긴천식을치유하는효과를관찰함으로써 CEC의천식증상에관한치료효과가있을것이라는가설을세우고이를증명하고자한다. - 9 -
실험재료및방법 1. 실험동물및실험군 실험동물은 7주령암컷 BALB/c 마우스를 KoaTech(Seoul, Korea) 에서구입하여사용하였다. 동물실험계획은서울대학교실험동물자원관리원 (IRB No. SNU-09-0115-4) 과서울대병원전임상실험실의승인 (IACUC No. 10-0211) 을받았고, 마우스는각각서울의대기초연구동의동물실험실과서울대병원의생명연구원전임상실험실에서 specific pathogen-free(spf) 상태로사육하면서실험하였다. 마우스는실험목적에따라서 3~5개의실험군에각각 5마리씩구성하였다. 실험 1(Exp. 1) 은 OVA를이용하여마우스에천식을유발시킬때다양한농도의 CEC를한달간처리하여천식을완화시키기에적합한 CEC의농도를찾고자실시하였다 (Fig. 1A). 실험 2(Exp. 2) 는실험 1에서확인한천식완화효과를가지는적정농도의 CEC를복강내주사와비강내주입, 두종류의다른방법으로투여하여천식완화에좀 - 10 -
더효과적인투여경로를찾고자하였다 (Fig. 1B). 실험 3(Exp. 3) 은동일실험기간내에투여되는 CEC의총량이천식완화효과와총량이같을경우어느시간간격으로투여하는것이천식완화에더효과적인지알아보기위해다양한시간간격으로투여방침을정해실험을하였다 (Fig. 2A). 마지막으로실험 4(Exp. 4) 에서는천식에이환된마우스를 OVA로지속적으로자극함으로써환자의실질적인상황과비슷한환경을만들고, 지속적인 OVA 자극하에서 CEC를투여하였을경우천식완화효과가있는지를확인하고자하였다 (Fig. 2B). 2. 예쁜꼬마선충조항원제작및준비 예쁜꼬마선충의조항원 (crude extract of C. elegans, CEC) 을제작하기위해예쁜꼬마선충 N2 주를 Escherichia coli OP50이포함된배지에서배양하였다. 18 o C를유지중인배양기에서 4기유충을 5마리씩새배지에옮겨 7일간배양한후동일한성장단계의충체을얻기위하여충란이포함된배지의일부를잘라새배지에옮겼다. 이후 3일간배양한뒤배지를 3차증류수로 5차례씻어내어동일한성장과정의성충을회수하였다. 회수한성충을 4 o C, 3,000 rpm으로 - 11 -
15분간원심분리하고 phosphate buffered saline(pbs) 을첨가하여회전식균질기와초음파분쇄기를사용하여분쇄하였다. 충체균질액은 4 o C, 15,000 rpm으로 20분간원심분리한후상층액을분리하고 Polymixin B(Pierce, Rockford, IL, USA) 를사용하여내독소 (endotoxin, LPS) 를제거하였다. 내독소제거처치후예쁜꼬마선충조항원에서측정된내독소농도는 0.1 mg의 CEC 당 1 ng 미만이었다. 또세균및바이러스의오염을배제하기위해 0.22 mm filter로여과하고분주하여 -70 o C에보관하였다. 3. 천식유발및 CEC 투여 BALB/c 마우스에천식을유발시키기위해서 OVA(Sigma, St. Louis, MO, USA) 75 mg을 3 mg의 aluminum hydroxide(alum, Sigma) 와혼합하여실험첫날과 7일째에복강내로주입하였다. 실험 14, 15, 21일째에 OVA 50 mg을비강내로투여하였고, 23일째에 Flexivent(Scireq, Montreal, Canada) 를이용하여기도과민성을측정하여천식이성공적으로유도되었는지를확인하였다. 이후실험목적에따라서천식에이환된마우스를실험목적에따라각각 - 12 -
3~5 개실험군으로나누어실험에사용하였다. 음성대조군의경우에는 OVA 대신 PBS 로실험첫날과 7 일째에복강내로주사하여감작하였고, 실험 14, 15, 21 일째에비강내로 PBS 를투여하여자극하였다. 실험 1은음성대조군과양성대조군을제외한세군의실험군마우스에다양한농도의 CEC(CEC10군 : 10 mg, CEC25군 : 25 mg, CEC50군 : 50 mg) 를 OVA와함께처리한후기도과민성, 폐포세척액내염증세포수및싸이토카인분비양상과천식의병리학적, 면역학적소견을관찰하여세종류의 CEC 농도중천식완화효과를보이는적정농도를결정하였다 (Fig. 1A). 실험 2는투여경로에따른 CEC의천식완화효과를비교하기위해실험 1에서결정된 CEC의적정농도를마우스의복강내 (CECip군) 또는비강내 (CECin군) 로투여하여 CEC의천식완화효과를관찰하였다 (Fig. 1B). 실험 3에서는천식증상을효과적으로완화시킬수있는 CEC 투여간격을결정하기위해천식이발생된실험군마우스에적정농도의 CEC를투여한후천식병리의완화정도를관찰하였다 (Fig. 2A). 실험 2에서투여경로에관계없이 CEC효과를보여복강내자극을실시하였다. PBS군을제외한나머지마우스를 OVA로감작및자극하여 - 13 -
천식을유도한후기도과민성을측정하여천식이확실히유도되었음을확인한후천식이유도된마우스를 6개의실험군으로나누어한달간동량의 CEC를처리할시어떤시간간격으로 CEC를투여했을때천식완화에효과적이었는지확인하고자하였다. 음성대조군 (PBS군) 은 PBS로감작및자극을실시한후한달동안 PBS를매주 1회씩처치하였으며, 양성대조군 (OVA군) 은 OVA로천식을유도시킨후 OVA로한번의재감작을실시하였다. CEC W25군은매주 1회 CEC 25 mg을한달간투여하여총 100 mg을투여하였고, CEC M100군은 CEC 100 mg을한번투여하여한달뒤천식완화효과를관찰하였다. CEC W25*2군은매주 2회 CEC 25 mg을한달간투여하여총 200 mg을투여하였고, CEC W50군은매주 1회 CEC 50 mg을한달간투여하여총 200 mg을투여하였다. CEC W50*2군은매주 2회 CEC 50 mg을한달간투여하여총 400 mg을투여하였고, CEC W100군은매주 1회 CEC 100 mg을한달간투여하여총 400 mg을투여하였다. 실험 4에서는실험 3과같은실험방법으로마우스에천식을유도한뒤천식에이환된마우스를다음한달동안 OVA로지속적으로자극하는상태에서 CEC를투여하였을경우천식완화효과가있는지확인하였다 (Fig. 2B). PBS군은음성대조군으로서 PBS로자극하였고, - 14 -
OVA군은양성대조군으로천식이완전히이환된후 OVA로계속자극하였다. OVA+CEC군은처음한달간 OVA로천식을유도한뒤다음 4주동안매주 2회씩 OVA 50 mg과 CEC 25 mg을같이처리하였다. CEC군은천식유도후 4주동안매주 2회씩 CEC 25 mg만처리하였다. 4. 기도과민성측정 OVA로감작시킨마우스에서천식이유도되었는지확인하기위하여 OVA로마지막자극을실시한다음날혹은치료목적으로 CEC를투여하기전날 methacholine을농도별로처리하여기도과민성을측정하였다. 마우스를 zoletile(virbac, Carros, France) 로마취한후기도를확보하여 flexivent 시스템 (Scireq) 과연결한후기도를통해 PBS를폐에분사시켜기본값을측정하였다. 이후 methacholine의농도를 6.25, 12.5, 25 mg/ml 순서로점차증가시키며폐에분무시켜 methacholine 유발성호흡장애를측정함으로써마우스의기도과민성을측정하였다. - 15 -
