임상병리검사과학회지 : 제 28 권제 1 호 PCNA 와 Cytokeratin 단클론항체를이용한 2 중변역조직화학염색방법에관한연구 원광대학교의과대학병리학교실 한국생명공학연구소 * 문형배 노종섭 곽효일 유대열 . 한용만 . 이경광 * The Study on

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1 임상병리검사과학회지 : 제 28 권제 1 호 PCNA 와 Cytokeratin 단클론항체를이용한 2 중변역조직화학염색방법에관한연구 원광대학교의과대학병리학교실 한국생명공학연구소 * 문형배 노종섭 곽효일 유대열 *. 한용만 *. 이경광 * The Study on the Method of Double Immunohistochemical Stain using Monoclonal PCNA and Cytokeratin Antibodies Moon, Hyung-Bae., Roh, Jong-Sup., Kwak, Hyo-Il Yu, Dae - Yeul *., Han, Y ong - Mahn *., Lee, Kyung - K wang* Dept. of Pathl 여 O 앓 ~ Wonkwang University Sch 이 of Medicine, Iksan, Korea Korea Research Institute of Bioscienæ and Biotechn 여탱 y*, Taejon, Korea A comparative study of double immunohistochemical stain using monoclonal proliferating cell nuclear antigen(pcna) and cytokeratin(ck) antibodies in the same tissue was applied to a tissue from colonic adenocarcinoma which was fixed by formalin and embedded in paraffin. The expression of the PCNA and CK antigen in the same slide was excellent in the tissue by the first application of PCNA stain and later applification of the CK stain, but expression of PCNA was poor in the tissue by the first application of CK stain and later application of the PCNA stain. All three observers prefer to the combination of the red colored expression of PCNA antigen by AEC in the nuclei and bluecolored ex pression of cytoplasmic CK antigen by fast blue than that of blue-colored nuclei and red - colored cytolasm. Above results indicate that the immunostains needed pretreatment of proteolytic enzyme might by stained after the immunostains needed no pretreatment of proteolytic enzyme for the detection of antigens in the double immunohistochemical stain. Key words : double immunohistochemistry - PCNA - cytokeratin - proteolytic enzyme 이논문은 1996 년도원광대학교교비지원에의하여연구됨

