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1 J Korean Soc Transplant 2009;23: DOI: /jkstn Review Article 이종이식거부반응의조절 : 이종췌도이식을중심으로 서울대학교의과대학의학연구원장기이식연구소 1, 서울대학교병원장기이식센터및장기이식연구소 2, 서울대학교의과대학신장내과및장기이식연구소 3 김화정 1 ㆍ양재석 2 ㆍ안규리 3 Current Strategies for Successful Islet Xenotransplantation Hwajung Kim, Ph.D. 1, Jaeseog Yang, M.D. 2 and Curie Ahn, M.D. 3 Transplantation Research Institute, Seoul National University Medical Research Center 1, Transplantation Center Seoul National University Hospital, Transplantation Research Institute, Seoul National University Medical Research Center 2, Division of Nephrology, Transplantation Research Institute, Seoul National University Medical Research Center 3, Seoul, Korea Diabetes mellitus is increasing all over the world and is a serious health problem. Pancreatic islet transplantation is promising treatment for diabetes mellitus, but an imbalance between deceased pancreas donors and recipients limited the widespread clinical application. Therefore, pig islets could be used as an alternative islet source in transplantation. However, a big hurdle to clinical application of islet xenotransplantation is the instant blood mediated inflammatory reaction (IBMIR), which is characterized by activation of the coagulation cascade, platelets and complement systems. Innate immune cells infiltrate the islets in the process of IBMIR and thereby accelerate the early graft loss. Characteristics of IBMIR in islet xenotransplantion are very different from the rejection in solid organ xenotransplantation. Therefore, we focus on the molecules for surmounting IBMIR in order to accomplish successful islet xenotransplantation. To prevent the IBMIR in islet xenotransplantation, development of genetic modified pigs containing anti-coagulant, anti-thrombosis and complement regulatory genes, or capsulation of islet with biomaterials for blocking immune response around islet surface can be tried. Gα-Gal knockout pigs and the diverse transgenic pigs for complement regulatory protein or anti-coagulant genes have been developed for xenotransplantation. This review summarized on characteristics of rejection in islet xenotransplantation and discusses the strategies for overcoming the rejection. Key Words: Islet xenotransplantation, Instant blood mediated inflammatory reaction (IBMIR), Genetically modified pig, Diabetes mellitus 중심단어 : 이종췌도이식, 급성혈액매개성염증반응, 형질전환돼지, 당뇨병 이종췌도이식의역사 당뇨병은당대사장애이외에도말기신부전증, 망막질환, 말초혈관질환, 신경질환등다장기합병증이수반되어환자의삶의질을낮추고, 많은경제적부담을갖게한다. 수명의증가와식생활의변화로당뇨병의유병률이전세계적으로크게증가하고있고, 특히아시아지역의유병률은서구사회보다도빠르게증가하고있다.(1) 국내당뇨병유병률은 2005년조사결과 30세이상성인에서당뇨병유병률은 9.1% (256만명추정 ) 이며남자가 책임저자 : 안규리, 서울시종로구대학로 103 서울대학교의과대학신장내과및장기이식연구소, Tel: , Fax: curie@snu.ac.kr 접수일 : 2009 년 12 월 28 일, 게재승인일 : 2009 년 12 월 28 일 10.2% 로여자 7.9% 에비해더높고연령이증가함에따라유병률은더증가하였다.(2) 당뇨병의치료는경구혈당강하제및인슐린을이용한치료방법과췌장및췌도를이용한이식이있다. 경구혈당강하제는췌장에서인슐린분비를증가시키거나인슐린에대한내성을개선시켜서제2형당뇨병초기에치료제로서이용이가능하나, 인슐린분비가되지않는제1형당뇨병과진행된제2형당뇨병의치료에는이용이제한적이다. 인슐린치료제는다양한인슐린제형이개발되어많이사용되고있지만, 잦은인슐린의투여로당뇨병환자의삶의질이감소되며, 저혈당발생으로인한위험증가가동반될뿐아니라, 인슐린치료만으로는합병증을근본적으로예방하지못하여우리나라의경우에도최근당뇨병성신병증이말기신부전증의주요원인으로그빈도는 45% 에달한다 (2007년도대한신장학회통계 ). J Korean Soc Transplant December 2009 Volume 23 Issue 3