5. 폐포세척액수집과세포수측정 폐포세척액 (bronchoalveolar lavage fluid) 을얻기위해마우스를 zoletile로마취시킨다음마우스의기관지에카테타를삽입, 고정한후 0.5 ml의 PBS로세번씩세척하여최종적으로 1.4~1.5 ml의폐포세척액을모았다. 이후 4 o C, 1,200 rpm으로 10분간원심분리한후상층액은싸이토카인측정을위해 -70 o C에서보관하였고, 침전물은 100 ml의 PBS를섞은후혈구계산기를이용하여전체세포수를계수하였다. 또혈구감별계산을하기위하여 cytospin을이용해슬라이드위에세포를붙인뒤 giemsa 염색을하여염증세포비율을측정하였다. 6. 폐포세척액내싸이토카인분비측정 폐포세척액내싸이토카인분비량은 sandwich ELISA kit(ebioscience, San Diego, USA) 를사용하여각각 IL-4, IL-5, IL- 12p70, IL-13, 그리고 IFN-g 정량을측정하였다. 각각의싸이토카인측정한계수준은 IL-4, IL-5, IL-13은 4 pg/ml, IL-12p70과 IFN-g는 15 pg/ml이었다. - 16 -
7. 혈청내총 IgE 항체가측정 마취된마우스의심장천자를통해채취한혈액을원심분리하여 혈청을모아 -20 o C 에보관하였다. 혈청내총 IgE 항체가를 sandwich ELISA(Bethyl, Montgomery, USA) 를통해측정하였다. 8. 병리학적관찰 마우스에서폐를제거하여 10% phosphate-buffered formalin에 24시간고정시킨후고정된조직을파라핀으로포매한다음 3~5 mm의두께로조직절편을제작하였다. 준비된조직절편을 hematoxylin과 eosin으로염색한뒤폐조직의염증, 염증세포의침윤등병리학적소견을광학현미경으로관찰하였다. 9. 통계분석 - 17 -
모든자료는 student t-test 를사용하여실험군사이에통계적 유의성을비교하였으며, 95% 신뢰구간 (P < 0.05) 또는 99% 신뢰구간 (P < 0.01) 에서통계학적유의성을판정하였다. - 18 -
Fig. 1. The schematic protocols for asthma induction in BALB/c mice and for investigation of the suppressive effect of C. elegans crude extract (CEC) on airway inflammation depending on doses (A) or administration routes (B) of CEC. Mice were sensitized with PBS, OVA, or CEC mixed with aluminum hydroxide on days 0, 1 and then boosted on day 7. Then, they were challenged with OVA by intranasal injection on days 14, 15, 21 and 22. Airway hyper-responsiveness (AHR) to various concentrations of methacholine was measured on day 23. After 24 hr, bronchoalveolar lavage (BAL) fluid and blood were collected from anesthetized mice, and lungs were isolated for histological observation. Each group was consisted of 5 mice. Arrows, OVA sensitization; arrow heads, OVA challenge; open circle, measurement of AHR; filled circle, sacrifice of mice. - 19 -
Fig. 2. The schematic protocols for observation of the suppressive effect of C. elegans crude extract (CEC) on airway inflammation after establishment of asthma by OVA sensitization and challenge in BALB/c mice. (A) Suppressive effect of CEC was investigated in asthma-induced mice according to the various doses and intervals of CEC administration. (B) Suppressive effect of CEC was also investigated in mice after establishment of asthma using various doses and intervals of CEC administration. Asthma was established in mice by sensitization and challenge with OVA using the same protocol in Fig. 1A, and mice were treated with different doses or intervals of CEC administration according to the experimental schemes; arrow heads, challenge with PBS, OVA and/or CEC; open circle, measurement of AHR; filled circle, sacrifice of mice. - 20 -
결 과 1. CEC 의천식억제효과및적정농도확인 천식에이환된마우스에다양한농도의 CEC를처리하여천식완화에적합한 CEC 농도를찾고자하였다 (Fig. 1A). 각실험군마우스의폐조직을관찰한결과 PBS군에서는염증반응이관찰되지않은반면, OVA군에서는주로호산구로구성된염증세포들이기관지주변으로현저하게침윤되어있는것을관찰하였다 (Fig. 3). 그러나 OVA와 CEC를함께감작한세군의실험군 (CEC10군, CEC25군, CEC50군 ) 에서는양성대조군인 OVA군에비해염증세포의침윤이현저하게감소되었다 (Fig. 3). 실험 23일째에다양한농도의 methacholine을처리하여실험군 마우스의기도과민성을측정한결과 OVA군은 methacholine 농도가 증가할수록 기도과민성이 증가하였다 (Fig. 4A). CEC를 처리했을 경우에는 methacholine 농도가증가할수록기도과민성도증가하긴 하였으나 OVA군의경우에비해유의하게감소되었으며 (P < 0.01), CEC의농도가높을수록기도과민성의증가도현저하게억제되었다. 즉 methacholine 최고농도인 25 mg/ml에서 CEC10군은 4.7±0.4, - 21 -
CEC25 군은 3.0±0.6, CEC50 군은 2.3±0.5 의기도과민성을보였으며, CEC50 군은음성대조군인 PBS 군과비슷한수준의기도과민성을 보였다 (Fig. 4A). 폐포세척액내의염증세포를계수한결과 OVA군에서는 16.0±0.15 x 10 6 로 PBS군의 3.8±0.1 x 10 6 에비하여현저하게증가되어있음을확인하였다 (Fig. 4B, P < 0.01). OVA와 CEC를함께감작한세군의실험군 (CEC10군, CEC25군, CEC50군 ) 에서는기도과민성의경우와같이 CEC 농도가증가할수록염증세포의수가감소하여, CEC10군은 10.4±0.1 x 10 6, CEC25군은 7.0±0.1 x 10 6, CEC50군은 3.7±0.2 x 10 6 로계수되었다. 염증세포에대한감별계수결과양성대조군인 OVA군에서관찰된염증세포중호산구가주요세포임을확인하였으며, PBS군과 CEC를처리한세실험군에서모두호산구의수가현저하게감소하였다 (Fig. 4C, P < 0.01). CEC의천식억제현상을이해하기위해폐포세척액내의싸이토카인분비량을측정하여비교한결과 OVA군에서는 Th2 싸이토카인인 IL- 4와 IL-13의분비량이 PBS군에비하여유의하게증가하였다 (Fig. 5, P < 0.01). 반면, Th1 싸이토카인중 IL-12는 OVA군에서 PBS군에비하여유의하게감소하였으나 IFN-g의분비량은증가하였다 (Fig. 5, P - 22 -