2 1. 서론 면역조직화학염색은, 조직내에존재하지만 일반적인 hematoxylin eosin 염색및조직화학 적염색으로는확인되지않는특수한항원을 면역학적인방법을이용하여조직내에서발현 시킴으로서조직및세포의형태를유지하면서 조직및세포의기원뿐아니라여러종류의혼 합된세포중에서도관찰하고자하는특정세포 의상태를알수있으며, 각종질환의진단, 병인연구, 치료방법제시및예후추정등에이 용할수있으므로외과병리학분야뿐만아니 라기초자연과학분야에서도많이이용하고 있다 1-5) 그러나일반적으로이용되고있는면 역조직화학염색방법은조직표본에서한가지 항원만을파악하는데그치지만, 동일조직표본 내에서두가지이상의항원을확인해야할경 우가생기므로서다중면역조직화학염색법즉, 2 중또는 3 중면역조직화학염색방법을개발 하여이용하고있다 6-9) 그러나 2 중또는 3 중 면역조직화학염색법은대중화되어있지않으 며염색방법에따른염색효과가다양하게나 타나므로 2 중또는 3 중면역조직화학염색법 에대한연구가더욱필요한실정이다. 이에 핵에서발현되는항원과세포질에서발현되는 항원을동일조직표본내에서발현시키는방법 을이용하여, 염색순서및사용되는발색제를 서로다르게적용함으로인해나타나는서로 다른염색결과를비교관찰하여가장좋은면 역조직화학염색법을개발하고자본연구를시 도하였다. 1. 연구대상조직편 II. 연구대상및방법 면역조직화학염색에서항원의발현은대상 조직의고정방법, 항원의종류및염색방법에 따라다르게나타나는데, 일반적으로동결절편 을이용하는것이포르말린고정파라핀포매 조직보다양호하다고알려져있다 10-12) 따라서 본연구에서는임상적으로가장많이이용하고 있는포르말린고정파라핀포매조직편을이 용하였으며, 대상조직은원광대학교의과대학 부속병원에서대장선암종으로인해절제된대 장에서육안적으로선암종조직및정상대장 조직이함유되었다고판단한조직편을 10% 중성포르말린용액에적정시간고정한후, 통 상적인방법으로제작한파라핀포매괴에서 4 때두께의연속절편을채취하여본연구에이 용하였다. 2. 면역조직화학염색 본연구에이용한일차항체들은핵내에존 재하면서세포주기에관여한다고알려져있는 proliferating cell nuclear antigen(pcna) 항원 (PCNA DO-10, DAKO, CA, USA, 1: 20으 로희석하여사용 ) 과상피세포의세포질에서 염색되는 cytokeratin 항원 (CK-19, DAKO, CA, USA, 1 : 50 으로희석하여사용 ) 을이용하 였다. 본연구에이용한면역조직화학염색방 법을간략하게설명하면다음과같다. 4 tjffi 두 께로박절한조직편을상용화된 probe on plus slide(fisher Scientific, Pittsburg, USA) 에부 착하여 58 0 C 오븐에서 30 분동안방치한후, xylene 으로파라핀을제거하고무수알코을 4 회 거친후증류수로세척하였다. Auto blocker (Research genetics, AL, USA) 로 10 분간 endogenous peroxidase를저지시킨후 lmmuno/ DNA 완충액 (Research genetics, AL, USA) 으 로 4 회세척한다음 protein blocker(research genetics, AL, USA) 를실온에서 10 분간처치 하였다. 적당한농도로희석한일차항체를실 온에서 30 분간작용시킨후 lmmuno/dna 완 충액으로 4 회세척하고 2 차항체로서 universal secondary antibody(dako, CA, USA) 를실온 에서 15 분간작용시킨후 lmmuno/dna 완충 액으로 4 회세척하였다. Streptavidin AP detectlon system 또는 streptavidin HRP detec

3 tion system(dako, CA, USA) 들은각각실온 에서 15 분간작용시켰으며, 그후 lmmuno/ DNA 완충액으로 4 회세척하고, 두가지항원 의 발현을구별하기위하여 HRP system 을이 용한경우발색제는적갈색으로염색되는 amlnoethyl carbaz 이 e(aec) 과 AP detection system 을이용한경우푸른색조로염색되는 fast blue(research genetics, AL, USA) 를이용하 였으며, 각발색제는실옴에서 10 분간작용시 켰다. 2 중면역조직화학염색을시행한경우 한가지염색을종료한후슬라이드를실온에서 1 시간동안 double stain enhancer( Zymed, CA, USA) 에담궈둔후다른종류의면역조직 화학염색을시행하였으며, 염색이종료된다음 lmmuno/dna 완충액및증류수로 4 회씩세척하고 universal mount(research genetics, AL, USA) 로봉입하였다. PCNA 및 CK 에 대한염 색방법은 CK 염색을실시하기전 45 0 C 에서 4 분동안 pepsm 용액 (Research Genetics, AL, USA) 에 슬라이드를처치한후염색을진행시 킨점외에는동일하였다. blue, PCNA - AEC, CK -fast blue 및 CK AEC 조합으로염색하였다. 4. 실험군 a) PCNA - blue/ck - AEC blue군 PCNA 에대한염색을 fast blue 발색제를이용하여먼저시행하고, 나중에 CK에대한염색을 AEC 발색제를이용하여시행하였다. b) PCNA-AEC/CK-blue군 PCNA 에대한염색을 AEC 발색제를이용하여먼저시행하고, 나중에 CK 에대한염색을 fast blue 발색제를이용하여시행하였다. c) CK - blue/pcna - AEC군 CK에대한염색을 fast blue 발색제를이용하여먼저시행하고, 나중에 PCNA에대한염색을 AEC 발색제를이용하여시행하였다. d) CK-AEC/PCNA-blue군 CK 에대한염색을 AEC 발색제를이용하여먼저시행하고, 나중에 PCNA에대한염색을 fast blue 발색제를이용하여시행하였다. 3. 대조군 ID. 성적및고찰 2중면역조직화학염색에서사용하였던 PCNA 및 CK에대한염색을시행하였으며, 발색제는 fast blue 및 AEC를이용하여 PCNA-fast 1. 조직편의탈락여부 면역조직화학염색에서흔히문제되는점은 Table 1. General consideration of the each methods on double immunohistochemical stain for the proliferating cell nuclear antigen(pcna) in the nuclei and cytokeratin(ck) antigen in the cytoplasms. PCNA - AECj PCNA - bluej CK-AECj CK-bluej CK-blue CK-AEC PCNA-blue PCNA-AEC Attachment of tissue on slide Good Good Good Good Nonspecific staining reactions No No No No Sensitivity of antigenic expression Good Good Poor Poor Specificity of antigenic expression Good Good Poor Poor Contrast between nucleus & cytoplasm Good Moderate Poor Poor PCNA - AECjCK - blue means the first PCNA stain using AEC as detections system and later CK stain using the fast blue as detection system. PCNA - bluejck - AEC, CK - AECjPCNA - blue and CK - bluejpcna - AEC are same sequencial application as above PCNA - AECjCK - blue

