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1 대한생식의학회지 : 제 36 권제 3 호 2009 관동대학의과대학제일병원산부인과 1, 고려대학교의과대학산부인과학교실 2, 생식생물학물불임연구실 3, 진단병리과 4, 임상병리과 5 이현주 1 김선행 2* 양광문 1 박동욱 3 박찬우 1 홍성란 4 조동희 5 송인옥 1 궁미경 1 강인수 1 Expression of Decidual Natural Killer (NK) Cells in Recurrent Abortion Patients with Increased Peripheral NK Cells Hyun Joo Lee 1, Sun Haeng Kim 2*, Kwang Moon Yang 1, Dong Wook Park 3, Chan Woo Park 1, Sung Ran Hong 4, Dong Hee Cho 5, In Ok Song 1, Mi Kyoung Koong 1, Inn Soo Kang 1 1 Department of Obstetrics & Gynecology, Cheil General Hospital & Women's Healthcare Center, 2 Department of Obstetrics and Gynecology School of Medicine, Korea University, 3 Laboratory of Reproductive Biology & Infertility, 4 Department of Pathology, 5 Department of Clinical Pathology Objectives: The purpose of this study was to compare the decidual NK cell populations between increased pre-conceptional peripheral NK cell population and normal pre-conceptional peripheral NK cell population in women with a history of recurrent abortion. Methods: Fourteen women with history of recurrent abortion and elevated pre-conceptional peripheral NK cell, above 15% of peripheral lymphocyte population were included in this study. As a control, twelve women with history of recurrent abortion and their peripheral NK cell percentage showed below 15% were included. Distribution of CD56 + and CD16 + NK cells in paraffin embedded decidual tissues including implantation sites were examined by immunohistochemical staining using anti-cd56, 16 monoclonal antibodies. After immuohistochemical staining, the numbers of decidual NK cells were counted and compared these results between study and control groups. Results: There was significant difference in decidual CD56 + NK cell count (170.1±132.1 vs. 68.3±66.1, p=0.02) between increased peripheral CD56 + NK cell group and control group. But, there showed no statistically significant correlation between decidual CD56 + NK cell count and peripheral CD56 + NK cell percentage (r=0.229, p=0.261). Also there was no statistically difference decidual CD16 + NK cell count between study and control group (25.70±11.72 vs ±22.67), and no correlation between decidual CD16 + NK cell and peripheral CD16 + NK cell percentage (r=-1.40, p=0.535). Conclusions: This study shows that decidual CD56 + NK cell are significantly increased in decidua of women exhibiting a history of recurrent abortion with increased CD56 + peripheral NK cell. This study suggests that the percentage of peripheral NK cell reflect the expression of decidual NK cell. Consequently, pre-conceptional peripheral blood NK cell population can be the useful marker for detecting the risk of subsequent miscarriage. [Korean. J. Reprod. Med. 2009; 36(3): ] Key Words: Peripheral CD56 + NK cell, Decidual CD56 + NK cell, Recurrent abortion 주관책임자 : 김선행, 우 ) 서울특별시성북구안암동 5 가 126-1, 고려대학교의과대학산부인과학교실 Tel: (02) , Fax: (02) , ksh5335@kumc.or.kr

