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1 대한안과학회지 2017 년제 58 권제 5 호 J Korean Ophthalmol Soc 2017;58(5): ISSN (Print) ISSN (Online) Original Article 연옥이첨가된콘택트렌즈보관용기의항아메바효과 Amoebicidal Effect of Nephrite-containing Contact Lens Storage Case 정재우 1 이종헌 1 박성희 2 유학선 2 김윤경 3 이지은 1,4 Jae Woo Jung, MD 1, Jong Heon Lee, MD 1, Sung Hee Park, BS 2, Hak Sun Yu, PhD 2, Yoon Kyung Kim, PhD 3, Ji-Eun Lee, MD, PhD 1,4 부산대학교의학전문대학원양산부산대학교병원안과학교실 1, 부산대학교의학전문대학원기생충학교실 2, 부산여자대학교안경광학과 3, 부산대학교의학전문대학원양산부산대학교병원의생명융합연구소 4 Department of Ophthalmology, Pusan National University Yangsan Hospital, Pusan National University School of Medicine 1, Yangsan, Korea Department of Parasitology, Pusan National University School of Medicine 2, Yangsan, Korea Department of Optometry, Busan Women s College 3, Busan, Korea Research Institute for Convergence of Biomedical Science and Technology, Pusan National University Yangsan Hospital, Pusan National University School of Medicine 4, Yangsan, Korea Purpose: To compare the amoebicidal effects of nephrite containing contact lens (CL) storage cases with conventional CL storage cases. Methods: Acanthamoeba lugdunensis were inoculated onto 5% nephrite containing CL storage cases as well as conventional CL storage cases both with and without silicone hydrogel contact lenses (SHCLs). Then the amount of Acanthamoeba proliferation on CL storage cases and the number of adherent Acanthamoeba on SHCLs were determined and compared. The effects of multipurpose solution (MPS) with and without 1% or 5% nephrite solution on Acanthamoeba adhesion were analyzed. Results: Nephrite containing CL storage cases showed more inhibitory effects on Acanthamoeba proliferation (p = 0.02) and significantly reduced the number of adherent Acanthamoeba on SHCLs compared with conventional CL storage cases, regardless of SHCLs generation (p = 0.001, p = and p < 0.001, respectively). The number of adherent Acanthamoeba on the first generation of SHCLs was significantly reduced by MPS with 1% and 5% nephrite solutions (p = 0.03 and p = 0.004, respectively), but the numbers for the second and third generation SHCLs were not. Conclusions: Nephrite could be used as a new additive component for CL storage cases and multipurpose solutions to improve the disinfection effects on Acanthamoeba. J Korean Ophthalmol Soc 2017;58(5): Keywords: Acanthamoeba, Contact lens storage case, Multipurpose solution, Nephrite 가시아메바각막염은드물게발생하는질환이었으나, 전세계적으로콘택트렌즈사용이보편화되면서발생빈도가 Received: Revised: Accepted: Address reprint requests to Ji-Eun Lee, MD, PhD Department of Ophthalmology, Pusan National University Yangsan Hospital, #20 Geumo-ro, Mulgeum-eup, Yangsan 50612, Korea Tel: , Fax: Jiel75@hanmail.net 급격하게증가하게되었다. 1,2 가시아메바각막염은모든종류의콘택트렌즈사용자에서발생할수있으며, 심각한통증을동반할뿐만아니라시력상실을초래할수있는심각한감염성안질환으로, 3,4 감염초기에적절한진단과효과적인치료가매우중요하다. 가시아메바에의한콘택트렌즈의오염은렌즈보관용기와보존액등의부적절한관리로인해소독이충분히이루어지지않아발생하게되는데, 5 소독액으로살충되지않은가시아메바가보관용기안에서콘택트렌즈에부착된후렌 c2017 The Korean Ophthalmological Society This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 509