염색시행도중여러가지화학물질에노출되며, 간혹가열을하여야하는경우가있어슬라이드에부착한조직편이탈락되는경우가있는데, 본연구대상인 2중면역조직화학염색의경우에는통상한번만시행되는면역조직화학염색에비하여 2배이상화학물질에노출되기때문에조직편의탈락이염려되었으나염색도중조직편이탈락된경우는없었다 (Table 1 ).

A photomicrograph shows the expression of PCNA in the nuclei by the AEC as a detection system.

대조군의항원발현도는 PCNA의경우암종세포대부분에서발현되었으며, 핵이다양한형태로변한경우핵의모양에따라 PCNA의발현양상도바뀌었으며, 암종이침범하지않은주위점막에서는점막에위치하는세포의일부에서양성반응을나타내었다 (Figure 1). 대조군의 CK 발현은모든상치세포에서양성반응을나타내었으나암종세포에서더욱강하게발현되었다 (Figure 2).

4 염색시행도중여러가지화학물질에노출되며, 간혹가열을하여야하는경우가있어슬라이드에부착한조직편이탈락되는경우가있는데, 본연구대상인 2중면역조직화학염색의경우에는통상한번만시행되는면역조직화학염색에비하여 2배이상화학물질에노출되기때문에조직편의탈락이염려되었으나염색도중조직편이탈락된경우는없었다 (Table 1 ). 이는조직편의탈락을예방하기위하여특수처리된 probe one plus 슬라이드를사용하였기때문으로생각되었다. 2. 비특이성염색반응면역조직화학염색에서문제시되는비특이성반응은 2중면역화학염색을시행하여도출현하지않았다 (Table 1). 3. 염색의민감도및특이도 Flgure 1. A photomicrograph shows the expression of PCNA in the nuclei by the AEC as a detection system. The positivity of the PCNA expression is more frequent in the adenocarcinoma (right) than in the normal nucosal(left) of the colon. Single immunohistochemical stain for PCNA. 대조군의항원발현도는 PCNA의경우암종세포대부분에서발현되었으며, 핵이다양한형태로변한경우핵의모양에따라 PCNA의발현양상도바뀌었으며, 암종이침범하지않은주위점막에서는점막에위치하는세포의일부에서양성반응을나타내었다 (Figure 1). 대조군의 CK 발현은모든상치세포에서양성반응을나타내었으나암종세포에서더욱강하게발현되었다 (Figure 2). 2중면역조직화학염색을시행한모든실험군에서 CK 염색도대부분의상피세포에서발현되었으나, 점막하층, 근육충등의비상피세포에서는발현되지않아민감도및특이도가매우높음을알수있었으며, 이는대조군의성적과동일하였다. 2중면역조직화학염색을시행한경우 PCNA 염색을먼저시행한 PCNA-fast bluejck-aec 및 PCNA - AECjCK - blue군의 PCNA 항원은 PCNA 염색만시행한대조군과비슷한염색양상을보여민감도및특이도가매우높음을알수있었다 (Figure 3). 그러나 CK 에대한염색을 Figure 2. A photomicrograph shows the expression of cytokeratin(ck) in the cytoplasm by the fast blue as a detection system. The intensity of the stain was stronger in the adenocarcinoma (right) than in the normal mucosal(left) of the colon. Single immunohistochemical stain for cytokeratin

Figure 3. A photomicrograph shows good expres- Figure 5.