< 0.01). CEC를 OVA와함께감작시킨세군의실험군 (CEC10군, CEC25군, CEC50군 ) 에서는 OVA군과비교하여 Th2 싸이토카인의분비가현저하게감소하였고 (P < 0.01), Th1 싸이토카인의분비는유의하게증가하였다 (P < 0.01). 이러한현상은 CEC의농도가증가할수록더확연하게관찰되었다. 마우스혈청내총 IgE 항체가는 OVA 군에서 26.7±1.5 ng/ml 로다른실험군에비해높은항체가를보였다 (Fig. 6). 반면 PBS 군에서는 1.3±0.9 ng/ml 로가장낮은항체가를보였고, CEC 농도가높을수록총 IgE 항체가가 OVA 군과비교하여점차낮아지는양상을보여 CEC10 군은 16.0±7.7 ng/ml, CEC25 군은 9.7±5.2 ng/ml, CEC50 군은 4.0±1.5 ng/ml 의항체가를보였다. - 23 -
Fig. 3. Histopathological observation of the lungs of mice sensitized with PBS, OVA, or OVA+CEC. Infiltrations of inflammatory cells around the airways were remarkably decreased in CEC-treated mice compared to that in OVA-sensitized and challenged mice. PBS (n=5), mice sensitized with PBS and challenged with OVA; OVA (n=5), mice sensitized and challenged with OVA; CEC10 (n=5), mice sensitized with OVA and CEC 10 mg, and challenged with OVA; CEC25 (n=5), mice sensitized with OVA and CEC 25 mg, and challenged with OVA; CEC50 (n=5), mice sensitized with OVA and CEC 50 mg, and challenged with OVA. (X100) - 24 -
Fig. 4. (A) Airway hyper-responsiveness to increasing concentration of methacholine presented as airway resistance. (B) Total cell counts in bronchoalveolar lavage (BAL) fluid collected from mice. (C) Differential cell counts of inflammatory cells in BAL fluid. Numbers of eosinophils were significantly reduced by treatment with various doses of CEC. Data from individual mice are presented as arithmetic means in histograms. PBS (n=5), mice sensitized with PBS and challenged with OVA; OVA (n=5), mice sensitized and challenged with OVA; CEC10 (n=5), mice sensitized with OVA and CEC 10 mg, and challenged with OVA; CEC25 (n=5), mice sensitized with OVA and CEC 25 mg, and challenged with OVA; CEC50 (n=5), mice sensitized with OVA and CEC 50 mg, and challenged with OVA. *, P<0.01 vs. OVA group. - 25 -
Fig. 5. Effect of CEC treatment on cytokine production in BAL fluid. In contrast to OVA-treated mice, productions of Th2 type cytokines such as IL-4 and IL-13 were significantly decreased, but those of Th1 type cytokines such as IL-12 and IFN-g were significantly increased in BAL fluid of CEC-treated mice. The effect of CEC on cytokine production depended on the dose of CEC. Data from individual mice are presented as arithmetic means in histograms. PBS (n=5), mice sensitized with PBS and challenged with OVA; OVA (n=5), mice sensitized and challenged with OVA; CEC10 (n=5), mice sensitized with OVA and CEC 10 mg, and challenged with OVA; CEC25 (n=5), mice sensitized with OVA and CEC 25 mg, and challenged with OVA; CEC50 (n=5), mice sensitized with OVA and CEC 50 mg, and challenged with OVA. *, P<0.01 vs. OVA group. - 26 -
Fig. 6. Total IgE titers in sera of mice with OVA-induced airway inflammation. The production of total IgE was significantly decreased by CEC treatment, which was positively correlated with the dose of CEC. Data from individual mice are presented as arithmetic means in histograms. PBS (n=5), mice sensitized with PBS and challenged with OVA; OVA (n=5), mice sensitized and challenged with OVA; CEC10 (n=5), mice sensitized with OVA and CEC 10 mg, and challenged with OVA; CEC25 (n=5), mice sensitized with OVA and CEC 25 mg, and challenged with OVA; CEC50 (n=5), mice sensitized with OVA and CEC 50 mg, and challenged with OVA. *, P<0.01 vs. OVA group. - 27 -