2 - 대한안과학회지 2017 년제 58 권제 5 호 - 즈착용시각막에침투하여감염성각막염을일으키는것으로알려져있다. 6 특히감염에노출될수있는콘택트렌즈의반복적착용은가시아메바각막염의발생빈도를높일수있게되므로, 7 이러한감염발생을줄이기위한노력으로살충효과를가지는보관용기및보존액을개발제작하여제품화하고자하는연구들이보고되고있다. 국외에서는보관용기의제품화과정에서자체적인항균효과를가지게하여사용자의안전성뿐아니라편의성을확보할수있도록한연구들이진행되고있는데, 특히은 (Ag) 은광범위한항균작용과포유류의세포에독성이적은장점을가지고있어, 8-10 은이포함된콘택트렌즈보관용기의효과를보고한바있으나, 11,12 국내에서는이에대한연구가전무한실정이다. 보존액의경우최근사용자의편의를극대화하면서살균효과를가진다목적용액 (multipurpose contact lens care solutions, MPSs) 이보편적으로사용되고있으나, 여러보고에서다목적용액을사용했음에도불구하고잠재적감염가능성을제시한바있어, 이에대한사용자의주의가여전히필요한실정이다. 16 옥 (jade) 은경옥 (jadeite) 과연옥 (nephrite) 으로구별할수있는데특히연옥의경우인체에유익한 3가지광물즉, 칼슘, 철분, 마그네슘등을주성분으로하고있어연옥착용시고혈압, 당뇨병등의심혈관계질환에서유익할수있음이밝혀져있고, 17 연옥수와연옥분을이용한실험에서염증유발생체인자들의유전자발현을억제하는약리효과가보고된바있다. 18 특히세균성내독소인 lipopolysaccharide (LPS) 로유발된염증에서유도되는 IL-1B, TNF-a, COX-2 및 inos 유전자발현을모두현저하게억제하는효과는연옥을콘택트렌즈보관용기나다목적용액에첨가하였을때광범위한항균효과가더해짐을기대해볼수있게한다. 18 이에본연구에서는국내에서최초로구입이비교적용이하고가공하기쉬우며인체에무해한것으로알려진연옥 (nephrite) 이콘택트렌즈보관용기와보존액의항균첨가물로서가능성이있는지에대해알아보고자하였다. 였으며, 이와비교를위한연옥수는나노크기의미세입자로가공한연옥분을 phosphate buffer saline (PBS) 에 100 mg/ml 농도로준비하여 ph 7.2로보정한후 24시간보관하고, 이후 2,500 rpm에서 5분간원심분리하여얻은상층액을취한후 1% 및 5% 로각각희석된용액을만들었다. 가시아메바각막염의가장흔한균종으로 Acanthamoeba castellani 및 A. polyphaga가보고되어있지만, 8 국내에서는 A. lugdunensis가콘택트렌즈보관용액에서가장흔히발견되는균종이므로이를사용하였다. 9 콘택트렌즈사용으로인해발생한가시아메바각막염환자에서배양된 A. lugdunesis를이용하여 Riboprinting 및 18S rdna sequence analyses 방법으로균주를확인한후, 9-12 실온에서 peptone-yeast extract/glucose (PYG) 배지가든플라스크에서배양하였다. 연옥이포함된용기와일반콘택트렌즈보관용기에서가시아메바접종후증식정도를비교하기위하여균주 /ml 가포함된 PBS 용액 2 ml를각보관용기에접종한후, 실온에서 24시간동안배양시켰다. 배양후상층액을제거하였으며, 보관용기의바닥에부착된가시아메바를 PBS 1 ml를분주하여떼어낸후혈구계산판 (hematocytometer) 을이용하여그수를측정하였다. 가시아메바가콘택트렌즈에부착되는정도를비교하기위해, 각각의보관용기에 1, 2 및 3세대렌즈를위치시킨후, 2 ml PBS를채워넣고 /ml의가시아메바를분주하였다. 실온에서 18시간동안배양된렌즈를 PBS로세척한뒤각렌즈를위상차현미경을이용하여 16군데촬영한후렌즈에부착된가시아메바수를측정하였다. 대상과방법 콘택트렌즈보관용기는전통적인폴리에틸렌재질의보관용기를사용하였으며, 전체무게의 5% 의연옥이균질하게포함되게보관용기를제작하여비교하였다. 콘택트렌즈는 1세대 (Air Optix, Alcon, Fort Worth, TX, USA), 2세대 (Acuvue Oasys, Johnson & Johnson, Jacksonville, FL, USA) 및 3세대 (Biofinity, Cooper vision, Southampton, UK) 실리콘하이드로겔렌즈를구분하여사용하였다. 다목적용액은 Opti-Free Express (Alcon, Fort Worth, TX, USA) 를이용하 Figure 1. Number of Acanthamoeba on contact lens (CL) storage cases. CL storage cases containing 5% nephrite significantly reduced the numbers of Acanthamoeba compared with traditional CL storage cases. 510

- 정재우외 : 연옥콘택트렌즈보관용기의항아메바효과 - 연옥수를포함한다목적용액과기존의다목적용액의항아메바효과를비교하기위해각각의용액을기존의콘택트렌즈보관용기에 2 ml씩채운후각각의용기에 1, 2 및 3세대렌즈를넣고 1 10 4 /ml의가시아메바를분주하였다. 실온에서 6시간동안배양한후, 상기와동일한방법으로렌즈에부착된가시아메바의수를비교하였다.