2 1990년 Scharp 등 (3) 은혈당조절을위하여인슐린치료가필요한당뇨병환자들에게공여자의췌장을췌도단위로분리한후이식하는방법, 즉췌도이식을시도하였다 (Fig. 1). 췌도이식은췌장이식에비해시술이간단하고수술합병증이적으며반복적인이식이가능하다는장점을가지고있어서, 제1형당뇨병환자에적용가능한유망한치료법이다.(4) 2000년에이르러 Shapiro (5) 에의해동종췌도이식의방법이임상적으로응용할수준으로까지상당히개선되었다. 그러나한명의당뇨병환자를치료하기위해서는두명이상의뇌사자로부터췌도를공급받아야하지만뇌사자장기의공급에는제한이있으므로급증하는이식수요를충족시키는데한계가있다. 그러므로췌도의안정적인공급이가능한방법으로써돼지의췌도를이용한이종췌도이식에대한관심이급증하고있다.(6) 한편이종이식은장기이식분야를중심으로발전하였다. 이종장기나세포의공급원으로는이종장기이식이처음시도되던 1963년경에는바분원숭이와같은영장류가많이사용되었다. 특히이종이식의아버지라불리는 Reemtsma 는침판지의신장을신부전환아에게이식하여최장 9개월의생존을유지하였으며 (7) 1984년에는 Bailey에의해서선천성심장질환이있는 Baby Fae에게바분원숭이의심장을이식하여 20일간유지한바있다.(8) 그러나영장류는멸종위기종인데다가번식이어렵고, 이종장기공급원으로활용하기에는사회적거부감이높으며, 높은감염의위험성을가지고있어서, 검토결과미니돼지가적절한이종장기공급원으로선정되었다. 미니돼지는사람과같은크기의돼지로서장기의크기가사람과유사하고, 임신기간이 127일로번식이용이하며, 사람과오랜시간을같이지낸동물이어서사람에게치명적인감염원을보유할가능성이낮다. 또한, 형질전환무균사육이가능하다는장점을가지고있을뿐아니라, 돼지의인슐린은아미노산염기서열이사람과 85.5% 가유사하기때문에장기뿐아니라췌도공급원으로서돼지의활용가능성역시매우높게평가되어서이종췌도이식은자연스럽게돼지췌도를대상으로개발되었다. 한편, 돼지의장기를인체에이식할경우, 동종이식보다훨씬심각한이종이식거부반응이일어난다. 이러한이종이식거부반응을극복하고이식초기의급격한이종장기손상을방지하여안정적인이식기능을유지하도록하기위하여, 면역거부반응을조절하여면역관용을유도하거나장기의생존율증가를유도할수있는유전자를삽입또는제거한형질전환돼지의개발이필수적이다. 본종설에서는이종이식시에발생하는거부반응의특성 과이를극복하기위해개발된다양한형질전환돼지및그한계점에대해알아보고, 성공적인이종췌도이식을위하여어떠한추가적인전략이필요한지에대해기술하고자한다. 이종이식거부반응의 3단계이종장기이식을하는경우에는동종이식보다강하고빠른거부반응이일어나는데, 이종장기이식거부반응은발생순서에따라초급성거부반응 (hyperacute rejection, HAR), 급성혈관성거부반응 (acute vascular rejection, AVR), 급성세포매개성거부반응 (cell mediated rejection, CMR) 으로분류한다 (Fig. 2). 초급성거부반응은돼지에존재하는항원에대해인간이이미가지고있는자연항체 (xenoreactive natural antibody, XNR) 가반응함에따라발생하는거부반응이다. 이거부반응은 ABO 혈액형부적합장기이식에서관찰되는급격한초급성거부반응과유사해서이식후수분또는수시간내에이식된장기혈관에혈전이생성되고괴사에이르는심한손상이발생한다. 이초급성거부반응의주원인은 (1) 돼지내피세포에다량존재하는알파갈 (Galactose-α 1,3-Galactose, αgal) 에대한자연항체반응과 (2) 혈청보체조절인자 (complement regulatory proteins, CRPs) 의분자호환성결여에따른과도한혈청보체활성화에의한손상이다. 인간은돼지가가지고있는알파갈전이효소 (α-1,3-galactocyltransferase) 가없기때문에돼지에존재하는당단백질인알파갈에대한자연항체를가지고있어서, 돼지의장기가인간에게이식되었을경우격렬한항원-항체반응이일어난다. 이항원-항체반응이일어나면혈청보체가활성화되면서 2차적인조직손상이일어나는데, 혈청보체활성화를조절하는혈청보체조절인자가인간과돼지사이에종간호환성이없기때문에혈청보체활성화가지속적으로발생하여조직손상이가속화된다. 초급성거부반응을극복하기위해 human decay accelerating factor (hdaf) 유전자등의인간혈청보체억제유전자를발현시키거나,(9) 알파갈항원을결손시킨돼지가제작되었다.(10) 초급성거부반응은혈관접합이이루어지는고형장기이종이식에서는격렬하게나타나지만성체돼지의췌도에는알파갈발현이매우적은데다가, 장기이식에서처럼혈관을직접연결하지않기때문에췌도이식에서의초급성거부반응은다행히매우약한편이다. 급성혈관성거부반응은혈관내피세포가 2차적으로활성화되어선천성면역세포의침윤과함께혈액응고현상이나타나는것이특징이다. 알파갈이결손된상황이라 J Korean Soc Transplant December 2009 Volume 23 Issue 3

Fig. 1. Treatment of diabetes mellitus strength & weakness. Fig. 2.

(A) Hyperacute rejection: Hyperacute rejection of pig organs in humans is induced by binding of preformed human

Complement is activated by antibody binding, and triggers lysis of endothelial cells.

and host antibodies, macrophages, and platelets.

numbers of T cells), intravascular thrombosis, and fibrinoid necrosis of vessel walls.

3 Fig. 1. Treatment of diabetes mellitus strength & weakness. Fig. 2. Three stages of graft rejection in xenotransplantation. (A) Hyperacute rejection: Hyperacute rejection of pig organs in humans is induced by binding of preformed human 'natural' antibody, which is generally directed against Gala1-3Gal that exists on the surface of pig cells. Complement is activated by antibody binding, and triggers lysis of endothelial cells. (B) Acute vascular rejection: Acute vascular rejection is induced by interactions between the graft endothelial cells and host antibodies, macrophages, and platelets. The response is characterized by an inflammatory infiltrate of mostly macrophages and natural killer cells (with small numbers of T cells), intravascular thrombosis, and fibrinoid necrosis of vessel walls. (C) Acute cellular rejection: Cellular rejection is caused by cellular immunity i) Natural killer cells, which accumulate in and damage the xenograft, and ii) T-lymphocytes, which are activated by MHC molecules through both direct and indirect xenorecognition. Abbreviations: MHC, major histocompatibility complex. J Korean Soc Transplant December 2009 Volume 23 Issue 3