2 대한생식의학회지 반복유산 (recurrent spontaneous abortion, RSA) 은임신 20주이전에 2번 1 또는 3번이상 2 자연적으로임신이소실되는경우를말하며전체임산부의약 1% 의빈도로보고된다. 3 그원인으로는태아의염색체이상이 2.6~7.7%, 모체의해부학적이상이 15.4~27%, 내분비학적요인이 5.1~35%, 면역학적요인이 40%, 원인불명이 30~50% 를차지하고있다. 4 자연유산의원인중하나로거론되는면역학적요인은자신의세포혹은조직을공격하는특징을가진자가항체에의한자가면역과유전적으로반동종이식 (semi-allograft) 인배아를공격하는모체의면역세포들로이루어진동종면역이있는데최근에는동종면역거부반응에대한연구및중요성이점차증가되고있다. 한편반복유산의동종면역학적기전에관련된면역세포는자연살해세포 (Natural killer cell, NK cell), T 세포 (T cell), B 세포 (B cell), 대식세포 (Macrophage) 그리고수지상세포 (Dendritic cell) 등이있으며이들의세포는각각고유의역할을담당하면서상호보완또는경쟁적으로작용하여반이소성태아에대한모체의면역거부및면역관용에효과적인역할을수행한다. 5 반복유산의동족면역의기전으로자연살해세포의역할에대한보고가여러연구자에의해제시되었는데, Aoki 등 (1995) 은반복적으로유산을경험한여성에서말초혈액자연살해세포 (peripheral blood natural killer cell, pnk cell) 가증가되어있음을관찰하였으며, 임신전에말초혈액자연살해세포가증가되어있는경우다음임신시에도유산이될가능성이높다고보고하였다. 6 Yamada 등 (2003) 은화학적임신이나유산이되는환자군에서정상적으로분만을하는군보다임신전말초혈액자연살해세포가높아져있다고보고하였고, 7 Perricone 등 (2006) 은건강한비임신상태의여성보다반복유산군의비임신상태의여성이말초혈액자연살해세포가증가되어있음을보고하였다. 8 하지만여러연구에도불구하고말초혈액자연살해세포의증가가태아의소실을초래하는기전 에대해서는이견이많고고려해야될점이많다. 태아의융모막세포와모체혈액간의태반장벽의존재와, 말초혈액자연살해세포의표적세포에대한직접적인영향에대한연구가부족한점, 또한대부분이생체외실험의결과라는점등이말초혈액자연살해세포의증가가반복유산의직접적인원인으로인식되는데한계점이되어왔다. 따라서 extravillous trophoblast와직접접하게되는자궁내탈락막의면역세포의대부분 (75% 이상 ) 을차지하는자궁내자연살해세포의역할및그기원에대한연구가반복유산과의연관성하에많은연구자들에의해시행되었다. 말초혈액자연살해세포의대부분이 CD56, CD16 marker를발현하고강한세포독성 (cytotoxicity) 을가지는데비해 9,10 자궁자연살해세포는 CD56 marker 를강하게발현하며 CD16 marker를발현하지않으며, 세포독성은약하며다양한종류의싸이토카인을분비한다. 11,12 자궁자연살해세포는생리주기에따라그수와비율이변하여배란전의증식기에는거의보이지않다가배란후중기황체기에증가하기시작하여후기분비기에가장많이보인다. 13 임신이되지않고생리가시작되려하면자궁자연살해세포는세포자멸사 (apoptosis) 와유사한변화를보이며거의관찰되지않지만임신이될경우지속적으로존재하여태반이형성되는임신초기에는탈락막전체림프구의약 70% 를이루며임신 20주이후에는감소하기시작하여임신말기에는관찰되지않는다. 14 또한, 자궁자연살해세포는초기태반형성에관여하여반복착상실패 (recurrent implantation failure), 반복자연유산 (recurrent spontaneous abortion), 전자간증 (preeclampsia) 등에관계되어있을것이라는여러가설 15 들이발표되기는하지만검체채취의윤리성과검사방법의어려움으로아직까지이에대한연구가쉽지않은실정이다. 한편자궁내자연살해세포는그기원에있어첫째, 말초혈액자연살해세포가배란시기와임신초기에융모세포및자궁내막세포에화학적물질 (chemokine) 및세포접착물질 (cell adhesion mole

