= 증례보고 = 약물유도망막변성생쥐모델에서안구내면역세포에서의 F4/80 와 nestin 발현양상 대한안과학회지 2013 년제 54 권제 6 호 J Korean Ophthalmol Soc 2013;54(6):945-953 pissn: 0378-6471 eissn: 2092-9374 http://dx.doi.org/10.3341/jkos.2013.54.6.945 안상일 온영훈 박태관 순천향대학교의과대학부천병원안과학교실 목적 : MNU (N-methyl-N-nitrosourea) 로망막변성을유도한생쥐의섬모체와시신경에서 F4/80 와 nestin 의발현양상을알아보고자한다. 대상과방법 : 30 마리생쥐를대상으로 MNU (60 mg/kg) 를복강내주사한후 2 일, 4 일, 7 일, 30 일에각각안구를적출하여 Hematoxylin and eosin (H&E) 염색및 Terminal deoxyribonucleotidyl transferase mediated dutp nick end labeling (TUNEL) 염색, F/80 와 nestin 의면역조직형광염색을시행하였다. 결과 : MNU 주입후세포자멸사에의한광수용체의파괴가나타났다. 면역조직형광염색결과대조군에서는 F4/80 와 nestin 이함께발현되지않았으나, 실험군에서는시신경및섬모체내부에서 F4/80 의발현이증가하였고, 이세포들은 nestin 을함께발현하였다. 망막의외핵층에서도 F4/80 와 nestin 을함께발현하는세포가관찰되었다. 결론 : 망막의손상에반응하여 F4/80 및 nestin 을함께발현하는세포가섬모체와시신경을따라망막내부로이동하여일시적으로증식및활성화된다. < 대한안과학회지 2013;54(6):945-953> N-methyl-N-nitrosourea (MNU) 는일상환경에널리퍼져있는질소화합물로다양한동물들에서망막변성을유발하는알킬 (alkylating) 화합물이다. 1,2 생쥐의망막변성모델에서 MNU는광수용체의세포자멸사 (apoptosis) 를유발하며그손상은용량과시간에비례하는것으로알려졌다. 3 MNU에의한망막손상후세포잔해를제거하고이를복구하기위해면역세포의침윤및뮬러세포 (Müller cell) 의증식이일어난다. 4 단핵구 (monocyte) 는척추동물의면역시스템을담당하는백혈구의한종류이다. 골수에서생산되어혈액속에존재하는단핵구는염증이나감염에반응하여조직속으로이동하여각조직에특이적인대식세포 (resident macrophage) 로분화, 증식한다. 5 또한대식세포는중추신경계선천면역시스템의주작용세포로외상이나감염, 변성등의 Received: 2012. 7. 13. Revised: 2013. 1. 3. Accepted: 2013. 4. 10. Address reprint requests to Tae Kwann Park, MD, PhD Department of Ophthalmology, Soonchunhyang University Bucheon Hospital, #170 Jomaru-ro, Wonmi-gu, Bucheon 420-767, Korea Tel: 82-32-621-5426, Fax: 82-32-621-5435 E-mail: tkpark@schmc.ac.kr * 이논문은 2011 년도정부 ( 교육과학기술부 ) 의재원으로한국연구재단의지원을받아수행된기초연구사업임 (No. 2011-0014354). 손상에반응하여혈액을타고손상된조직으로이주하여사이토카인 (cytokines), 산화제 (oxidants) 등을분비하며면역반응을유발한다. 6 미세교세포 (microglia) 는신경조직의특수화된대식세포로교세포 (glia cell) 의한종류이다. 미세교세포는조혈모세포기원의단핵구에서유래하여섬모체나홍채, 망막혈관을타고망막주변부와시신경유두를통해망막으로유입된다. 7 망막의미세교세포는외부에서침입한미생물에대항하여숙주의방어기전에중요한기능을하는데, 정상성체망막에서는휴지상태에있다가변성, 염증, 외상등의다양한자극에반응하여활성화된다. 8 활성화된미세교세포는잔해물의탐식및재생기전을촉진시키는데관여한다. 9 이처럼망막에서골수구계세포 (myeloid cell) 들은혈액기원의혈관주위대식세포 (perivascular macrophage) 및망막실질의미세교세포 (resident microglia) 두가지형태로존재하게된다. 10,11 성체쥐의대식세포와미세교세포는분자량 160 kda의막단백질인 F4/80 항원을가지고있어이항원표지자를이용하여이들세포들을확인할수있다. 12-15 대식세포, 수지상세포 (dendritic cell) 등과같은다수의면역세포들이섬모체, 홍채, 맥락막등의혈관결합조직에존재하며안구의면역학적항상성을유지한다. 16-18 망막의손상이발생하면이세포들의망막내로이동이증가하는데, Kaneko et al 19 은망막손상에반응하여다수의골수기원세 945