4 고해도알파갈이외의돼지특이항원이존재하므로이에대한항체반응이미약하게유발되고, 허혈성손상, 분자적합성의결여등에의해서도혈관내피세포가활성화되어섬유소 (fibrin) 가침착되고, 선천성면역반응계세포의침윤이일어난다. 이를극복하기위해서는선천성면역반응조절과함께항응고유전자를과발현시키거나, 섬유소분해를촉진하는유전자를도입한형질전환돼지의개발이필요하다. 본연구팀은급성혈관성거부반응유도에돼지 CD40 (pcd40) 와사람 CD40 수용체 (hcd40l) 사이의역할을검토하였다. 췌도이식을포함한 (11) 대부분의이종장기이식에서는항-CD154 항체가사용되어왔으나, 그작용기전에대해서는정확히밝혀지지않았다. 돼지혈관내피세포의 pcd40를자극하면, NFκB 신호전달체계를통해서돼지의주조직복합체 (swine leukocyte antigen, SLA) 와접착분자의발현이증가하는한편, 특히 monocyte chemoattractant protein-1 (MCP-1), regulated upon activation, normal T cell expressed and secreted (RANTES), IL-10 같은 chemokine 분비가현저히증가하여이식한조직내로중성구, 단핵구등염증세포가침윤하게된다. (12) 그뿐아니라혈관내피세포의 Fgl2 발현이증가되어 (13) 혈액응고반응을촉진시킨다. 따라서이종이식거부반응에서의 pcd40-hcd40l 상호작용은이종이식의급성혈관성거부반응에서관찰되는선천성면역거부반응의주요매개기전이므로이를효과적으로차단하는것이이종이식장기생존에중요한역할을할것으로추정된다. 가장후기에관찰되는급성세포매개성거부반응은동종이식에서흔하게관찰되는세포매개성거부반응과유사하며, 임파구등면역세포에의해발생하는거부반응으로선천성면역반응보다는후천성면역반응이주로관여하며, 이식후부터수개월까지발생하는거부반응이라고알려져있다. 그러나이종이식시의급성세포매개성거부반응은동종이식보다강력할것으로예상된다. 예를들면, 혈관연결이필요없고, 알파갈항원이모두존재하는 rat to mouse 사이에서이종피부이식을해보면, 이종피부이식의생존기간이 major histocompatibility complex (MHC) 부적합마우스간의동종피부이식보다생존기간보다짧아짐을볼수있다. 그원인을살펴보면, 이종이식편에는보다심한중성구와단핵구의침착이관찰되며, IP-10, MCP-1, RANTES 등 chemokine의발현이동종이식에서보다현저히증가되어있고 (14) 매우심한 inducible nitric oxide synthase (inos) 의활성화가관찰된다.(15) 그리고돼지의 RANTES(16), IP-10(17) 는돼지와사람사이에분자서열이유사하여염증반응유도에관여 할것으로추정된다. 그뿐아니라 pcd86-hcd28 차단만으로는 T-림프구활성화를충분히차단할수없어서 (18) 이종이식에서의 T-림프구활성화와침윤과정에도역시선천성면역반응세포나 chemokine, 항산화분자같은면역매개반응이추가적으로관여할것으로추정되며, 이를극복하기위해서는다른차원의면역억제제개발이추가로필요할것이다. 이종췌도이식거부반응이종췌도이식에서는이종장기이식에서관찰되는초급성거부반응은미약하고, 급성혈관성거부반응은유사하게발생하지만, 혈액응고반응이심한급성혈액매개성염증반응 (instant blood mediated inflammatory reaction, IBMIR) 이라고하는독특한거부반응이관찰된다 (Fig. 3). IBMIR에관여하는기전은크게 (1) 혈액응고시스템의활성화및혈소판활성화 (2) 혈청보체의활성화로인한세포용해 (3) 선천면역세포의췌도내침윤등으로구분할수있다. IBMIR은이식된췌도가혈액에직접노출될경우, IgG, IgM 항체가췌도표면에결합하여혈청보체와조직인자 (tissue factor, TF) 를활성화하고, 활성화된조직인자에의해트롬빈이형성되고, 이와같은혈액응고계및혈소판, 혈청보체활성화는결국췌도주위로혈액응고와섬유소침착, 염증매개세포의침윤을유발하여췌도가급격히파괴되는현상으로서 (19) 역시조직인자가핵심역할을하는혈액응고계와선천면역반응이주로관여하지만, 일부저산소증같은비면역적요인에의해서도진행된다.(20) 이러한 IBMIR는이식초기췌도의급격한소실을초래하므로이를조절하기위해서는 IBMIR 를조절할수있는항응고, 항혈소판, 항보체, 항염증유전자가도입된돼지의개발이추가로필요할것으로생각된다. 1) 혈액응고 1999년에 Bennet는췌도를간문맥으로이식할경우, 췌장이식에비해이식성적이월등히떨어지는것을확인하였고, 이러한원인은췌도와혈액사이에발생하는염증반응때문임을최초로보고하였다.(21) 그후이들은사람이나돼지의췌도에동종의혈액을흘려주었을때, 혈소판이활성화되고선천면역세포들이췌도내로침윤하며, 췌도주변에혈액이응고되는것을관찰하였다. 이는췌도를특히간문맥으로이식할때혈액응고시스템과혈소판의활성화를피할수없음을보여준다. 또한돼지의췌도를영장류에간문맥으로이식한후 2일째에간을 J Korean Soc Transplant December 2009 Volume 23 Issue 3

Fig. 3. Putative model for the IBMIR. (A) In contact with blood, IgG and IgM antibodies bind to the islet surface and activate complement which leads to deposition of C3b/iC3b to the surface.