3 제 36 권제 3 호, 2009 이현주 김선행 양광문 박동욱 박찬우외 5 인 cule) 의작용에의해자궁내로이동하며그표현형이세포독성이강한 CD56 dim CD16 + 에서세포독성이약하고사이토카인등융모막침습과혈관생성에필수적인물질을분비하는역할을가진 CD56 bright 16 - 세포로진화한다는것과이시기에불완전한진화와동반된말초혈액자연살해세포의자궁내로의이동은반복유산의원인이된다는설, 둘째, 혈액내의전구세포에서기원한다는설, 셋째, 임파구내의 CD56 bright 16 - 자연살해세포가이동한다는설, 넷째, 줄기세포에서의기원설등여러가지학설이제시되었는데이중말초혈액의자연살해세포의기원및진화에관한학설이가장많이연구되어왔다. 16~19 따라서본연구에서는말초혈액의자연살해세포가증가된경우자궁내로이동하는자연살해세포의수적증가가있으며특별히세포독성이없는표현형으로의진화가부진한경우반복유산의원인이된다는가설하에반복자연유산환자에서자궁내탈락막자연살해세포의발현양상을관찰하여그양상이말초혈액자연살해세포의증가양상과어떠한관련이있는지를보고자하였다. 연구대상및방법 1. 연구대상 2005년 3월 1일부터 2008년 6월 30일까지관동의대제일병원습관성유산클리닉을방문한여성중임신 20주이전에 2번이상의반복유산을경험한유산환자를대상으로하였다. 습관성유산의원인을규명하기위한여러가지검사 ( 자궁난관조영술, 복강경및자궁경검사, 자궁경부균배양검사, 부부염색체검사, 자가항체검사 (anticardiolipin antibody, antinuclear antibody, antimicrosomal antibody, antithyroglobulin antibody, lupus anticoagulant), 동종면역진단을위한말초혈액자연살해세포의백분율측정, 배란 7일후황체호르몬검사, 자궁내막검사를시행하여그중원인이자연살해세포의증가에기인한면역학적요인의경우를실험군에포함하 였다. 자연살해세포의증가에의한반복유산진단시정의는임신전말초혈액을채취하고자연살해세포의백분율을측정하여 15% 이상이되는경우로정의하였다. 20 반복유산진단후임신 6~12주사이에다시계류유산이된환자들을대상으로자궁경부확장배출술과배아염색체검사를시행하였고이중임신전말초혈액자연살해세포의백분율이 15% 이상증가된 14명을실험군으로선택하였으며, 대조군으로는같은검사를시행한군가운데말초혈액에서자연살해세포의백분율이 15% 미만인 12명을포함하였다. 이들의계류유산검체에서착상부위를포함한자궁탈락막조직을확인하고, 탈락막조직이매우작거나융모만있는경우그리고내막조직만있는경우는연구에서제외하였다. 이연구는제일병원의연구윤리위원회의심의를거쳐수행되었다. 2. 말초혈액자연살해세포아형검사 (peripheral NK cell subtype test) 각각의환자로부터말초혈액 10 ml를채취한후 F-H gradient solution 2000 μl를이용하여림프구나큰단핵세포를세척, 분리하였다. CD16 + 및 CD56 + 자연살해세포에간접형광염색하여유세포분석기 (FACscan Becton-Dickinson Co, mautainview, USA) 를사용하여분석하였다. 항체는 CD16 - FITC/ CD56 - PE를사용하였다. 간접형광염색법에서는모두 FITCIgG1/PE-IgG2를음성대조물질로사용하였다. 즉, sodium heparin 진공시험관에채취한전혈 50 μl에형광색소가부착된해당세포의항체 20 μl 를첨가하고잘혼합한후실온암소에서 15분간반응시켰다. 적혈구용혈용액 2 ml를넣고소용돌이반응 (vortex) 를시키고실온암소에서 10분간반응시켰다. 5분간원심분리하여상층액은버리고 PBS로 1회세척한후 1% paraformaldehyde 용액을넣어잘섞어준후분석에사용하였다. 결과분석은 LYSISTMII sort program 을이용하였으며백분율을구하였다

대한생식의학회지 A B C D 400 400 400 400 Figure 1. Immunohistochemical staining for CD56 + NK cells in deciduas.