결과 실온에서 24시간동안배양한결과, 5% 연옥이포함된보관용기에서일반콘택트렌즈보관용기보다가시아메바수가 A B C D E F Figure 2. Phase contrast microscopic images of adherent Acanthamoeba trophozites.

The number of Acanthamoeba adhering to contact lenses.

3 - 정재우외 : 연옥콘택트렌즈보관용기의항아메바효과 - 연옥수를포함한다목적용액과기존의다목적용액의항아메바효과를비교하기위해각각의용액을기존의콘택트렌즈보관용기에 2 ml씩채운후각각의용기에 1, 2 및 3세대렌즈를넣고 /ml의가시아메바를분주하였다. 실온에서 6시간동안배양한후, 상기와동일한방법으로렌즈에부착된가시아메바의수를비교하였다. 통계처리는 SPSS 18.0 for Windows (SPSS Inc., Chicago, IL, USA) 통계프로그램을이용하였으며, 가시아메바의개 체수를 t 검정방법및 analysis of variance (ANOVA) with the Scheffe test를이용하여비교하였다. 유의도는 p값이 0.05 미만인경우를통계학적으로유의한것으로간주하였다. 결과 실온에서 24시간동안배양한결과, 5% 연옥이포함된보관용기에서일반콘택트렌즈보관용기보다가시아메바수가 A B C D E F Figure 2. Phase contrast microscopic images of adherent Acanthamoeba trophozites. (A-F) These pictures show the decreased number of Acanthamoeba adhering to all generations of silicone hydrogel contact lenses in contact lens (CL) storage cases containing 5% nephrite. Figure 3. The number of Acanthamoeba adhering to contact lenses. The number of Acanthamoeba adhering to contact lenses in contact lens storage case containing 5% nephrite was significantly reduced compared with traditional contact lens storage case regardless of the generation of silicone hydrogel contact lenses. A B C Figure 4. Phase contrast microscopic images of adherent Acanthamoeba trophozoites. (A-C) The Opti-free express contatining 1% or 5% nephrite solutions significantly reduced the adherence of Acanthamoeba trophozoites on Air-Optix compared with Opti-free express only. 511

4 - 대한안과학회지 2017 년제 58 권제 5 호 - Figure 5. Relationship of Acanthamoeba trophozoites adherence among contact lenses treated with multipurpose solution with and without 1% or 5% nephrite solution. In case of Air Opitx, the effect of Opti-free express containing nephrite solutions on disinfection in terms of Acanthamoeba trophozoites were superior to that of Opti-free express only. Acanthamoeba trophozoite did not adhere to Oasys and Biofinity regardless of multipurpose solution with and without nephrite solutions. 유의하게더감소하였다 (Fig. 1, p=0.02). 각각의보관용기에 1, 2 및 3세대렌즈를위치시킨다음가시아메바를분주하여배양한결과, 일반콘택트렌즈보관용기보다 (Fig. 2A-C) 5% 연옥이포함된보관용기에서 (Fig. 2D-F) 콘택트렌즈에부착된가시아메바의수가낮게측정되었다 (Fig. 3, 각각 p=0.001, p=0.001 및 p<0.001). 다목적용액을처리한콘택트렌즈중 1세대렌즈의경우 1% 및 5% 연옥수를포함한다목적용액에서기존의다목적용액을처리한경우보다부착된가시아메바의수가각각유의하게낮은수준으로측정되었으며 (Fig 4, 각각 p=0.03 및 p=0.004), 2세대 ( 각각 p=0.59 및 p=3.612) 및 3세대 ( 각각 p=0.450 및 p=0.462) 렌즈에서는연옥수포함여부에관계없이부착된아메바수가적었고유의한차이는관찰되지않았다 (Fig. 5). 고찰 가시아메바각막염은드물게발생하는질환이지만외상이나다른원인이없이도콘택트렌즈착용자에서비교적흔히병발되고있다. 19 적절히관리함에도불구하고살충되지않은가시아메바가보관용기에잔존해있다가콘택트렌즈에흡착될수있으며, 이후착용과정에서각막내로침투하여각막염을일으키게되는데, 가시아메바각막염이발생하게되면심한통증과눈부심이발생하게되고적절한시 기에진단과치료가이루어지지않으면실명까지도초래할수있다. 3 콘택트렌즈사용에대한교육과세정및보관용액의발달로이전에비해콘택트렌즈와관련된감염성각막염의빈도는현저하게줄었으나, 콘택트렌즈착용과연관된감염성각막염은여전히보고되고있고, 사용자가적절한방법으로취급을하더라도콘택트렌즈보관용기를통한감염에서자유로워질수없음이보고된바있다. 20 가시아메바가각막에침투하는가장중요한단계는콘택트렌즈표면에가시아메바가부착되는과정으로, 렌즈에부착될수있는가시아메바를미리살충하거나콘택트렌즈표면처리를통해가시아메바부착정도를낮출수있다면, 가시아메바각막염을효과적으로예방할수있을것이다. 각막에부착되는가시아메바의감염을줄이기위해표면특성을달리한여러세대의실리콘하이드로겔렌즈가개발되었는데, 2세대및 3세대렌즈가 1세대보다함수율과산소투과율이높고, 부드러운표면을가지고있어가시아메바영양형의흡착이유의하게감소됨이확인된바있다 보관용기에서도은이첨가된보관용기가상품화되어영국에서는 2004년부터사용되어왔으며, 미국에서는 2005년부터식품의약청의승인을받아서판매되고있는실정이다. 그러나국내에서는살균효과를위한콘택트렌즈뿐아니라보관용기에대한연구는전무한실정으로, 본연구에서는기존에항균효과가보고된바있는연옥을콘택트렌즈보 512