Thrombin subsequently generates fibrin and activates platelets. (C) Platelet activation increases the affinity of the integrins GPIIb-IIIa and a2b1 for fibrin and collagen, respectively.

(D) Amplified by platelets, thrombin generates more fibrin creating a capsule containing platelets, PMNs, and monocytes surrounding the islets. Chemotactic factors (e.g., C5a and IL-8) that were released as a consequence of IBMIR or released directly from the islets (e.

Abbreviations: IBMIR, instant blood mediated inflammatory reaction; IL-8, Interleukin-8; PMNs, polymorphonuclear leukocytes; MCP-1, monocyte chemoattractant protein-1. Source: Nilson B.

5 Fig. 3. Putative model for the IBMIR. (A) In contact with blood, IgG and IgM antibodies bind to the islet surface and activate complement which leads to deposition of C3b/iC3b to the surface. (B) Tissue factor (TF) activates the coagulation system via the extrinsic pathway. As a consequence of extrinsic pathway activation, prothrombin is cleaved into thrombin. Thrombin subsequently generates fibrin and activates platelets. (C) Platelet activation increases the affinity of the integrins GPIIb-IIIa and a2b1 for fibrin and collagen, respectively. Activated platelets bind to fibrin and collagen on the islet surface. (D) Amplified by platelets, thrombin generates more fibrin creating a capsule containing platelets, PMNs, and monocytes surrounding the islets. Chemotactic factors (e.g., C5a and IL-8) that were released as a consequence of IBMIR or released directly from the islets (e.g., MCP-1, IL-8 etc.), exert their action on PMNs and monocytes that infiltrate the islets in large numbers after 30 min. Abbreviations: IBMIR, instant blood mediated inflammatory reaction; IL-8, Interleukin-8; PMNs, polymorphonuclear leukocytes; MCP-1, monocyte chemoattractant protein-1. Source: Nilson B. The instant blood-mediated inflammatory reaction in xenogeneic islet transplantation. Xenotransplantation 2008;15:96-8. p.97. 액응고 조절에 관여하는 주요 유전자로는 tissue factor pathway inhibitor (TFPI), TM, Antithrombin (AT), CD39 (ectoatpase, CD39) 등이 있다. 이 중 TFPI는 FVIIa와 FXa에 결합하여 조직인자에 의해 유도되는 혈액응고시스 템의 초기단계를 방어하게 된다.(24) 분리된 췌도의 표면 에는 조직인자가 다량 노출되어 있으므로, 췌도 표면에 TFPI의 발현을 높여줄 경우 외인성 응고 기전을 방어하 는데 효과적일 수 있다. 그러나 췌도 표면에 노출된 조직 인자와 인간 혈액내 순환하는 FVIIa와의 결합에 의해 혈 액응고시스템이 활성화되어 트롬빈이 소량이라도 활성화 생검하였을 때, 췌도 주변에 혈전이 형성되고 췌도의 괴 사가 일어난 것이 관찰됨으로써, 이종이식의 경우 혈액응 고계의 활성화가 더욱 격렬할 것임을 시사하였다.(22) 한편, 혈액응고시스템은 외인성 경로와 내인성 경로에 의해 활성화되며(Fig. 4), 혈액 내 순환하는 혈청 단백질 또는 효소원에 의해 철저하게 조절된다.(23,24) 췌도를 간문맥으로 이식했을 경우, 췌도 표면에 잔류되어 있는 콜라젠과 음전하를 띤 유전자에 의해 내인성 응고반응이 진행될 뿐만 아니라, 분리된 췌도의 표면에 노출된 조직 인자로 인한 외인성 응고반응이 모두 작용하게 된다. 혈 J Korean Soc Transplant December 2009 Volume 23 Issue 3