(A), H-E staining in decidua of study group; (B), anti-cd56 positive staining in serial section of (A); (C) H-E staining

Expression of Decidual Natural Killer (NK) Cells in Recurrent Abortion Patients with Increased Peripheral NK Cells.

Red arrows indicate positive stained CD16 + NK cells.

in decidua of control group; (D), anti-cd16 positive staining in serial section of (C). Hyun Joo Lee.

탈락막자연살해세포의아형검사 (Decidual NK cell subtype test) 1) 탈락막자연살해세포의면역조직화학염색 (Immunohistochemical staining) (1)

(2) 면역조직화학염색연구에사용한일차항체로는 CD56과 CD16에대한 mouse monoclonal antibody인항체 (novocastra TM, Vision BioSystems Ltd.

4 대한생식의학회지 A B C D Figure 1. Immunohistochemical staining for CD56 + NK cells in deciduas. Red arrows indicate positive stained CD56 + NK cells. (A), H-E staining in decidua of study group; (B), anti-cd56 positive staining in serial section of (A); (C) H-E staining in decidua of control group; (D), anti-cd56 positive staining in serial section of (C). Hyun Joo Lee. Expression of Decidual Natural Killer (NK) Cells in Recurrent Abortion Patients with Increased Peripheral NK Cells. Korean J Reprod Med A B C D Figure 2. Immunohistochemical staining for CD16 + NK cells in deciduas. Red arrows indicate positive stained CD16 + NK cells. (A), H-E staining in decidua of study group; (B), anti-cd16 positive staining in serial section of (A); (C) H-E staining in decidua of control group; (D), anti-cd16 positive staining in serial section of (C). Hyun Joo Lee. Expression of Decidual Natural Killer (NK) Cells in Recurrent Abortion Patients with Increased Peripheral NK Cells. Korean J Reprod Med 탈락막자연살해세포의아형검사 (Decidual NK cell subtype test) 1) 탈락막자연살해세포의면역조직화학염색 (Immunohistochemical staining) (1) 조직선택계류유산된연구대상에서자궁경부확장배출술로얻은조직을 formalin으로고정후 paraffin으로포매한 (embed) 검체를대상으로, hematoxylin-eosin 으로염색하여착상부위의탈락막을확인하였다. (2) 면역조직화학염색연구에사용한일차항체로는 CD56과 CD16에대한 mouse monoclonal antibody인항체 (novocastra TM, Vision BioSystems Ltd.) 를 1:200으로희석하여이용하였다. 대표적인조직절편을선택한후 5 μm 두께로잘라 xylene으로탈파라핀화 (deparafinization) 를하고 graded alcohol로 rehydration을했다. 10 mm citrate buffer (ph 6.0) 에서 5분간 microwave 를이용하여항원을추출하고, 1차항체를 4시간동안반응시킨후 LSAB kit과 DAB solution (DAKO cytomation, Carpinteria, CA) 을이용하여발색시켰다. (3) 면역조직화학염색의판독검경은 400배배율로탈락막부위 4부위를검경하여갈색으로염색된 CD56 양성반응, CD16 양성반응세포의수를각각세었다. 한편, 판독시가능한개인의오차범위를최대한줄이기위해숙련된병리학전공의사와각각분리하여 2회실시후그결과를처리하였다 (Figure 1, Figure 2). 2) 통계학적분석연구결과는 SPSS version 12.0 통계패키지 (Windows, Microsoft.WA, U.S.A) 를사용하여통계학적으로분석하였다. 두군간의비교는 Student's t-test를이용하였고상관관계에는 Pearson's corre