2. 투여경로에따른 CEC 의천식병변완화효과 실험 2에서는마우스를 OVA로감작시켜천식을유도할때같이주입되는 CEC의투여경로에따른천식억제효과를관찰하였다 (Fig. 1B). OVA군에서는 PBS군에비해기관지주변으로염증세포의침윤이현저하게증가되어있었으나 OVA와 CEC를함께감작한 CECin(intranasal injection) 군과 CECip(intraperitoneal injection) 군의경우투여경로에상관없이두군모두 OVA군에비해염증세포의침윤이현저하게감소되었다 (Fig. 3). Methacholine에대한기도과민성역시 OVA군에서는 methacholine 농도가증가할수록증가하여최고농도인 25 mg/ml에서 8.9±0.3의기도과민성이관찰되어다 (Fig. 8A). 그러나 CEC를처리했을경우에는투여경로에상관없이 CECin군과 CECip군모두기도과민성의증가도현저하게억제되어음성대조군인 PBS군과비슷한수준을보였다. 즉 methacholine 최고농도인 25 mg/ml에서 PBS군은 2.4±0.6, CECin군은 2.5±0.3, CECip군은 2.3±0.3의기도과민성이관찰되었다 (Fig. 8A). CEC투여경로에따른폐포세척액내염증세포의수역시 CEC를두가지다른경로로처리했을시 OVA군의 16.0±0.9 x 10 6 개에비하여유의하게감소하여 CECin군은 5.7±1.2 x 10 6, CECip군은 5.7±0.9 x - 28 -
10 6 개의세포가계수되었다 (Fig. 8B, P < 0.01). OVA 군에서관찰된염증 세포의대부분을차지하는호산구역시 OVA 군에비해 PBS 군과 CEC 를 처리한실험군에서모두현저하게감소하였다 (Fig. 8C, P < 0.01). 싸이토카인분비양상도 OVA군에서는 IL-4와 IL-13의분비량이 PBS군에비하여유의하게증가한반면 (Fig. 9, P < 0.01), IL-12는 PBS군에비하여유의하게감소하였으나 IFN-g의분비량은증가하였다 (Fig. 9, P < 0.01). 그러나 CEC를 OVA와함께감작시킨 CECin군과 CECip군의경우투여경로에상관없이두군모두 OVA군에비해 Th2 싸이토카인의분비가현저하게감소하였으며 (P < 0.01), Th1 싸이토카인의분비는유의하게증가하였다 (P < 0.01). 총 IgE 항체가는 OVA군에서 924.5±240.9 ng/ml로가장높게관찰되었으나 PBS군, CECin군과 CECip군모두 OVA군에비해유의하게감소되었다 (Fig. 10, P < 0.01). - 29 -
Fig. 7. Histopathological observation of the lungs of mice with OVAinduced asthma. Infiltrations of inflammatory cells around the airways were significantly decreased in CEC-treated mice compared to that in mice with OVA-induced asthma, irrespective of administration route of CEC: intraperitoneal or intranasal injection. PBS (n=5) mice sensitized with PBS and challenged with OVA; OVA (n=5), mice sensitized and challenged with OVA; CECin (n=5), mice sensitized with OVA together with CEC 50 mg by intranasal injection and challenged with OVA; CECip (n=5), mice sensitized with OVA together with CEC 50 mg by intraperitoneal injection and challenged with OVA. (X100) - 30 -
Fig. 8. (A) Airway hyper-responsiveness to increasing concentration of methacholine. (B) Total cell counts in BAL fluid. (C) Differential cell counts in BAL fluid. Data from individual mice are presented as arithmetic means in histograms. PBS (n=5) mice sensitized with PBS and challenged with OVA; OVA (n=5), mice sensitized and challenged with OVA; CECin (n=5), mice sensitized with OVA together with CEC 50 mg by intranasal injection and challenged with OVA; CECip (n=5), mice sensitized with OVA together with CEC 50 mg by intraperitoneal injection and challenged with OVA. *, P<0.01 vs. OVA group. - 31 -
Fig. 9. Effects of CEC treatment on cytokine production in BAL fluid according to the route of CEC administration. Data from individual mice are presented as arithmetic means in histograms. PBS (n=5) mice sensitized with PBS and challenged with OVA; OVA (n=5), mice sensitized and challenged with OVA; CECin (n=5), mice sensitized with OVA together with CEC 50 mg by intranasal injection and challenged with OVA; CECip (n=5), mice sensitized with OVA together with CEC 50 mg by intraperitoneal injection and challenged with OVA. *, P<0.01 vs. OVA group. - 32 -