6 Fig. 4. Schematic representation of the coagulation pathways. 'a' present the activated clotting factors. Briefly, coagulation is initiated when islet-expressed tissue factor (TF) is exposed to the blood in IBMIR. TF then complexes with VIIa and enhances its activity. This sequence of coagulation activation is known as the extrinsic pathway. The complex of VIIa/TF activates factors IXa and Xa, which mediate the conversion of prothrombin into the active thrombin. Nevertheless, the small quantity of thrombin formed is sufficient to activate XIa, which reinforces thrombin generation by activating the intrinsic pathway. Furthermore, the intrinsic pathway can be activated by collagen residues or other negatively charged molecules on the islet surface. Thrombin, a potent platelet activator, cleaves fibrinogen into fibrin monomers, and activates the coagulation factor that cross-links fibrin monomers into an insoluble thrombus (XIIIa, not shown). Coagulation systems can be modulated by anti-coagulant molecules as TBM (thrombomodulin), TFPI (tissue factor pathway inhibitor), AT (antithrombin) and CD39 (ectoatpase). Source: van der Windt DJ, Bottino R, Casu A, Campanile N, Cooper DK. Rapid loss of intraportally transplanted islets: an overview of pathophysiology and preventive strategies. Xenotransplantation 2007;14: p.289. 되면, 트롬빈활성화에의한혈액응고시스템이자체적으로증폭되어혈소판이활성화되고, 섬유소생성이증가한다. 따라서, 초기의혈액응고시스템에만관여하는 TFPI 를조절하는것은전체적인혈액응고시스템을방어하기에는충분치않으며혈액응고시스템을효과적으로억제하기위해서는혈액응고관련유전자뿐아니라, 트롬빈활성화및혈소판활성화에관여하는유전자를모두조절하는것이중요할것으로생각된다. 혈소판은혈액응고시스템에의해활성화되는경우외에도다양한기전을통해서활성화되기때문에이종췌도이식에서발생하는 IBMIR에중요한역할을한다. Lin의보고에따르면, 돼지내피세포와인간의혈청을공동배양하였을때, 돼지내피세포에서분비되는조직인자는혈청 보체의존적임을확인하였으나, 인간의혈청없이, 인간의혈소판과돼지내피세포를공동배양하였을때는, 돼지내피세포와인간의혈소판모두에서조직인자가분비되는것을확인하였다.(25) 뿐만아니라, 분리된췌도의표면에잔존하는콜라젠이인간혈액에노출될경우, 혈액내 von Willebrand factor (vwf) 와결합하여혈소판활성화를유도한다.(23) 따라서혈소판활성화를억제하기위한형질전환돼지의췌도개발은물론혈소판억제제사용이필요하다. Thrombomodulin (TBM) 은 protein C 기전의한가지구성성분으로서, 혈액응고를방지하고염증을억제하는역할을한다.(26) 구체적으로는 TBM의특정부위가트롬빈의활성화에중요한역할을하는부위를폐쇄하여트롬빈을 J Korean Soc Transplant December 2009 Volume 23 Issue 3

7 불활성화시키고, protein C를활성화하여혈액내순환하는 FVa와 FVIIIa에결합함으로써혈액응고시스템을효과적으로억제한다. 특히 protein C는내피세포표면에존재하는 protein C 수용체 (endothelial cell PC receptor, EPCR) 과결합할경우강력한항응고능을가지는활성화된 protein C의양을 20배이상증가시킨다.(27,28) 그러므로 protein C의기전을조절하는형질전환돼지의개발은이종췌도특이적인거부반응을줄이는좋은방안이될수있다. Antithrombin (AT) 은간에서합성되는세린프로테아제억제제로서혈소판과 FXa를억제하여효과적인항응고기능을갖는다.(29) CD39 (ectoatpase, CD39) 는혈소판의길항제로알려져있는 ATP와 ADP를 AMP로전환하여혈소판의활성화를억제하는항응고능을갖는다.(30) 특히, ATP와 AMP는 neutrophil의활성화에서로상반된기능을가지기때문에, CD39가혈소판의길항제로써역할을할뿐아니라, 염증반응에도관여할것이라는관심이증가하였다. 최근에는 CD39가염증반응을조절하고면역억제기능을갖는등다양한기능을가지고있음이보고되었으므로이종췌도이식을위한유용한타겟유전자라생각된다.(31) 앞에언급한항응고또는항혈소판유전자는돼지췌도표면에일부존재하지만, 이식초기단계의복합적인스트레스에의해그기능이현저히떨어지게되며, 특히이러한유전자의이종간부적합성으로인해응고기전이더욱가속화된다.(23) 돼지의 TBM은인간의트롬빈에접합하기는하지만, 강력한항응고능을가지는활성화된 protein C의생성에는관여하지못하며, 항혈소판기능을갖는 CD39의발현은이식초기의스트레스로인하여발현이급격히감소되는것이보고되었다.(32) 또한혈소판활성화를유도하는 vwf의경우는종간부적합성이없기때문에, 이종이식시에혈소판을활성화시키는데기여하게된다.(23) 이와같이돼지췌도내의내재적인보호효과가있는유전자의발현이소실되거나제기능을하지못하게되고손상을유도하는유전자는그대로기능을유지하기때문에이러한종간부적합성에의해이종이식시에는보다심한혈액응고현상이관찰된다. 따라서, 이종췌도이식시에가장큰문제가되는 IBMIR을극복하기위해서는항응고및항혈소판유전자를과발현하거나응고를유도하는유전자의발현을억제할수있는형질전환돼지의개발이필요하다. 2) 항체순환및보체활성화보체가활성화되는경로는고전경로, 대체경로, 렉틴경로라불리는세가지이다. 이세가지과정은보체활성 화의초기단계인 C3 convertase ( 전환효소 ) 를활성화시키는경로만다를뿐그이후의과정은모두같다. 보체가활성화되면결과적으로이종췌도의세포표면에막공격복합체 (membrane attack complex, MAC) 가형성되어세포가용해되므로, 이종췌도이식에있어서큰장애가된다 (Fig. 5). 고전경로에의한보체의활성화는체액성면역반응의주요한작용기전이되며, 항원-항체복합체가형성되어, 혈청속의 C1 단백질과반응하게되어 C1을활성화시키고, 이활성화된 C1에의해 C2, C4, C3가연쇄적으로활성화된다. 돼지의고형장기이종이식의경우, 이식초기에돼지내피세포에존재하는알파갈전이효소와인간에존재하는알파갈항원-항체사이의격렬한항원-항체반응에의한초급성거부반응이나타나는것이특징이다.(33) 하지만돼지췌도의표면에는알파갈전이효소가거의없는것으로알려져있기때문에 (34,35) 이종췌도이식시에알파갈에대한항원-항체반응에의한보체활성화는거의발생하지않으며이보다는비알파갈항원-항체반응이나이종 MHC 타입에대한항원-항체반응에의한보체활성화에의한손상이주로작용할것으로생각된다.(5,36,37) 그러므로알파갈전이효소를제거한형질전환돼지의도입은고형장기의초급성거부반응을억제하는데는효과적이겠지만이종췌도이식에서의역할은불분명하다. 그러나이종췌도이식시유도되는보체의활성화는비알파갈항원-항체반응에의한것외에도혈소판에의해 C5a 가전환되어보체를활성화시키므로, 보체활성화와혈액응고시스템사이에새로운상호작용이있을것임을시사하기때문에혈청보체활성화조절역시이종췌도이식조절에필수적인역할을할것으로예측한다.(38) 이러한보체의활성화를조절하는타겟유전자로는 C1 inhibitor, complement receptor (CR1), CD46 (membrane cofactor pretein, MCP), CD55 (decay accelerating factor, DAF) 와 CD59 등이있다. C1 inhibitor는 C1의분해를억제하여, 항원-항체복합체에의한보체의활성화를막을수있으므로, 과다한보체의활성화를억제하며, type 1 complement receptor (CR1) 와 CD55는생성된 C4b에결합하여 C2a를제거하거나, C3b에결합하여 Bb를제거함으로써항보체기능을갖게된다. CD46은 C3b와 C4b의불활성화를촉진하여보체가활성화되는것을억제함으로써보체에의한세포파괴를방지할수있고, CD59는 C7, C8과 C5b6의결합을방해하여막공격복합체 (MAC) 의형성을방지함으로써강력한항보체능을갖는다.(39) CD46, 55, 59 등과같이보체의활성화를효과적으로조절할수있는유전자를가진형질전환돼지는이미개 J Korean Soc Transplant December 2009 Volume 23 Issue 3