5 제 36 권제 3 호, 2009 이현주 김선행 양광문 박동욱 박찬우외 5 인 Table 1. Characteristics of patients with elevated peripheral NK cell group and normal peripheral NK cell group Peripheral NK cell <15% Peripheral NK cell 15% Significance Age (years) 32.0± ±3.6 a NS b Gravidity 3.6± ±0.7 NS Parity 0.2± ±0.3 NS No. of SA c 3.0± ±0.7 NS Peripheral CD56 + NK cell 12.3± ±6.3 p<0.00 Peripheral CD16 + NK cell 10.4± ±7.2 p=0.004 a; mean ± SD (standard deviation) b; NS, not significant c; SA, spontaneous abortion p<0.05 mean significant Hyun Joo Lee. Expression of Decidual Natural Killer (NK) Cells in Recurrent Abortion Patients with Increased Peripheral NK Cells. Korean J Reprod Med lation test를이용하였다. p<0.05인경우통계적으로유의하다고정의하였다. 결 과 1. 대상환자의특성 말초혈액의 CD56 + CD16 + NK cell이증가되어있는반복유산군 14명과증가되지않은반복유산군 12명의환자군사이에는평균연령 (33.4±3.6, 32.0 ±2.8), 임신력 (2.7±0.7, 3.6±1.8), 출산력 (0.1±0.3, 0.2±0.4), 평균자연유산횟수 (2.4±0.7, 3.0±1.4) 에차이가없었고말초혈액 CD56 + 자연살해세포의평균은각각 24.3±6.3, 12.3±1.8(%) 이고 CD16 + 의평균은각각 18.4±7.2, 10.4±3.9(%) 이었다 (Table 1). 2. 탈락막조직에서의면역염색말초혈액자연살해세포가증가되어있는반복유산환자군과대조군사이에탈락막 CD56 + 자연살해세포의숫자에있어말초혈액자연살해세포가증가된군에서 170.1±132.1이며대조군에서 68.3± 66.1를보여통계적으로유의한차이를보였다 (p= 0.02, Figure 3). 반면, 탈락막조직의 CD16 + 자연살해세포는말초혈액자연살해세포가증가된군과대조군사이에유의한차이를볼수는없었다 (25.70±11.72 vs ±22.67, p=0.49, Figure 4). Figure 3. Comparison of total count of decidual CD56 + NK cell in each group. Box and whisker plot showing quantification of total decudual CD56 + NK cells. Mean values represented in each boxes (p=0.02). Hyun Joo Lee. Expression of Decidual Natural Killer (NK) Cells in Recurrent Abortion Patients with Increased Peripheral NK Cells. Korean J Reprod Med 말초혈액자연살해세포와탈락막자연살해세포의상관관계 말초혈액자연살해세포와탈락막자연살해세포와의상관관계분석결과, 말초혈액 CD56 + 자연살해세포의백분율과자궁내 CD56 + 자연살해세포의총수와는직접적인상관관계를볼수없었다

대한생식의학회지 Figure 4. Comparison of total count of decidual CD16 + NK cell in each group. Box and whisker plot showing quantification of total decidual CD16 + NK cells.

6 대한생식의학회지 Figure 4. Comparison of total count of decidual CD16 + NK cell in each group. Box and whisker plot showing quantification of total decidual CD16 + NK cells. Mean values represented in each boxes (p=0.49). Hyun Joo Lee. Expression of Decidual Natural Killer (NK) Cells in Recurrent Abortion Patients with Increased Peripheral NK Cells. Korean J Reprod Med Figure 6. Correlation between decidual CD16 + NK cell and peripheral CD16 + NK cell. Scatter plot showing negative correlation between the peripheral and decidual CD16 + NK cell population, which was not statistically significant Pearson's correlation coefficient (r)=-1.40, r 2 =0.02, p=0.535 Hyun Joo Lee. Expression of Decidual Natural Killer (NK) Cells in Recurrent Abortion Patients with Increased Peripheral NK Cells. Korean J Reprod Med (r=0.229, p=0.261, Figure 5). 또한말초혈액 CD16 + 자연살해세포와탈락막 CD16 + 자연살해세포와도통계적으로유의한상관관계가없었다 (r=-1.40, p=0.535, Figure 6). 고 찰 Figure 5. Correlation between decidual CD56 + NK cell and peripheral CD56 + NK cell. Scatter plot showing positive correlation between the peripheral and decidual CD56 + NK cell population, which was not statistically significant Pearson's correlation coefficient (r)=0.229, r 2 =0.052, p=0.261 Hyun Joo Lee. Expression of Decidual Natural Killer (NK) Cells in Recurrent Abortion Patients with Increased Peripheral NK Cells. Korean J Reprod Med NK cell은선천면역계 (innate immune system) 의핵심적인요소로서각종바이러스, 박테리아와같은외부항원들에대응하여초기의면역반응을유발하는데중요한역할을하며, 표적세포에대한세포살해기능및 cytokine을포함한여러가지단백질을분비하는기능을갖고있다. 9,10 말초혈액자연살해세포 (peripheral NK cell) 는혈액내백혈구의 10% 정도를차지하며유세포분석에서대부분이 CD56 marker가약하게염색되며 CD16 marker가양성인세포 (CD56 dim CD16 + ) 소견을보이며강한세포독성을가지는것으로알려져있