Fig. 10. Effects of CEC treatment on serum total IgE titers of mice with OVA-induced asthma according to the route of CEC administration. Data from individual mice are presented as arithmetic means in histograms. PBS (n=5) mice sensitized with PBS and challenged with OVA; OVA (n=5), mice sensitized and challenged with OVA; CECin (n=5), mice sensitized with OVA together with CEC 50 mg by intranasal injection and challenged with OVA; CECip (n=5), mice sensitized with OVA together with CEC 50 mg by intraperitoneal injection and challenged with OVA. *, P < 0.01 vs. OVA group. - 33 -
3. CEC 투여방법에따른기도염증완화효과 실험 3은한달동안마우스를 OVA로감작및자극시켜천식을유발시킨후다음한달동안 CEC를처리하여천식완화효과가있는지를알아보고, 한달동안투여한총 CEC의양과투여방법에따른천식완화효과를관찰함으로써천식완화에가장효율적인 CEC 투여방법을찾고자하였다 (Fig. 2A). 폐조직에서병리학적관찰을실시한결과 CEC를처치한실험군에서 OVA군에비해염증세포의침윤이현저하게감소되긴하였으나여전히기도주위염증이지속되고있었다 (Fig. 11). 또, 한달간투여총량이같은 CEC W50*2군과 CEC W100군의병리학적소견이큰차이를보이지않았다 (Fig. 11). Methacholine 최고농도인 25 mg/ml에서 PBS군의기도과민성은 2.4±0.0이었고, OVA군은 12.9±0.6이었다 (Fig. 12A). CEC를투여한실험군에서는 CEC의투여용량을늘릴수록기도과민성억제효과가더컸으며, 한달간투여한 CEC 최종농도가같은경우에는투여빈도는기도과민성억제에유의한차이를보이지않았다 (Fig. 12A). 즉한달간총 100 mg의 CEC를투여한 CEC W25군과 CEC M100군의기도과민성은각각 3.5±SD, 4.4±0.5이었으며, 한달간총 200 mg의 CEC를투여한 CEC W25*2군과 CEC W50군의기도과민성은 4.7±SD 및 3.1±SD이 - 34 -
었다. 또한달간총 400 mg 의 CEC 를투여한 CEC W50*2 군과 CEC W100 군의기도과민성은각각 2.3±0.7, 2.5±1.1 이었다. 폐포세척액내염증세포의수역시 OVA군의염증세포수에비해 CEC를투여한실험군에서한달간투여된 CEC의총용량에비례하여감소하였으나투여빈도는유의한차이를유발하지못하였다 (Fig. 12B). 호산구역시 OVA군에비해 CEC를투여한실험군에서모두현저하게감소하였다 (Fig. 12C, P < 0.01). 싸이토카인분비양상도 OVA군에서관찰된 Th2 싸이토카인 (IL-4, IL-13) 의증가와 Th1 싸이토카인 (IL-12, IFN-g) 의감소현상이 CEC 를투여한실험군에서는정반대로관찰되어 Th2 싸이토카인의감소와 Th1 싸이토카인의증가양상을관찰할수있었다 (Fig. 13). 혈청내총 IgE 항체가역시 OVA군에비해 CEC를투여한실험군에서는유의하게감소하였다 (Fig. 13, P < 0.01). - 35 -
Fig. 11. Histopathological observation of the lungs of mice treated with different doses and intervals of CEC administration after establishment of asthma. PBS (n=5) mice challenged with PBS biweekly after establishment of asthma; OVA (n=5), mice challenged with OVA biweekly after establishment of asthma; CEC M100 (n=5), mice challenged with CEC 100 mg once after establishment of asthma; CEC W50*2 (n=5), mice challenged with CEC 50 mg two times a week for one month after establishment of asthma; CEC W100 (n=5), mice challenged with CEC 100 mg weekly for one month after establishment of asthma. (X200) - 36 -
Fig. 12. (A) Airway hyper-responsiveness to increasing concentration of methacholine in experimental mice. (B) Total cell counts in BAL fluid. (C) Differential cell counts in BAL fluid. PBS (n=5) mice challenged with PBS biweekly after establishment of asthma; OVA (n=5), mice challenged with OVA biweekly after establishment of asthma; CEC W25 (n=5), mice challenged with CEC 25 mg weekly after establishment of asthma; CEC M100 (n=5), mice challenged with CEC 100 mg once after establishment of asthma; CEC W25*2 (n=5), mice challenged with CEC 25 mg two times a week for one month after establishment of asthma; CEC W50 (n=5), mice challenged with CEC 50 mg weekly after establishment of asthma; CEC W50*2 (n=5), mice challenged with CEC 50 mg two times a week for one month after establishment of asthma; CEC W100 (n=5), mice challenged with CEC 100 mg weekly for one month after establishment of asthma. *, P < 0.01 vs. OVA group. - 37 -
Fig. 13. Effect of CEC treatment on cytokine production in BAL fluid according to various treatment regimens after establishment of asthma in mice. PBS (n=5) mice challenged with PBS biweekly after establishment of asthma; OVA (n=5), mice challenged with OVA biweekly after establishment of asthma; CEC W25 (n=5), mice challenged with CEC 25 mg weekly after establishment of asthma; CEC M100 (n=5), mice challenged with CEC 100 mg once after establishment of asthma; CEC W25*2 (n=5), mice challenged with CEC 25 mg two times a week for one month after establishment of asthma; CEC W50 (n=5), mice challenged with CEC 50 mg weekly after establishment of asthma; CEC W50*2 (n=5), mice challenged with CEC 50 mg two times a week for one month after establishment of asthma; CEC W100 (n=5), mice challenged with CEC 100 mg weekly for one month after establishment of asthma. *, P < 0.01 vs. OVA group. - 38 -