- 대한안과학회지 2013 년제 54 권제 6 호 - 포들이섬모체와시신경, 망막의혈관을따라망막내로이주하여미세교세포로분화한다고하였다. 아울러이러한섬모체기원의면역세포들이 nestin 등의원시신경세포표지자를발현하는것이보고되었다. 20 중간섬유중의하나인 nestin은중추신경계의원시신경세포표지자로알려졌는데, 21 망막에서는뮬러세포및성상세포 (astrocyte), 미세교세포에서발현되며 22-27 혈관내피세포 (vascular endothelial cell) 와혈관주위세포 (pericyte) 등에서도발현된다. 28 이번연구에서는생쥐에서 MNU로망막변성을유도한후시간경과에따른세포자멸사를포함한망막의조직병리학적변화를관찰하고, 대식세포및미세교세포의표지자인 F4/80와원시신경세포표지자로알려진 nestin의발현양상을알아보고자한다. 대상과방법 대상외견상외안부질환이없고각막, 수정체의혼탁이없는 8주령체중 22-24 g 가량의 C57Bl/6N CrljOri mice( 오리엔트바이오, 성남, 경기도, 대한민국 ) 를대상으로하였다. 실험동물을이용한모든생체실험은안과및시과학연구회 (Association for Research in Vision and Ophthalmology) 에서규정하고있는동물사용에대한지침을준수하였다. 실험방법 1. 망막변성의유도와약물의주입 MNU (Sigma Aldrich, St. Louis, MO, USA) 를 -20 의암실에서보관하였으며, 사용직전 0.05% acetic acid 가함유된생리식염수에 MNU를용해시켰다. MNU 는 60 mg/kg 의용량을단회복강내주사하였으며, 약물주입후시간을기록하였다. 약물주입전과약물주입후 2일, 4일, 7일, 30일을각군으로나누어, 각군당 6마리 12안, 총 30 마리 60안에대해실험을하였다. 2. 조직의고정및표본제작생쥐를희생시킨후, 안구를적출하여 4% paraformaldehyde 용액에서 4시간고정시켰다. 적도부위에서안구를절개하고수정체를포함한전안부와유리체를제거한후, 냉동절편형성시발생가능한조직의손상을방지하기위하여 10, 20% 의 sucrose 용액에각각 1시간씩방치한후 30% 의 sucrose 용액에서 12시간방치하였다. OCT (Optical Cutting Temperature, SaKura Finetek, Torrance, CA) 용액에포매시킨다음 4 μm 두께의조직절편을제작하여 polylysine-coated 유리슬라이드에표본제작하였다. 3. H&E 및 TUNEL 염색조직학적변화를관찰하기위해 Hematoxylin and eosin (H&E) 염색및세포자멸사의확인을위해 Terminal deoxyribonucleotidyl transferase mediated dutp nick end labeling (TUNEL) 염색을하였다. Apoptosis 염색 (TAKARA BIO INC, Shiga, Japan) 을사용하여제조회사에서제공하는실험방법에의해 TUNEL 염색을하였다. 원리를간략히요약하면 Terminal deoxynucleotidyl transferase를사용하여 apoptosis의결과로생긴 nucleosome-sized DNA fragment의양끝에 digoxigeninlabeled deoxyuridine triphosphate를연결한후 anti-digoxigenin peroxidase를적용하여항원 -항체반응을일으키고 0.05% diaminobenzidine 을가하여발색반응을일으켰다. 4. 면역조직형광염색조직절편을 PBS (0.1M PBS+0.1% Triton X-100) 용액으로 5분씩 3번씻은다음 blocking을위해 0.1M Phosphate buffer에 5% goat serum과 0.5% Triton X-100를첨가한용액에담가상온에서 1시간처리하였다. 1차항체로서 mouse monoclonal anti-nestin antibody (1:1000; Chemicon, Temecula, CA, USA) 및 rat monoclonal anti-f4/80 antiserum (1:1000; AbD Serotec, Oxford, UK) 을 5% goat serum에희석하여준비하였다. 각각의일정에서얻어진조직절편들을두가지의일차항체가섞여있는반응액에노출시킨다음섭씨 4도의조건에서 16시간반응시킨다음, 0.1% PBS 용액으로 5분씩 3번씻은후각각의일차항체와반응하는 2차항체로서 goat anti-mouse IgG Alexa Fluor 488 (molecular Probes, Grand Island, NY) 및 goat anti-rat IgG Alexa Fluor 568 (molecular Probes, Grand Island, NY) 를 5% goat serum에 1:1000으로희석하여실온에서 1시간처리하였다. 면역형광염색반응을마무리한다음 4,6-diamidino-2-phenylindole (DAPI) (D9542; Sigma, St Louis, MO) 을이용하여세포내핵의대조염색을마무리하였다. 5. 이미지분석조직의면역형광염색을형광현미경 (Axioplan microscope; Carl Zeiss, Oberkochen, Germany) 을이용하여관찰하였으며, image-capture software (Axiovision; Carl Zeiss Inc., Oberkochen, Germany) 로사진을분석하였다. 946