7 제 36 권제 3 호, 2009 이현주 김선행 양광문 박동욱 박찬우외 5 인 다. 하지만말초혈액 NK cell의일부 (5% 미만 ) 의경우 CD56 marker가강하게나타나는반면 CD16 marker가보이지않는세포 (CD56 bright CD16 - ) 가관찰되는데이들은세포독성은거의없으며여러종류의 cytokine을분비하는기능이있는것으로보고되었다. 9~12 말초혈액 NK cell과습관성유산과의관련성에대해서는여러논문이발표되었는데, Kwak (1995) 등은 RSA 환자의임신전과임신중말초혈액을관찰하여 CD56 + CD16 + NK cell이정상군에비해서증가되었음을보고하였고, 임신전말초혈액자연살해세포의활동성 (NK cell activity) 의증가를보이는반복유산환자에서다음임신시유산율이유의하게높음을보고하였다. 21 Yamada (2003) 등은말초혈액내 CD56 + NK cell의 activity와백분율을각각 46% 와 16.4% 로 Cut-off value를정했을때화학적임신및자연유산의위험도가 3.6배및 4.9배로증가한다고보고하였으며, 7 차등 (2005) 20 은말초혈액 CD56 + NK cell 백분율 15% 를 cut-off value로하여조사해본결과반복유산을진단하는 sensitivity와 specificity가각각 68.8%, 92.9% 이며위험도는 3.4배증가한다고보고하였으며 20 저자들은위논문을본실험의실험군과대조군의설정의기준으로정하였다. 자궁내 NK cell은 CD56 marker 뿐아니라 CD9 를일정하게발현하며, 22 태아의융모막세포와의결합에작용하는 KIR 수용체 (CD19) 를특징적으로발현하는것으로알려져있다. 즉, 태아의 extravillous trophobalst의 nonclassical MHC Ib와상호작용하여국소적면역관용 (local immune tolerance) 상태를만들어융모막세포의침윤을가능하게한다. 이러한 Semi-allograft인배아의착상을가능하게하는탈락막의 CD56 + CD16 - NK cell은비임신시나증식기에는관찰되지않다가착상을준비하는후기분비기나태반이형성되는초기임신시에급격히증가하여그기원과분화에관해서여러가지가설이제시되고있다. 즉, 자궁내 NK cell의기원에대한가설에는혈액내의전구세포 (progenitor cell) 에서기원한다는설 22 과줄기세포 (stem cell) 에서기원한다는설등의여러가지의견이있지만, 현재로서는말초혈액 NK cell에서자궁내로이동, 분화한다는연구결과에무게가실리고있다. Heuvel 등 (2008) 은 circulating CD56 + NK cell은배란기및임신초기의두기간에걸쳐자궁조직으로이동하는데첫째는배란시기의호르몬에영향에의한 CXCR3 ligand를통하며두번째는 trophoblast 에서분비하는 SDF-1b가 NK cell의 CXCR4 ligand 에작용하는것과같은화학주성을통하여이루어진다고보고하였다. 16 한편, Xia Wu 등 (2005) 은임신제 1분기시탈락막의 CD56 bright CD16 - NK cell은 CXCR4, CXCR3라는 receptor를발현한다고하였으며 trophoblast는그의 ligand인 CXCL12/stromal cellderived factor 1을발현하여 NK cell의이동을유도하는세포주화성인자 (chemoattractors) 로서작용한다고하였다. 17 다른연구에의하면 Claudia Carlino 등 (2008) 은임신제 1분기의말초혈액 NK cell이 receptor들을비임신시의여성이나남성보다더많이발현하며이동능력이더높다고보고하였으며, 18 또한 Keskin 등 (2007) 은배양된탈락막기질세포에서분비되는 TGF-β가말초혈액유래 CD16 + NK cell을 CD16 - NK cell로분화시킴을보고하였다. 19 결과적으로다수의연구결과가말초혈액 NK cell이임신시탈락막과 trophoblast가분비하는여러싸이토카인에반응하여자궁내로이동한다는가설즉, 임신시자궁내막에존재하는낮은세포독성을갖는 CD16 - NK cell의기원이말초혈액의 CD16 + NK cell임을시사한다. 본연구에서는말초혈액 NK cell이증가되어있는반복유산환자에서자궁내 NK cell의수를분석하였다. 그결과말초혈액과탈락막의 NK cell 수간의일치되는상관관계를볼수는없었고, 탈락막내 CD16 + 를가진 NK cell의수는말초혈액에서검사된 CD16 + 의수와는상관관계또한볼수없었으나이는자궁내막의 NK cell의대부분이 CD56 + CD16 - 인점을본다면말초혈액에서자궁내막조직으로침습한 CD56 + CD16 + NK cell이여러요인에의해 CD56 + CD16 - NK cell로분화되어 CD16 + 표현형의경우상관관