Fig. 14. Serum total IgE titers in mice according to various treatment regimens after establishment of asthma in mice. PBS (n=5) mice challenged with PBS biweekly after establishment of asthma; OVA (n=5), mice challenged with OVA biweekly after establishment of asthma; CEC W25 (n=5), mice challenged with CEC 25 mg weekly after establishment of asthma; CEC M100 (n=5), mice challenged with CEC 100 mg once after establishment of asthma; CEC W25*2 (n=5), mice challenged with CEC 25 mg two times a week for one month after establishment of asthma; CEC W50 (n=5), mice challenged with CEC 50 mg weekly after establishment of asthma; CEC W50*2 (n=5), mice challenged with CEC 50 mg two times a week for one month after establishment of asthma; CEC W100 (n=5), mice challenged with CEC 100 mg weekly for one month after establishment of asthma. *, P < 0.01 vs. OVA group. - 39 -
4. 천식이환후 OVA 노출시 CEC 의효과 실험 4는천식에이환된마우스가 OVA 항원에계속자극받는환경에서 CEC를투여하였을때천식완화효과가있는지를확인하고자실시하였다 (Fig. 2B). CEC를 OVA와함께또는단독으로천식에이환된마우스에투여했을때 OVA군에서관찰되었던염증세포의기도주위침윤은현저하게감소하였으나, 염증반응이여전히지속되고있음이관찰되었다 (Fig. 15). 그러나기도과민성은 OVA군에비해유의하게감소하여 (P < 0.01) methacholine 25 mg/ml에서 OVA와 CEC를함께 4주간투여한 OVA+CEC군은 3.2±0.8이었다 (Fig. 16A). 또, CEC만단독으로투여한 CEC군은 2.4±0.1로 PBS군 (2.0±0.0) 과비슷한수준의기도과민성을보였다. 폐포세척액내염증세포의수도 CEC를 OVA와함께또는단독투여한두실험군에서모두 OVA군에비해유의하게감소하였으며 (Fig. 16B, P < 0.01), 염증세포중호산구수도유의하게감소한반면단핵구세포가증가하는양상을보였다 (Fig. 16C). Th2 싸이토카인의분비량은 CEC를 OVA와함께또는단독투여한두실험군에서모두 OVA군에비해유의하게감소하였으나 (Fig. 17, P < 0.01), Th1 싸이토카인의경우에는 CEC를단독투여한 CEC군에서만 OVA군에비해유의하게증가하였다 (Fig. 17, IL-12, P < 0.01; IFN-g, - 40 -
P < 0.05) 또, 혈청내총 IgE 항체가는 CEC 를 OVA 와함께또는단독 투여한두실험군모두 OVA 군에비해유의하게감소하였다 (Fig. 18, P < 0.01). - 41 -
Fig. 15. Histopathological observation of the lungs of mice with established asthma. PBS (n=5) mice challenged with PBS after establishment of asthma; OVA (n=5), mice challenged with OVA after establishment of asthma; OVA+CEC (n=5), mice challenged with OVA+CEC after establishment of asthma; CEC (n=5), mice challenged with CEC after establishment of asthma. (X200) - 42 -
Fig. 16. (A) Airway hyper-responsiveness to increasing concentration of methacholine. (B) Total cell counts in BAL fluid. (C) Differential cell counts in BAL fluid. Data from individual mice are presented as arithmetic means in histograms. PBS (n=5) mice challenged with PBS after establishment of asthma; OVA (n=5), mice challenged with OVA after establishment of asthma; OVA+CEC (n=5), mice challenged with OVA+CEC after establishment of asthma; CEC (n=5), mice challenged with CEC after establishment of asthma. *, P < 0.01 vs. OVA group. - 43 -
Fig. 17. Effects of CEC treatment on cytokine production in BAL fluid of mice with established asthma. Data from individual mice are presented as arithmetic means in histograms. PBS (n=5) mice challenged with PBS after establishment of asthma; OVA (n=5), mice challenged with OVA after establishment of asthma; OVA+CEC (n=5), mice challenged with OVA+CEC after establishment of asthma; CEC (n=5), mice challenged with CEC after establishment of asthma. *, P < 0.01 vs. OVA group. **, P < 0.05 vs. OVA group. - 44 -