8 Fig. 5. Schematic representation of the complement cascades. The membrane attack complex is initiated when C5 convertase cleaves C5 into C5a and C5b. After C6, binds to C5b, the C5bC6 complex is bound by C7. This junction alters the configuration of the protein molecules exposing a hydrophobic site on C7 that allows the C7 to insert into the phospholipid bilayer of the pathogen. Similar hydrophobic sites on C8 and C9 molecules are exposed when they bind to the complex, so that they can also insert into the bilayer. The ring structure formed by C9 is a pore in the membrane that allows free diffusion of molecules in and out of the cell. If enough pores form, the cell is no longer able to survive. There is a membrane bound complement regulator such as DAF (CD55), CD59, and MCP (CD46). CD46 is an inhibitory complement receptor and decay accelerating factor (CD55) is a 70 kda membrane protein that regulates the complement system on the cell surface. CD59 inhibits the complement membrane attack complex by binding C5b678 and preventing C9 from binding and polymerizing. Abbreviations: MBL, Mannose binding lectin; MASP, MBL-associated serine protease; DAF, decay accelerating factor; MCP, membrane cofactor pretein. Source: van der Windt DJ, Bottino R, Casu A, Campanile N, Cooper DK. Rapid loss of intraportally transplanted islets: an overview of pathophysiology and preventive strategies. Xenotransplantation 2007;14: p.290. 발되었다.(40,41) 그러나 CD55 형질전환돼지에서내피세포에서는 CD55의발현이높은반면, 췌도세포내에서는발현이극히미약하므로 (42) 이종췌도이식을위한목적유전자로서는부적절하다. 반면에 CD46은돼지췌도에도높은발현을보였고, 돼지췌도영장류이식실험에서장기생존성적으로보였다.(43) 3) 염증세포침윤이종이식시혈액응고시스템과염증반응은밀접하게상호작용을한다.(44) 이식초기에췌도표면에염증성사이토카인의분비가증가하면, 염증세포가췌도내로침윤하여췌도의손상을유도한다. 또한강력한염증유도인자중하나인 tumour necrosis factor (TNF) 의분비가증가하여세포사멸이유도되기도하며, 췌도주변에활성화된선천면역세포들이항원제시를함으로써적응면역의단계에이르게된다. 췌도이식초기에 IBMIR가진행되면활성화된혈소판 표면에중성구나단핵구에대한수용체로서작용하는 p-selectin의발현이증가한다.(45) 또한트롬빈이활성화되어혈액내의과립구와단핵구표면에존재하는 protease activated receptor (PAR) 을자극하여다양한사이토카인분비를유도하여 (46) neutrophilic granulocyte와거식세포등이췌도내에침윤한다.(47) 췌도이식시수용성보체활성화의산물인 C3a와 C5a의증가는중성구나거식세포에대해강력한 chemoattractant로작용한다.(21) 2002년 Ozmen 등은분리된췌도에트롬빈억제제인 melagatran을처리했을때 IBMIR가억제되어면역세포의췌도내침윤이감소되는것을관찰함으로써 (48) 혈액응고반응을조절하는것이염증반응을억제하는데제일중요한과정임을보고하였다. 췌도를이식하기전이나췌도를분리하는과정에서도기계적손상과산화적손상이발생하여췌도표면에서 MCP-1를비롯한 IL-1b, TNF-a, IFN-c, IL-6,IL-8 등의염증매개유전자가분비된다.(49,50) 이러한다양한면역 J Korean Soc Transplant December 2009 Volume 23 Issue 3