- 안상일 외 : 망막변성 생쥐에서 F4/80와 nestin의 발현 - 결 용체층(photoreceptor layer)과 외핵층(outer nuclear lay- 과 er)의 배열이 무질서해지고, 색소상피층(retinal pigment epithelium)의 파괴가 나타났다. MNU 주입 4일 후에는 광 조직학적인 변화 수용체층과 외핵층의 파괴가 더욱 진행되었고, MNU 주입 MNU 주입후 시간 경과에 따른 망막의 조직병리학적인 7일 후에는 광수용체층 및 색소상피층이 완전히 소실되었 변화를 H&E 염색을 통해 관찰하였다. MNU 주입 2일 후엔 으며 외핵층은 1-2층만이 남아있고 내핵층(inner nuclear 대조군과 비교하여 망막의 두께가 다소 감소하였으며 광수 layer) 또한 얇아졌다. 30일 후에는 외핵층도 완전히 사라 A B C D 20 µm Control MNU 2 days MNU 7 days MNU 30 days Figure 1. H&E staining results of the retina. (A) Normal. (B) 2 days after M NU treatment, the retinal thickness is slightly decreased and the arrangement of PRL and ONL is distorted. (C) 7 days after M NU treatment, the depletion of PRL and RPE are noticed and the ONL is reduced to a few layers of cells. (D) 30 days after M NU treatment, the ONL completely disappeared (RPE = retinal pigment epithelium; PRL = photoreceptor layer; ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer; IPL = inner plexiform layer; GCL = ganglion cell layer). A B C MNU 2 days MNU 7 days D 20 µm Control MNU 30 days Figure 2. TUNEL staining results of the retina. (A) No TUNEL+ cells are observed in the control group. (B) Large number of TUNEL+ cells are shown in the ONL at 2 days after M NU treatment. (C) Seven days after M NU treatment, small num ber of TUNEL+ cells are remained in the ONL and GCL. (D) Thirty days after M NU treatment, no TUNEL+ cells are observed as the ONL disappeared (RPE = retinal pigment epithelium; PRL = photoreceptor layer; ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer; IPL = inner plexiform layer; GCL = ganglion cell layer). 947
- 대한안과학회지 2013년 제 54 권 제 6 호 - 져서 내핵층의 일부와 얇아진 내망상층(inner plexiform 일부 남아 있으며, 신경절세포층에서도 TUNEL 양성세포 layer), 신경절세포층(ganglion cell layer)만이 남아있다 가 보이고 있다. 주입 30일째는 외핵층이 완전히 소실됨에 (Fig. 1). 따라 TUNEL 양성세포들도 관찰되지 않았다(Fig. 2). 망막세포의 세포자멸사를 관찰하기 위해 시행한 TUNEL 염색에서 대조군에서는 TUNEL 양성세포가 보이지 않으나 면역조직형광 염색 MNU 주입 2일째는 다수의 TUNEL 양성세포들이 외핵층 내에 나타났으며, 신경절섬유층에도 일부 나타났다. 4일째 1. 