Fig. 18. Serum IgE titers in mice with established asthma. Data from individual mice are presented as arithmetic means in histograms. PBS (n=5) mice challenged with PBS after establishment of asthma; OVA (n=5), mice challenged with OVA after establishment of asthma; OVA+CEC (n=5), mice challenged with OVA+CEC after establishment of asthma; CEC (n=5), mice challenged with CEC after establishment of asthma. *, P < 0.01 vs. OVA group. - 45 -
고 찰 본연구에서는천식억제효과를가진다고보고된구충과같은기생충을실험대상으로사용하는대신자유생활을하는예쁜꼬마선충의조항원을대상물질로선택하여실험을진행하였다. 예쁜꼬마선충은생활사를실험실에서쉽게유지할수있고, 생명체중맨처음으로전게놈서열이밝혀지는등신경과학, 생태발생학, 노화등과같은다양한연구분야에서매우유용하게이용되고있는자유생활선충이다 (42). 예쁜꼬마선충은수명이대략 2~3주로길지않고 OP50이라는대장균이포함된액체또는아가배지에서배양이용이하므로실험실내생활사영위가쉽지않은기생충보다는실험에사용될대상물질을다량으로쉽게얻을수있는장점이있다. 또전유전자서열이규명되어있기때문에예쁜꼬마선충의물질중염증억제효과를가진물질을찾을수있다면, 재조합단백질제작등후속연구가훨씬더원활해질것으로기대된다. 이번연구결과에서예쁜꼬마선충의조항원 (CEC) 이천식마우스 모델에서 OVA 에의해유도되는천식의발병을효과적으로억제할뿐만 - 46 -
아니라이미천식이발병한마우스에서도천식완화효과가있음을관찰하였다. 이동물모델에서유발되는숙주면역반응을관찰한결과양성대조군인 OVA군에서는기관지주위의염증세포의침윤, 호산구- 매개천식의특징적인소견인기도과민성증가, 폐포세척액내총세포수및호산구의증가, Th2 싸이토카인의증가, 알레르기성천식환자들에게서확인되는대표적인현상인혈청내총 IgE 항체가의증가등이관찰되었으나, CEC를처치한실험군에서모두공통적으로기도주위염증세포의침윤을비롯한병리소견이많이완화되었으며, methacholine에대한기도과민성이현저하게억제되었고, 폐포세척액내총염증세포의수와호산구의수도유의하게감소하였다. 또 IL-4와 IL-13 등 Th2 싸이토카인의분비가유의하게감소한반면 Th1 싸이토카인인 IL-12와 IFN-g의 분비는증가하였으며, 혈청내총 IgE 항체가도유의하게감소하는양상을보였다. 급성천식을유도하면서 OVA와 CEC를함께처리했을때 CEC의농도의존적으로천식유도가억제되었고 CEC의투여경로에상관없이천식완화효과를보였다. 또한천식을먼저유도한마우스에치료목적으로 CEC를처리하였을때는투여되는최종용량이증가할수록천식완화효과도증가하였고, 마우스가천식에이미이환된후 OVA로계속자극받는상황에서 CEC를투여하여도천식완화효과가있음을확인하였다. - 47 -
기도과민성은천식의특징중대표적인것으로규명되어있다 (43). 본연구에서는현재기도과민성등기도의기능을평가하기위해서주로이용되고있는 penh method와 flexivent system을이용한측정방법중 flexivent system을이용하여측정하였다. 그이유로과거에는 penh method가비용을최소화하고시간을절약하며희생되는마우스수를줄일수있고, 사용하기에도용이해서보편적으로폐기능측정에사용되었으나 (44, 45) 최근 penh 값이기도저항성을항상정확하게반영하는것은아니라는증거들이밝혀지면서기도과민성을더욱더정확하게측정할수있는방법의필요성이대두되었고 (44, 45) flexivent system은기도에직접관을삽입한후컴퓨터를이용하여기도과민성을측정하기때문에기도과민성을정확하게측정할수있는방법으로인정되고있다. 한편폐의염증세포중특히호산구의증가는기도과민성을증가시키는데중요한역할을한다고보고되었다 (46). 본연구결과, 양성대조군에서관찰되었던폐포세척액내의호산구증가와기도과민성의증가가 CEC를처리했을시현저하게감소하는것을확인하였다. 또한폐조직의병리학적소견을관찰하였을때기관지주변의염증세포의침윤이현저하게감소한것이확인되었다. 이는 CEC가천식표현형의대표적인증상인염증세포즉, 호산구증가와그로인해유도되는기도과민성을억제함으로써천식을완화시킨다는 - 48 -
것을시사할수있다. IL-4는 Th2 반응을대표하는싸이토카인으로 B세포로부터의 IgE 생성을활성화시키고호산구의침윤을촉진하며미경험 T세포를 Th2 면역반응관련세포로분화를유도하는등염증반응에서매우중요한역할을한다고알려졌다 (47). IL-13 역시 IL-4와함께 Th2 반응에있어대표적인싸이토카인이다. 본연구에서 CEC를처리하였을시 OVA로유도된천식의특징적인반응인 Th2 싸이토카인 IL-4와 IL- 13의분비양상이억제되는것을확인하였다. 반면 CEC를처리한실험군모두 Th1 싸이토카인인 IL-12와 IFN-g가증가하였다. 이번연구에서시료물질로사용된예쁜꼬마선충에서분비되는단백분획중하나인 cystatin은사람의말초혈액단핵구의 IFN- g와 IL-12 생산을증가시키고카텝신 B를억제하여 Th1 세포의발현을활성화시킨다고알려졌다 (48). 이러한연구결과는예쁜꼬마선충의조항원이 Th1 반응을강화하여 Th2 반응으로인해유발되는알레르기성기도염증반응을억제할수있을것이라는가능성을제시해준다 (49). 이러한추론은최근에발표된연구결과에의해증명되었는데, 예쁜꼬마선충의조항원을천식모델마우스에 OVA와함께감작시켰을때천식의유발이억제됨이확인되었다 (41). - 49 -
IgE는알레르기성천식을유도하고발전시키는데매우중요한역할을하는요소로알려져있다 (50). 혈청내증가된 IgE는비만세포와호염기성세포표면에있는 FcεReceptor I에결합하게되고 (51) 이러한결합으로인해싸이토카인과케모카인으로잘알려진히스타민, 프로스타글란딘, 류코트라이엔의분비를촉진한다 (52, 53). 이렇게분비된히스타민은염증을유도하는싸이토카인과 B세포의항체를생성하는데영향을미친다고알려졌다 (54). 본연구에서 CEC를처리했을시양성대조군에비교하여혈청내총 IgE의항체가가현저하게감소한것을확인하였다. 이번결과역시 CEC의투여가천식의대표적인증상인 IgE의증가를억제하여천식을완화시킨다는것을증명해주며 IgE 증가와관련된싸이토카인인 IL-4의감소결과역시이결과와부합한다는것을설명하고있다. 본연구에서예쁜꼬마선충배양시 OP50이라는대장균을영양원으로사용하기때문에예쁜꼬마선충조항원을제작할때내독소 (LPS) 의오염가능성을배제할필요가있다. 따라서조항원을제작할때충체를증류수로 5차례이상세척한후균질액을만들었으며, 내독소제거젤을사용하여 CEC에오염되었을지도모르는내독소를최대한제거하고자하였다. 이후 CEC 내의내독소농도를측정한결과 - 50 -