This doube immunohistochemical stain sion of CK in the cytoplasm.

by the AEC and later applica by the fast blue as the detection system.

Double immunohistochemical stain for the PCNA and cytokeratin 먼저시행하고 PCNA 에대한염색을나중에 시행했던 CK - bluejpcna - AEC 및 CK AECjPCNA - blue 군은 CK 에대한염색은양 호하였으나 PCNA 에대한염색은매우불량하 여 CK

5 Figure 3. A photomicrograph shows good expres- Figure 5. A photomicrograph shows poor expressions of the PCNA in the nuclei and CK in the sion of the PCNA in the nuclei and good exprescytoplasm. This doube immunohistochemical stain sion of CK in the cytoplasm. This doube immuno was done in order of the first application of histochemical stain was done in order of the first PCNA by the AEC and later application of CK application of CK by the AEC and later applica by the fast blue as the detection system. tion of CK by the AEC and later application of Double immunohistochemical stain for PCNA and PCNA by the fast blue as the detection system. cytokeratin. Double immunohistochemical stain for the PCNA and cytokeratin 먼저시행하고 PCNA 에대한염색을나중에 시행했던 CK - bluejpcna - AEC 및 CK AECjPCNA - blue 군은 CK 에대한염색은양 호하였으나 PCNA 에대한염색은매우불량하 여 CK 염색을먼저시행한경우 PCNA 에대 한판독이어려웠다 (Figure 4, 5, Table 1). 이 상의소견은 CK 에대한염색과정중 pepsm 전처리를시행해야하므로 13) pepsm 전처리가 PCNA 에대한염색반응을억제하였을것으로 Figure 4. A photomicrograph shows poor expression of the PCNA in the nuclei and good expression of CK in the cytoplasm. This doube immunohistochemical stain was done in order of the first application of CK by the fast blue and later application of PCNA by the AEC as the detection system. Dωble immunohistochemical stain for the PCNA and cytokeratin 사료되어 pepsm 이나 trypsin 등의단백분해효 소의전처리가필요한염색과단백분해효소의 전처리과정이필요하지않은염색을같이시 행해야하는경우단백분해효소의처리가필요 한염색을나중에시행해야함을알수있었 다

This double immunohistochemical stain was done in order of the first application of PCNA by the fast blue and later application of CK by the AEC as the detection system.

6 Figure 6. A photomicrograph shows good expression of the PCNA in the nuclei and CK in the cytoplasm, but the differentiation between nuclear and cytoplasmic antigen is not so good to compare with the Figure 3. This double immunohistochemical stain was done in order of the first application of PCNA by the fast blue and later application of CK by the AEC as the detection system. Double immunohistochemical stain for the PCNA and cytokeratin 방법은핵과세포질의대비는비교적양호하였으나세포질내 CK항원을붉은섹조로염색하고, hematoxylin으로핵을대조염색한통상적인단일면역조직화학염색표본과비슷하여서로혼동할우려가있었으며, hematoxylin - eosm 염색과도핵과세포질의염색대비가비슷하여판독자들의선호도가감소하지않았나생각된다 (Figure 6, Table 1). 그러나핵및세포잘에존재하는다른항원을발현시키는데이용되는발색제는상호간에구분이명확한발색제를이용하는경우판독자의취향에따라선호도가다른것으로생각되었다. 이상의성적및고찰을종합하면 2중면역조직화학염색을시행할때단백분해효소의전처치가필요한염색은단백분해효소의전처치가필요없는다른종류의면역조직화학염색을한후에시행해야할것으로사료되었으며, 핵에존재하는항원의발현은붉은색을이용하고세포질에존재하는항원은푸른색의발색제를사용하는것이가장권장할만한방법으로사료되었다. N. 결론 4. 발색제조합선호도면역조직화학염색에서발색제의선택은 2차항체와발색제간의화학적반응도와면역조직화학염색을판독하는판독자의취향에따라서선택되는데, 본연구에서는 hematoxylin - eosin 염색에서이용되는붉은색조와푸른색조의조합을사용하였으며, 2중면역조직화학염색결과핵에위치하는 PCNA항원이붉은색으로염색되고세포질에있는 CK항원은푸른색으로염색되는방법을 3명의판독자모두가가장우수하다고하였는데, 그이유로핵과세포질의염색상대비가다른방법에비해뚜렷하다고판정하였닥. 한편핵내의 PCNA항원을푸르게염색하고, 세포질내 CK항원을붉게염색한 핵에존재하는 PCNA 항원과세포질의 CK 항 원의동일조직내 발현양상및핵과세포질의 발색제조합선호도를관찰하기위하여, 포르 말린고정파라핀포매된대장선암조직을이 용하여 2 중면역조직화학염색을시행한결과 다음과같은결과를얻었다. 1. PCNA 염색을먼저시행하고 CK 염색을 나중에시행한경우 PCNA 및 CK 염색은 매우양호하였으나, CK 염색을먼저시행 하고 PCNA염색을나중에시행한경우 CK염색은비교적양호하였으나 PCNA 염색이매우불량하였다. 2. PCNA 는 AEC 를이용하여붉게, CK 는 fast blue 를이용하여푸르게염색하는조 합을가장선호하였다. 이상의 소견으로 2 중면역조직화학염색을