8 대한생식의학회지 계를관찰할수없었던것이아닌가유추해볼수있다. 19 반면, 말초혈액 CD56 + NK cell이증가되어있는군에서자궁내 CD56 + NK cell이유의하게증가되어있는결과를볼수있었으며, 이는말초혈액의 NK cell이자궁내탈락막의 NK cell의발현을반영한다고볼수있다. 본연구에서는연구대상군과대조군에서염색체정상인군과비정상인군을따로분류하지않은상태에서실험을진행하였으며통계분석결과에서도염색체가정상인군과비정상인군간에통계적으로유의한차이를보이지않았다. 이는태아염색체이상이있는군에서도말초혈액 NK cell의백분율이높으면면역학적요인이있다고분류하였기때문이다. 결론적으로, 말초혈액 CD56 + NK cell의백분율이높은반복유산환자군에서자궁내 CD56 + NK cell 도역시증가되어있다는본연구의결과는, 말초혈액의 NK cell의백분율이자궁내 NK cell의발현을반영한다는근거를마련해주는연구라볼수있다. 하지만이미밝혀진 NK cell의다른 marker 들을비롯하여염색체이상이면역학적기전에미치는영향을배제하지않은것은본연구의한계점이라할수있으며, 연구대상군의수가많아져말초혈액과탈락막 NK cell간의상관관계를볼수있다면말초혈액의 NK cell이자궁내탈락막 NK cell의기원이라는가설을뒷받침할수있을것이라본다. 앞으로도본연구를뒷받침하는반복유산의동종면역학적요인인 NK cell의기원과세포살해능, 분비능에관한지속적인연구가필요하다고사료된다. 참고문헌 1. Hogge WA, Byrnes AL, Lanasa MC, Surti U. The clinical use of karyotyping spontaneous abortions. Am J Obstet Gynecol 2003; 189: Hannes M, Engler J, Gotilieb W, Dupont E. Recurrent spontaneous miscarriage. Rev Med Brus 1992; 13: Regan L. Recurrent miscarriage. Br Med J 1991; 302: Stray-Pedersen B, Stray-Pedersen S. Etiologic factors and subsequent reporductive performance in 195 couples with a prior history of habitual abortion. Am J Obstet Gynecol 1984; 148: Choudhury SR, Knapp LA. Human reproductive failure I: Immunological factors. Hum Reprod Update 2001; 7: Aoki K, Kajiura S, Matsumoto Y, Ogasawara M, Okada S, Yagami Y, et al. Preconceptional natural-killer-cell activity as a predictor of miscarriage. Lancet 1995; 345: Yamada H, Morikawa M, Kato EH, Shimada S, Kobashi G, Minakami H, et al. Preconceptional natural killer cell activity and percentage as predictors of biochemical pregnancy and spontaneous abortion with normal chromosome karyotype. Am J Reprod Immunol 2003; 50: Perricone R, Muzio G, Perricone C, Ciacomelli R, De Nardo D, Fontanna L, et al. High levels of peripheral blood NK cells in women suffering from recurrent spontaneous abortion are reverted from high-dose intravenous immunoglobulins. Am J Reprod Immunol 2006; 55: Lanier LL, Le AM, Ding A, Evans EL, Krensky AM, Clayberger C, Phillips JH. Expression of Leu-19 (NKH-1) antigen on IL2-dependent cytotoxic and non-cytotoxic T cell lines. J immunol 1987; 138: Cooper MA, Fehniger TA, Caligiuri MA. The biology of human natural killer-cell subsets. Trends Immunol 2001; 22: Orange JS, Ballas ZK. Natural killer cells in human health and disease. Clin Immunol 2006; 118: Cooper MA, Fehniger TA, Turner SC, Chen KS, Ghaheri BA, Ghayur T, et al. Human natural killer cells: a unique innate immunoregulatory role for the CD56(bright) subset. Blood 2001; 97: King A, Burrows T, Verma S, Hiby S, Loke YW. Human uterine leukocytes. Hum Reprod Update 1998; 4: Trundley A, Moffett A. Human uterine leukocytes and pregnancy. Tissue Antigens 2004; 63: Loke YW, King A. Human implantation: cell biology and immunology. Cambridge: Cambridge University Press 1995; van den Heuvel MM, Hatta K, Peralta CG, Han VK, Clark DA. CD56 + cells are recruited to the uterus in two waves: at ovulation and during the first 2 weeks after missed menses