마우스감작시사용되는 10 mg 의 CEC 내에 1 endotoxin unit/mg 미만임을확인하여내독소가본실험결과에미치는영향은무시해도 좋은것으로판단되었다. 천식모델마우스에서기도과민성, 기도주위염증, 호산구증가, IgE 항체가의증가등천식의다양한표현형이지속되는기간은표현형의종류에따라차이가있다. 한연구결과에의하면 BALB/c 마우스에서 OVA로감작과자극을통해천식을유발한뒤 5, 7, 9, 12주후에다시 OVA로자극하여기도과민성, 호산구증가등다양한천식의표현형을관찰한결과기도과민성은첫 OVA 자극시점으로부터 7주까지유지된반면 (55) 호산구증가, 기도염증, 혈청내 OVA-특이 IgE 항체가증가등의표현형은첫자극으로부터 12주까지도지속됨이관찰되었다 (55). 이러한연구결과를바탕으로본연구에서는천식에이환된마우스에서최소 4주동안은기도과민성, 기도주위염증, 호산구증가, IgE 항체가증가등천식의표현형이지속될것으로생각하여실험 3에서마우스에 OVA를이용하여천식을유발시킨후한달동안추가적인 OVA 자극이없는상태에서 CEC의천식완화효과를관찰하였다. 그결과한달간투여된 CEC의총용량에비례하여천식의표현형이모두감소하였으나투여빈도는유의한차이를유발하지는못하였다. - 51 -
천식은알레르기항원에대한 Th2 반응이과도하게활성화되어유발되는질환이므로 Th1 반응을강화시키는방법으로알레르기성기도염증반응을억제할수있을것으로기대된다 (38). 따라서 Th1 반응을강화하는전략이알레르기성기도염증반응을개선하기에좋은치료법으로사용될수있다. BALB/c 마우스에천식을유발할때 OVA 감작 3일전부터감작하는동안 IFN-g를기도에분무하면 OVA-특이 IgE 항체의감소, OVA-특이 IgG2a의증가, OVA에대한피부반응감소와함께정상폐기능이관찰되었다 (44). 또, 1차 OVA 감작으로인해염증반응이잘유도된 26일 ~30일째에 IFN-g를처리하였을경우에도 OVA-특이 IgE 반응이억제되고, OVA-특이 IgG2a가증가하면서기도과민성유도가억제되었다 (44). 이러한결과는 IFN-g를기도에전달함으로써설령 OVA에의한 1차감작이성공적으로유도되었을지라도알레르기항원에대한이차감작을막아줄수있음을의미하며, 이러한효과에 CD4+ T 세포가주역할을할것으로판단된다 (56). 천식에대한 IFN-g의억제효과는 IFN-g knockout 마우스를사용한실험에서도증명되고있다. IFN-g knockout 마우스를 OVA로감작및자극하고 8주후에관찰한결과기도과민성, 폐포세척액내호산구증가, 폐조직내에 CD4+ T 세포가증가하였으나재조합 IFN- 을 OVA 자극후 4주째에시작하여 1주일동안치료하면정상 - 52 -
마우스수준으로회복되었다 (57). 이결과는 IFN- g 가알레르기항원으로인해유도된기도염증과기도과민성을정상수준으로회복하는데에중요한역할을함을의미하며, 천식을비롯한알레르기질환의치료에 IFN- g 를사용할수있음을시사해준다. 본연구에서 CEC가천식의전형적인면역반응인 Th2 싸이토카인의증가와 Th1 싸이토카인의감소현상을역전시켜천식완화효과를보이는데, IFN- g 를비롯한 Th1 면역반응이천식억제에매우중요한역할을함을시사한다. OVA로천식이유도된 OVA군에서폐포세척액내염증세포가 PBS군에비해현저하게증가하였는데주로호산구로구성되어있었다. 그러나실험 1~3에서 CEC를처치한실험군마우스에서는폐포세척액의염증세포가유의하게감소하긴하였으나, 염증세포중대부분이호산구대신대식세포로구성되어있었다 (Figs. 4C, 8C, 12C). 폐포내의대식세포는폐의선천면역반응에중요한역할을하는세포이지만, IFNg 와 IL-12와같은물질을분비하거나다른세포와직접상호작용함으로써알레르기성천식을억제할수있을것으로제안되고있기도하다 (58). OVA로감작시킨마우스에서폐포대식세포를제거하면 Th1 반응이억제되고 Th2 반응이더유도되어항원의비강내자극으로인해염증반응과기도과민성이심해진반면, 반대로 - 53 -
대식세포를이입해주면 Th1 반응을선택적으로촉진하고호산구증가를유발하지않음이보고된바있다 (59). 본연구에서 CEC를처리한실험군에서폐포세척액내호산구가감소하고대식세포가증가함으로써 IL-12 분비가증가하고 Th1 반응이유도되어천식의유발과진행을억제했을것으로추측되나대식세포의역할에대해서는추후연구를통해규명할필요가있다고생각된다. 기생충감염이알레르기성질환의발달을억제하는면역학적메커니즘을관찰할때 IL-10이나조절 T세포의변화가관찰되는데마찬가지로대식세포의활성또는면역억제성 B세포가포함된다 (1). 예를들면마우스에선충의하나인 Acanthocheilonema viteae 가감염되었을때분비되는시스타틴은마우스를알레르기성기도염증으로부터보호하고소듐덱스트란설페이트로유도된대장염에서는조절 T세포는변화가없지만대식세포에의한 IL-10의생산을 증가시켜질환을완화시킨다 (33). IL-10 에의해조절된알러르겐 유도성기도호산구증가는마우스에 OVA를기도감작시키기 4주혹은 8주전에 N. brasiliensis를감염시키면감소한다 (13). 그러나선행연구에서 CEC를처리한천식모델마우스그룹의폐포세척액내에서 IL-10의증가는관찰되지않은것으로볼때이과정에서 IL- - 54 -
10 이 CEC 에의한천식유발억제기전에중요한역할을하지않을 것으로추측된다. 향후연구에서는 CEC 에의해천식의유발이억제되는 기전에서 IL-10 의역할을명확히밝히는것이필요하다고생각된다. 이연구에서예쁜꼬마선충의조항원이급성천식을유도하면서 CEC를함께처리했을때와천식을먼저유도한마우스에치료목적으로 CEC를처리했을경우모두 Th2 반응을 Th 1반응으로유도하면서알레르겐특이적인 Th2 반응을억제한다는점이관찰되었다. 그러나기생충항원의종류에따라 Th1과 Th2의균형조절은다르게나타난다. 예를들어 A. viteae의물질중하나인 ES-62는기생충에서얻을수있는면역조절분자중최고로꼽히며 (1, 31) 마우스에서기도염증을억제시키고 (34) 콜라겐으로인해유도되는관절염에서는콜라겐특이적인초기염증 Th1 싸이토카인의분비를억제한다 (35). 반면돼지회충의두가지단백분획은마우스에실험적으로유도시킨천식에서반대되는영향을미치는결과를보인다고알려졌다 (27). 알레르기를유발하는단백분획인 APAS-3은기도내호산구침윤을유발하며 IL-4와 IL-5, 그리고 AHR 유도와같은 Th2 반응을활성화시키는반면억제단백분획인 PAS-1은 APAS-3으로유도된기도염증을감소시킨다 (27, 60). 본연구에서명확하게밝히지는 - 55 -
못했지만 CEC내에도기도염증을억제하는구성요소가있을것이라예상된다. 천식을억제시키는 CEC 내의후보물질로는프로테아제억제제인시스테인, 포스포콜린이포함된단백질, 글리칸과당지질등이있다 (29, 33). 향후연구에서는 CEC에서천식을억제하는물질이무엇인지명확하게밝혀내기위하여후보단백질을포함한여러물질로실험을진행할필요가있다. 이연구에서예쁜꼬마선충의조항원이마우스천식모델에서알레르기기도염증을완화시킨다는것을처음밝혔다. 그러나동물모델에서적용했던이러한결과를사람의질병에적용하여해석할때에는주의가필요하고, 마우스에게서발견되었던현상을사람의천식질환에응용하여시도하기까지는더많은연구가뒷받침되어야한다. 그럼에도불구하고본연구에서관찰된예쁜꼬마선충조항원의천식증상완화효과는기생충유래물질에의해유도되는숙주면역조절현상을이해할수있는기초지견을제공하였고, 예쁜꼬마선충조항원중천식완화기능을가진물질을더검색하여규명한다면천식질환을조절할수있는새로운후보물질을개발할수있는가능성을시사한다고판단된다. 본연구결과예쁜꼬마선충의조항원이천식모델마우스에서천식의 - 56 -
유발을억제시킬수있을뿐아니라이미천식이유도된상태에서도천식완화효과가있음을확인하였으며, 이러한효과는예쁜꼬마선충의조항원에의하여 Th1 반응이증가됨으로써천식으로유도된 Th2 반응이상대적으로억제되는것으로생각된다. - 57 -
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