5 - 정재우외 : 연옥콘택트렌즈보관용기의항아메바효과 - 관용기에일정농도로균질하게섞어제작하여그향균효과를알아보았고다목적용액에첨가하여항균효과가극대화되는지를비교해보았다. 본연구결과, 연옥이포함된보관용기에서가시아메바증식정도가유의하게감소되었을뿐아니라, 보관용기내 1, 2 및 3세대실리콘하이드로겔렌즈모두에서부착된가시아메바수가통계적으로유의하게감소된소견을보였다. 콘택트렌즈관련감염성각막염의기전은균이렌즈에부착되는과정을전제로하므로, 6 이러한점을고려한다면렌즈에부착되는균의개체수가유의하게감소된소견은콘택트렌즈착용으로인한가시아메바각막염의가능성을현저히낮춰줄수있는소견으로생각된다. 국외에서는은을첨가한콘택트렌즈보관용기를통해콘택트렌즈에의한감염성각막염을감소시켰다는보고들이있는데, 11,12,26 대표적으로 MicroBlock (CIBA Vision, Atlanta, GA, USA), i-clean (Sauflon Pharmaceuticals Ltd., London, UK) 및 Nano-case (Marietta Vision, Marietta, GA, USA) 가개발되어상품화되어있다. Dantam et al 27 은이들의항균효과를비교한연구에서 MicroBlock은스테노트로포모나스말토필리아 (Stenotrophomonas maltophilia) 를제외한대부분의그람음성균에서, i-clean은황색포도상구균 (Staphylococcus aureus) 을포함한그람양성균에서, 그리고 Nano-case는스테노트로포모나스말토필리아에서뛰어난항균효과를보였다고하였다. 콘택트렌즈에서의가시아메바에대한살충효과는습연멸균을통한방법이가장효과적이며, 28,29 과산화수소를통한방법도효과적이라고보고된바있지만, 15,30 최근에는구입이용이하고이용방법이간단하고편리한다목적용액을대부분사용하고있다. 다목적용액은항균물질, 표면활성제, 완충성분, 점성유지물질, 킬레이트성분등을포함하고있어감염성각막염을낮출수있는효과적인성분으로이루어져있는데, 31 최근에는가시아메바살충에대한효과들도보고되고있다 Lee et al 34 은실리콘하이드로겔렌즈에다목적용액을처리후 2세대및 3세대보다 1세대렌즈에서가시아메바부착이더심했으며, 여러종류의다목적용액중에서 myristamiddopropyl dimethylamine (MAPD) 을포함한 Opti-Free Express가 polyhexamethylene biguanide (PHMB) 나 4차암모늄중합물을포함한다목적용액들보다콘택트렌즈에부착되는가시아메바수를탁월하게감소시켰다고보고하였다. 그러나 Opti-Free Express를사용한경우라할지라도 1세대렌즈에서는여전히콘택트렌즈에부착되는가시아메바의수가 2세대및 3세대렌즈보다유의하게높았던제한점이있었으므로, 34 본연구에서는다목적용액중가시아메바의콘택트렌즈부착감소에가장효과 적이었던 Opti-Free Express를사용하면서연옥수를첨가하여 1세대렌즈에서가시아메바부착감소에증대효과가있는지를알아보았다. 1% 및 5% 연옥수가포함된경우 Opti-Free Express 단독사용보다 1세대렌즈에부착된가시아메바수가의미있게감소되었으며, 이는향후가시아메바살충을위한다목적용액의첨가성분으로서연옥수의가능성을제시한것으로사료된다. 연옥은각섬석군광물의변종인 acitinolite-tremolite 계열의광물로서조직이치밀하고강하지만경옥보다부드럽고세공하기쉬운특성이있다. 연옥분과연옥수가 TNF-a, COX-2 및 inos 유전자발현을억제하여염증반응을줄여주고, 특히 1 mg/ml의연옥은세균성내독소인 LPS로유발된염증에서유도되는 IL-1B, TNF-a, COX-2 및 inos 유전자발현을모두현저하게억제하여염증을억제하는효과가있음이보고된바있다. 17,18 또한 Han et al 35 은 12-O-tetradecanoylphorbol-acetate (TPA) 로유발한염증모델에서 10 mg 및 20 mg 농도의연옥이 indomethacin (0.5 mg/ 20 μl) 과유사한염증억제효과를보였으며연옥의농도가증가할수록높은효능을나타내고, 특히 10 mg 농도에서가장큰염증억제효능을나타냄을보고하였다. 이와같은연구를통해연옥이각종감염과관련된염증의예방과치료에적용가능할것이며, 콘택트렌즈보관용기나보존액등의구성물질로서도활용가능할것임을기대해볼수있겠다. 본연구의제한점으로는현재까지국내외에서연옥의가시아메바살충효과에대한연구가이루어진바가없어연구결과에대한비교가쉽지않으며, 실리콘하이드로겔렌즈는폴리머중합체로서다른물질과반응시그물질을흡수하거나방출할수있는것으로알려져있어연옥성분이콘택트렌즈에흡수될경우렌즈와직접맞닿는각막상피세포의안정성에대한평가가이루어진바가없어연옥의안정성에관한후속연구가진행되어야할것으로사료된다. 결론적으로, 콘택트렌즈사용자에서빈번하게발병하는가시아메바각막염의발생을감소시키기위한방법으로콘택트렌즈보관용기및다목적용액에연옥을첨가할경우가시아메바의증식및부착정도를감소시켜가시아메바살충효과가증대됨을확인할수있었다. 향후각막상피세포에대한연옥의안정성이확보된다면, 연옥을첨가한콘택트렌즈보관용기와다목적용액을통해가시아메바각막염의예방효과를개선할수있을것으로사료된다. REFERENCES 1) Naginton J, Watson PG, Playfair TJ, et al. Amoebic infection of the 513