9 매개물질들이증가되면췌도내에염증세포들이침윤하게되어세포사멸이진행된다. 중성구와거식세포는 T- 세포에대한항원을제시하는역할을담당한다. 따라서 IBMIR는선천면역반응으로부터적응면역반응을유도하거나증가시키는, 즉 innate to adaptive talk의과정이라고볼수있다.(21) 또한항산화유전자및항세포사멸유전자들을발현하는형질전환돼지를개발하게된다면췌도분리및췌도이식초기에분비되는면역매개반응을억제할수있으므로, 면역세포의췌도내침윤을억제하고나아가후천성면역반응을감소시킬수있을것이다. 이종췌도이식거부반응의극복이종췌도이식거부반응을극복하기위한방안으로는 (1) 췌도이식특이적인거부반응을억제하기형질전환돼지개발과 (2) 췌도피막화기술개발을통한면역거부반응차단방법이제시되어있다. 1) 형질전환돼지개발을통한거부반응극복현재까지알려진이종이식을위한형질전환돼지개발현황은 Fig. 6과같다. 형질전환동물을생산하여이종간의장기및세포이식을시도하는연구가본격적으로시작 된것은 1990년대부터이다. 이후체세포핵이식에의한동물복제기술이발달함에따라 1994년영국에서초급성거부반응을완화하기위해서 hdaf가도입된형질전환돼지가생산된것을시작으로 (51) 다양한장기이식용복제돼지가생산되었으며, 대부분은초급성거부반응을줄이려는시도로제작되었다.(52,53) 가장괄목할만한성과는 2002년미국에서개발된알파갈전이효소가제거된형질전환돼지이다.(54) 이돼지는유전자제거기술 (gene knock-out) 을이용하여, 인간체내에서초급성거부반응을일으키는주요항원을제거한것으로, 이돼지의개발로초급성거부반응은극복되었다.(55) 그러나알파갈전이효소가제거된형질전환돼지의심장을영장류에이식하였을때초급성거부반응은관찰되지않았으나, 장기주변의혈관에서혈소판이응집되고섬유화가진행되어있는미세혈관혈전증 (Thrombotic microangiopathy, TM) 의소견이관찰되었다. 뿐만아니라장기의허혈과괴사, 부분적출혈및면역억제제과다사용에의한감염등으로인하여괄목할만한이식생존율을얻지못했다.(32) 따라서, 알파갈이나혈청보체조절인자같은단일의유전자변형만으로는인간의복잡한면역체계를효율적으로회피하기에는부족하며, 추가적으로미세혈관혈전증을조절할수있는항응고및항혈소판유전자를함께발 Fig. 6. Transgenic pigs production for overcoming graft rejection in xeotransplantation. J Korean Soc Transplant December 2009 Volume 23 Issue 3

10 현하는다중유전자발현형질전환돼지의개발이시급하다. 항응고기능을가진형질전환돼지에대해서는 2008년세계이종이식학회에서 D'Apice, Cowan 등에의해혈액응고조절을위한 CD39 유전자삽입돼지가개발되어영장류신장이식에대한효과가보고된바있으며, TBM, TFPI에대한돼지가제작되었다는발표가있었으나이들유전자삽입돼지의효용성에대하여는검증을요한다. 최근이분야의선두주자라고할수있는미국, 호주및일본연구팀등은알파갈결손돼지를기반으로하여다양한다중의유전자를발현하는돼지를생산하는데성공하였다.(56,57) 그러나앞서말한바와같이현재까지의연구는대부분심장, 간, 폐, 신장등고형의장기이식을위한연구가대부분이며, 당뇨병치료를위해특화된형질전환돼지의생산, 즉 IBMIR을조절하기위한다중형질전환돼지생산은아직초기단계에머물러있다. 2) 췌도피막화췌도를피막화하면이종이식에서발생하는면역세포나항체의공격으로부터췌도를보호할수있기때문에, 최근이식시에좋은성적들이보고되고있으며많은연구와임상시험이진행중이다. 하지만이방법은피막이산소와영양물질의전달능력을저해하고인슐린의분비를방해하기때문에, 이러한부작용을최소화시키면서췌도의생존을증진시키는피막화기술의확보가필수적이다. 특히돼지췌도는내피세포와마찬가지로허혈성손상에민감한데허혈성자극은 reactive oxygen species (ROS) 계를활성화하여 JNK, p38 발현이증가되고세포사멸과면역세포침윤을유발한다. 한편 H 2O 2 자극에의해 VCAM-1 등접착분자의발현이증가함을확인되어 (58) 허혈성손상에내성을유도할수있는 HO-1, A20과같은유전자도입췌도를확보하는것이도움이될것이다.(59) 췌도피막화의가장중요한요소는췌도를감싸는피막재료라고할수있다. 가장널리이용되고있는피막재료는알지네이트 (alginate) 로 1980년 Sun 등에의해최초로알려진생체적합성고분자이다. 알지네이트를이용한췌도피막화기술은이미상당한수준에도달한상태로네덜란드의 de Vos는미세피막화췌도를실험용쥐에이식한후약 200일간혈당조절기능이원활함을확인하였다.(60) 또한미국의기업인 Microislet Inc., Cerco Medical 및호주의 Living Cell Technologies 등이이미임상시험을준비또는진행중인상태이다. 그러나췌도를직접피막화하여이식하는기존의기술 ( 미세피막화 ) 은이식후제거가쉽지않아안정성문제가꾸준히제기되어왔다. 알지네이트이외에도 polyethylene glycol (PEG) 와같은다양한물질들이췌도피막화연구에활용되고있으며, 췌도표면에 recombinant azido-thrombomodulin 같은특수물질을처리하여혈전형성을차단하는방법도개발되어있다.(61) 이에대한대안으로는큰구조체에췌도를부착한후이식하는 거대피막화 기술을들수있다. 거대피막화기술의경우미국앨버타대학의 Tatsuya Kin 그룹에서는상용화된생체내분해되는스캐폴드 (synthetic absorbable biodegradable scaffold, Ethisorb TM Dura Patch, approved by FDA) 를사용하여췌도를파종하고이를개의장막 (omentum) 에여러겹으로붙여이식을하는연구를수행하였다.(62) 이와비슷한연구로미국마이애미대학의 D. M. Berman 그룹에서는위와같은 Ethisorb TM Dura Patch 스캐폴드를이용하여원숭이에이식을한실험을수행하였고 (63) 캐나다앨버타대학의 J.M. Dufour 그룹에서도인공구조체인 Dura patch를이용하여췌도를이식하는연구를수행한바있다.(64) 이러한거대피막화기술은개발초기단계수준에머물러있고, 아직상품화단계까지는이루지못한실정이다. 이종췌도이식현황 결론적으로성공적인췌도이식을위해서는혈액응고반응과보체의활성화및염증반응등을포함하는췌도이식특이적 IBMIR를극복할수있어야하며, 췌도피막화기술을현실화할수있어야한다. 그러기위해서는기존알파갈결손형질전환돼지를기반으로적어도혈청보체와혈액응고반응을조절할수있는다중형질전환돼지를개발하여야하며, 이를췌도피막화기술과접목한다면, 생존율이높은이종췌도개발이가능할것이다. 지금까지영장류를이용한이종췌도이식연구결과는적어도 4개의연구실에서 6개월이상의췌도생존율이보고되었다. 2006년 Hering 등과 (65) Cardona 등은 (66) 각각상당량의면역억제제를사용하여간문맥을통한췌도이식을시행하여 6개월이상의췌도생존율을얻었으며, 2009년피츠버그의 Cooper 팀은 CD46 유전자삽입돼지를이용하여 1년이상의췌도이식생존율을보고하였으므로 (43) 간문맥을통한췌도이식분야에서남아있는과제는앞으로면역억제제를어떻게최소화할것인가이다. 한편 2006년 Dufrane은면역억제제를전혀사용하지않은상태에서췌도피막화기술을이용하여최대 354일까지의췌도생존율을보고하였다.(67) 췌도피막화를이용한임상시험은현재멕시코, 뉴질랜드, 중국, 러시아에서시도되고있고, 이에대해미국, 호주의 Food and Drug J Korean Soc Transplant December 2009 Volume 23 Issue 3