시신경 는 외핵층내 TUNEL 양성세포들의 비율이 감소하였으며, MNU를 주입하지 않은 정상 대조군 시신경의 내부에선 7일째는 1-2층만 남아 있는 외핵층에 TUNEL 양성세포가 F4/80 양성인 세포들이 일부 관찰되었고(Fig. 3A; arrow- A B C D E F G H I J K L Figure 3. Immunochemistry examination of the optic nerve head. Immunofluorescent labeling with F4/80 (red) and nestin (green). In the control optic nerve head, there are some of F4/80+ cells (A: arrowhead) and nestin+ cells (B: asterisk), but no cells co-expressed F4/80 and nestin (C: merged image of A and B). Two days after M NU treatment, some of F4/80+ cells are also positive for nestin (D-F: arrow). Four days after M NU treatment, large number of F4/80 and nestin co-expressing cells are observed (G-I: arrow). Seven days after M NU treatment, these cells are decreased (J-L). Scale bar: 100 μm. 948
- 안상일 외 : 망막변성 생쥐에서 F4/80와 nestin의 발현 - heads) nestin을 발현하는 세포도 보였으나(Fig. 3B; as- 3G-I; arrow). MNU 주입 7일째 시신경에서는 F4/80 또 terisks) F4/80 및 nestin을 함께 발현하는 세포는 관찰되 는 nestin을 발현하는 세포들이 감소하는 양상을 보였다 지 않았다(Fig. 3C). MNU 주입 2일째는 F4/80 양성인 세 (Fig. 3J-L). 포와 nestin을 발현하는 세포가 일부 보였으며 F4/80 및 nestin을 함께 발현하는 세포들도 관찰되었다(Fig. 3D-F). 2. 섬모체 이러한 변화는 MNU 주입 4일째 더욱 뚜렷해져서 시신경 정상 대조군 섬모체 조직에서 F4/80는 섬모체 상피에서 내부에서 F4/80 및 nestin을 발현하는 세포들이 다수 관찰 발현되고 있으나 nestin은 함께 발현하지 않았다(Fig. 되었고 이들은 F4/80 및 nestin을 함께 발현하였다(Fig. 4A-C; arrow). 섬모체 기질(stroma)은 비특이적으로 nes- A B C D E F G H I Figure 4. Immunochemistry examination of the ciliary body. Immunofluorescent labeling with F4/80 (red) and nestin (green). In the control ciliary body, F4/80 immunoactivity is seen in the ciliary epithelium, but nestin is not expressed (A-C: arrow). The stroma of ciliary body is non-specifically stained by nestin, but F4/80 is not expressed (B, E, H: asterisk). In the choroid, F4/80 and nestin co-expressing cells are observed (A-C: arrowhead). Two days after M NU treatment, F4/80 immunoactivity of ciliary epithelium is more increased (D: arrow) and there are some of cells which co-expressed F4/80 and nestin (D-F: arrowhead). Four days after M NU treatment, F4/80+ cells are seen in the ciliary epithelium (G: arrow), and F4/80 and nestin co-expressing cells are seen in the fibrovascular tissue of ciliray body (G-L: arrowhead). (CB: ciliary body). Scale bar: 100 μm. 949
- 대한안과학회지 2013 년제 54 권제 6 호 - A B C D E F G H I Figure 5. Immunochemistry examination of the retina. Immunofluorescent labeling with F4/80 (red) and nestin (green). In the control retina, there are no F4/80+ cells (A), but nestin is found in retinal blood vessels (B: arrowheads) and muller cells (B: asterisk). Two days after MNU treatment, F4/80 and nestin are co-expressed between INL and ONL (D-F: arrow). Four days after MNU treatment, F4/80 is also expressed in the ONL (G-I: arrow). Nestin immunoactivity of the muller cells was increased (E, H) (PRL = photoreceptor layer; ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer; IPL = inner plexiform layer; GCL = ganglion cell layer). Scale bar: 50 μm. tin에염색되고있으나 F4/80는발현하지않았다 (Fig. 4B, E, H; asterisks). 맥락막에서는 F4/80과 nestin을동시에발현하는세포가관찰되었다 (Fig. 4A-C; arrowhead). MNU 주입 2일째조직에서는섬모체상피에서 F4/80 양성세포들의발현이더욱증가하였으며 (Fig. 4D; arrow) 이세포들은 nestin은발현하지않았다. 반면에섬모체내부의섬유혈관조직에서 F4/80 및 nestin을동시에발현하는세포들이관찰되었다 (Fig. 4D-F; arrowhead). MNU 주입 4 일째및 7일째에도 2일째와마찬가지로섬모체상피에서 F4/80만을발현하는세포들이보이며 (Fig. 4G; arrow), 섬모체내부섬유혈관조직에서는 F4/80 및 nestin을동시에발현하는세포들이관찰되었다 (Fig. 4G-L; arrowhead). 3. 망막정상망막에서는 F4/80을발현하는세포가보이지않으나 (Fig. 5A) nestin은수평하게망막을가로지르는망막의혈관에서발현되고있으며 (Fig. 5B; arrowheads), 신경절 섬유층의성상세포와방사상으로망막에위치하는뮬러세포에서도발현되고있음을알수있다 (Fig. 5B; asterisks). MNU 주입후 F4/80 양성세포들이외핵층부위에나타났으며, 이세포들은 nestin도함께발현하였다 (Fig. 5D-F; arrow). 또한대조군보다성상세포와뮬러세포에서의 nestin 발현이증가되었는데 (Fig. 5E), 이러한변화는 MNU 주입 4일째최고조로나타났으며 7일째및 30일째는 F4/80 및 nestin의발현이감소하였다. 고찰 중추신경계와망막의손상에반응하여손상된세포잔해를제거하고조직을복구하기위해미세교세포및대식세포가활성화되어면역반응을유발한다. 7,8,29 손상된뇌에서의미세교세포및대식세포의활성화에대해서는많은연구가보고되어왔으며, 망막에서도시신경을일부절단하거나레이져로유발한망막손상에대한연구는많이보고된 950
- 안상일외 : 망막변성생쥐에서 F4/80 와 nestin 의발현 - 바있으나, 23,30-32 MNU 등의약물로유발된망막변성모델에서의미세교세포및대식세포의발현에대한연구는아직많지않은실정이다. 19 이에본연구에서는 MNU로유발된망막변성의시간경과에따른조직병리학적인변화와, F4/80 및 nestin 발현양상을함께보고자하였다. 발암물질로알려져있는 MNU는질소화합물로서 p-450 system의대사활성화없이직접적으로알킬화를일으키는강력한돌연변이원이다. 26,27,33 여러동물실험에서 MNU가망막변성을일으키는것이알려져, 안과영역에서는주로망막색소변성을연구하는실험에사용되고있다. 3,31,34 MNU 는광수용체세포에독성을나타내어선택적으로광수용체의세포자멸사를유발하여망막변성을일으키는데, 이러한 MNU의독성은광수용체핵에서의 DNA adduct 형성을억제하고 Bax 단백질의상향조절 (up-regulation) 과 Bcl-2 단백질의하향조절 (down-regulation) 그리고 caspase family의활성화에의해야기된다. 35 MNU의광수용체에대한독성은시간과용량에비례하여증가하는것으로알려졌다. 36,37 망막의조직염색소견에서 MNU 주입후 24시간후에망막두께의감소가관찰되었고, 세포자멸사를나타내는 TUNEL 양성세포들은약물주입후 12시간부터관찰되기시작하여 24시간후에최고조를이루었다고한다. 35 Nambu et al 3 은 MNU에의한망막변성의첫번째변화로약물주입후 3시간째에광수용체내 외분절 (photoreceptor inner and outer segments) 의무질서화 (disorientation) 가나타난다고하였다. 이후광수용체핵의파괴가진행되고 5일째가되면최종적으로광수용체의내 외분절이사라지고, 7 일째는외핵층이사라진다고하였다. 본연구에서도이와비슷한변화를확인할수있었는데, MNU 주입후망막의전반적인두께가감소하였으며, 망막의다른층에서보다주로광수용체층과외핵층의파괴가나타났고, 이러한변화는 MNU 주입 7일째극명해졌다. 또한세포자멸사가일어나고있음을나타내는 TUNEL 양성세포들은 MNU 주입후외핵층에서주로나타났다. 이는광수용체를선택적으로파괴하는 MNU의작용기전때문에주로광수용체층및외핵층에서세포자멸사가일어나고세포층의두께감소가일어난것으로보인다. 