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7 - 정재우외 : 연옥콘택트렌즈보관용기의항아메바효과 - = 국문초록 = 연옥이첨가된콘택트렌즈보관용기의항아메바효과 목적 : 연옥이첨가된콘택트렌즈보관용기의항아메바효과를일반콘택트렌즈보관용기와비교하였다. 대상과방법 : 5% 연옥을첨가하여제작된콘택트렌즈보관용기와일반보관용기에서가시아메바증식정도를비교하였고, 각각의보관용기에실리콘하이드로겔렌즈를보관한후가시아메바의부착정도를비교하였으며, 1% 및 5% 연옥수를포함한다목적관리용액에콘택트렌즈를보관한후가시아메바의부착정도를측정하여다목적관리용액단독에보관한경우와비교하였다. 결과 : 일반용기에비해연옥이포함된콘택트렌즈보관용기에서가시아메바의개체수가유의하게감소하였고 (p=0.02), 1, 2 및 3 세대렌즈를보관했을경우에도콘택트렌즈에부착된가시아메바수가각각유의하게감소하였다 ( 각각 p=0.001, p=0.001, 및 p<0.001). 1% 및 5% 연옥수를포함한다목적관리용액을사용하였을때다목적관리용액단독에비해 1 세대렌즈의경우부착된가시아메바개체수가유의하게감소하였으나 ( 각각 p=0.03 및 p=0.004), 2 및 3 세대렌즈에서는유의한차이를보이지않았다 ( 각각 p=0.59 및 p=3.612 와 p=0.450 및 p=0.462). 결론 : 연옥이포함된콘택트렌즈보관용기와연옥수는향후실리콘하이드로겔렌즈에의한가시아메바감염을효과적으로감소시킬수있는차세대물질로서가능성이있을것으로사료된다. < 대한안과학회지 2017;58(5): > 515

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04 JKOS hwp 대한안과학회지 2018 년제 59 권제 8 호 J Korean Ophthalmol Soc 2018;59(8):724-729 ISSN 0378-6471 (Print) ISSN 2092-9374 (Online) https://doi.org/10.3341/jkos.2018.59.8.724 Original Article 연옥이첨가된콘택트렌즈보관용기의항녹농균효과 Anti-pseudomonal