7 시행할때단백분해효소의전처치가필요한 염색은단백분해효소의전처치가필요없는 다른종류의면역조직화학염색을한후에시 행해야할것으로사료되었다. REFERENCES 1. Cuello AC. lmmunohistochemistry. Chichester, John Wiley & Sons, 1-8, Beesley JE. lmmunocytochemistry, a prac tical approach. Oxford, Oxford University Press, 1-3, Taylor CR, Kledzik G. lmmunohistologic techniques in surgical pathology. A spectrum of new" special stains. Human Pathol 191 : 12 : DeLellis RA. Basic techiniques of immunochemistry. Diagnostic lmmunohistochemistry, New York, Masson Publishing, 7-16, Banks PM. Diagnostic application of an immunoperoxidase method in hematopathology. J Histochem Cytochem 27: , Noorden SV, Stuart MC, Cheung A, Adams EF, Polak JM. Localization of human pituitary hormones by multiple immunoenzyme staining procedures using monoclonal and polyclonal antibodies. J Histochem Cytochem 34 : , Antica M, Heiss M, Kummer U, Munker R, Thiel R Thierfelder S. Simultaneous demonstration of two antigens on single T cells using antibodies with contrasting labels. J lmmunol Methods 87: , Shi Z-R Itzkowitz SH, Kim YS. A comparison of three immunoperoxidase techniques for antigen detection in colorectal carcinoma tissues. J Histochem Cytochem 36 : , Van der Loos SV, Das PK, Houthoff HJ. An immunoenzyme triple staining method using both polyclonal and monoclonal an tibodies from the same species. Application of combined direct, indirect and avidinbiotin complex(abc) technique. J lmmunochem Cytochem 35 : , Vandesande F. lmmunohistochemistry Cuello AD ed., New Y ork, Hohn wiley & sons , Ong SL, Steiner AL. Localization of cyclic GMP and cyclic AMP in cardiac and skeletal muscle lmmunocytochemical demonstration. Science 195 : 183-5, Vivien - Roles B, Pevet P, dubois MP, Arendt J, Brown GM. lmmunohistochemical evidence for the presence of melatonin in the pineal gland, the retina and the Harderian gland. Cell Tiss Res 217 : , Moll R, Franke WW, Schiller DL, Krepler R. The catalogue of human cytokeratins : Patterns of expression in normal epithelial, tumors and cultured cells. Cell 31 : 11-24,

Jkbcs016(92-97).hwp

Jkbcs016(92-97).hwp Expression of bcl-2 and Apoptosis and Its Relationship to Clinicopathological Prognostic Factors in Breast Cancer - A Study with Long Term Follow-up correlated with the survival rate.(journal of Korean