이러한망막의손상에반응하여망막에서면역세포들의활성화가일어나게되는데, 손상된망막으로이주하여증식하는세포가대식세포인지미세교세포인지에대해서는논란이있다. Kaneko et al 19 은 MNU로유도한생쥐의망막변성모델에서골수기원의세포들이섬모체와시신경, 망막혈관등을통해망막내로이주하여미세교세포로분화한다고하였고, Wohl et al 25,30 은시신경병변을유발한성체쥐에서망막과섬모체에서 F4/80 양성미세교세포들의발현 이증가한다고보고하였다. Thanos 38 는유전성망막이상증쥐에서손상된광수용체세포가미세교세포의활성화와이주를유도한다고하였고, Roque et al 39 은망막변성모델쥐에서광수용체세포의변성에반응하여이주하는세포들이대식세포가아니고망막의미세교세포라고하였다. 이와는달리망막손상에서혈액기원의대식세포의침윤이더우세하다고한연구도있는데, Caicedo et al 40 은레이져로생쥐망막에 CNV (choroidal neovascularization) 를유도한후, 혈액기원대식세포들이 CNV 부위로이주하여증식하며, 이대식세포들은뮬러세포의활성화와밀접한관련이있다고보고하였다. 이들은레이져로생쥐의망막에 CNV를유발한후 F4/80의발현정도를관찰하였는데, F4/80 를함께발현하는혈액기원의대식세포와망막의미세교세포를구분하기위해 GFP (green fluorescent protein) 로표지한골수를생쥐에게이식한후 GFP의발현유무를함께관찰하였다. 그결과 F4/80를발현하는세포의대부분이 GFP를함께발현하였는데, 이를토대로 CNV 유도쥐모델에서활성화되어망막으로이주한세포들이망막의미세교세포가아니라혈액의대식세포라고하였다. 그런데 Caicedo et al 40 의연구가미세교세포의이주를주장한연구들과다른점은쥐의망막변성을유발한다른연구와는달리 CNV를유발한쥐를대상으로한점이다. 망막변성과 CNV의병태생리차이가망막손상에반응하여이주하는주된면역세포종류의차이를야기하였을가능성이있으며, 이에대해서는좀더많은연구가필요하다고하겠다. Nestin 은 6번째중간섬유로서포유류의중추신경계및망막에서뮬러세포와미세교세포등의교세포와원시신경세포의표지자로이용되고있다. 41 최근연구에서성체망막에약물이나레이져로손상을유발하면분화된뮬러세포및미세교세포에서 nestin을발현한다고보고하였다. 42,43 그러나아직대식세포가 nestin을발현한다고보고된바는없다. 따라서본연구에서 F4/80 양성세포들이 nestin을함께발현하는것으로보아, MNU 에의해변성된망막에침윤한세포들은대식세포라기보다는미세교세포라고해석하는것이타당할것으로생각한다. 망막손상에서 nestin 양성세포의발현이의미하는바에대해서는아직논란이있다. 일부연구에서는 nestin 양성세포가손상된신경조직을재생시킬수있는능력이있는원시신경세포의특성의발현을의미한다고한반면에, 42,43 다른연구에서는 nestin의발현이단지망막손상에반응하여나타나는신경교증 (gliosis) 을의미한다고하였다. 22,43,44 본연구에서는정상대조군에서는망막과시신경및섬모체에 F4/80과 nestin을함께발현하는세포가존재하지않았으나, MNU 에의해망막변성을유도한후에는시신경 951
- 대한안과학회지 2013 년제 54 권제 6 호 - 내부및섬모체의섬유혈관조직에 F4/80의발현이증가하였으며이들은 nestin을함께발현하였다. 그리고망막의외핵층에서도 F4/80과 nestin을함께나타내는세포들의발현이증가하였다. 이는정상망막에서는존재하지않던 F4/80 및 nestin 양성세포들이망막변성에의해망막외에서망막내로의이동이유도되어섬모체및시신경을통해망막내로이동하여증식하였다고볼수있을것이다. 아울러이러한 F4/80 양성세포들이 nestin을함께발현하는것으로보아손상된망막신경의재생에관여하는원시신경세포의특성을일시적으로발현하는것으로유추해볼수있겠으나, 이에대해서는좀더연구가필요할것으로생각한다. REFERENCES 1) Herrold KM. Pigmentary degeneration of the retina induced by N-methyl-N-nitrosourea. An experimental study in syrian hamsters. Arch Ophthalmol 1967;78:650-3. 2) Nakajima M, Nambu H, Shikata N, et al. Pigmentary degeneration induced by N-methyl-N-nitrosourea and the fate of pigment epithelial cells in the rat retina. Pathol Int 1996;46:874-82. 3) Nambu H, Yuge K, Nakajima M, et al. Morphologic characteristics of N-methyl-N-nitrosourea-induced retinal degeneration in C57BL mice. Pathol Int 1997;47:377-83. 4) Nakajima M, Yuge K, Senzaki H, et al. 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