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13 54) Lai L, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, et al. Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002;295: ) Phelps CJ, Koike C, Vaught TD, Boone J, Wells KD, Chen SH, et al. Production of alpha 1,3-galactosyltransferase-deficient pigs. Science 2003;299: ) Tu CF, Hsieh SL, Lee JM, Yang LL, Sato T, Lee KH, et al. Successful generation of transgenic pigs for human decay-accelerating factor and human leucocyte antigen DQ. Transplant Proc 2000;32: ) Ramsoondar JJ, Macháty Z, Costa C, Williams BL, Fodor WL, Bondioli KR. Production of alpha 1,3-galactosyltransferase-knockout cloned pigs expressing human alpha 1,2-fucosylosyltransferase. Biol Reprod 2003;69: ) Lee S, Chung J, Ha IS, Yi K, Lee JE, Kang HG, et al. Hydrogen peroxide increases human leukocyte adhesion to porcine aortic endothelial cells via NFkappaB-dependent up-regulation of VCAM-1. Int Immunol 2007;19: ) Hancock WW, Buelow R, Sayegh MH, Turka LA. Antibody-induced transplant arteriosclerosis is prevented by graft expression of anti-oxidant and anti-apoptotic genes. Nat Med 1998;4: ) de Vos P, Faas MM, Strand B, Calafiore R, Alginatebased microcapsules for immunoisolation of pancreatic islets. Biomaterials 2006;27: ) Stabler CL, Sun XL, Cui W, Wilson JT, Haller CA, Chaikof EL. Surface re-engineering of pancreatic islets with recombinant azido-thrombomodulin. Bioconjug Chem 2007;18: ) Kin T, O'Neil JJ, Pawlick R, Korbutt GS, Shapiro AM, Lakey JR. The use of an approved biodegradable polymer scaffold as a solid support system for improvement of islet engraftment. Artif Organs 2008;32: ) Berman DM, O'Neil JJ, Coffey LC, Chaffanjon PC, Kenyon NM, Ruiz P Jr, et al. Long-term survival of nonhuman primate islets implanted in an omental pouch on a biodegradable scaffold. Am J Transplant 2009;9: ) Dufour JM, Rajotte RV, Zimmerman M, Rezania A, Kin T, Dixon DE, et al. Development of an ectopic site for islet transplantation, using biodegradable scaffolds. Tissue Eng 2005;11: ) Hering BJ, Wijkstrom M, Graham ML, Hårdstedt M, Aasheim TC, Jie T, et al. Prolonged diabetes reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed nonhuman primates. Nat Med 2006;12: ) Cardona K, Korbutt GS, Milas Z, Lyon J, Cano J, Jiang W, et al. Long-term survival of neonatal porcine islets in nonhuman primates by targeting costimulation pathways. Nat Med 2006;12: ) Dufrane D, Goebbels RM, Saliez A, Guiot Y, Gianello P. Six-month survival of microencapsulated pig islets and alginate biocompatibility in primates: proof of concept. Transplantation 2006;81: J Korean Soc Transplant December 2009 Volume 23 Issue 3

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

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