9 제 36 권제 3 호, 2009 이현주 김선행 양광문 박동욱 박찬우외 5 인 Am J Reprod Immunol 2008; 59: Xia Wu, Li-Ping Jin, Min-Min Yuan, Ying Zhu, Ming-Yan Wang, Da-Jin Li. Human first-trimester trophoblast cells recruit CD56 bright CD16 - NK cells into deciduas by way of expressing and secreting of CXCL12/stromal cell-derived factor 1. The Journal of Immunology 2005; 175: Carlino C, Stabile H, Morrone S, Bulla R, Soriani A, Agostinis C, et al. Recruitment of circulating NK cells through decidual tissues: a possible mechanism controlling NK cell accumulation in the uterus during early pregnancy. Blood 2008; 111: Keskin DB, Allan DSJ, Rybalov B, Andzelm MM, Stern JNH, Kopcow HD, et al. TGFβ promotes conversion of CD16 + peripheral blood NK cells into CD16 - NK cells with similarities to decidual NK cells. PNAS 2007; 104: 차선화, 김해숙, 김혜옥, 송인옥, 유근재, 궁미경, 강인수, 양광문. 습관성유산환자에서저용량면역글로블린치료와말초혈액내 Natural Killer(NK) 세포의임계치에관한연구. Kor J Fertil Steril 2005; 132: Kwak JY, Beaman KD, Gilman-Sachs A, Ruiz JE, Schewitz D, Beer AE. Up-regulated expression of CD56 +, CD56 + / CD16 +, and CD19 + cells in peripheral blood lymphocytes in pregnant women with recurrent pregnancy losses. Am J Reprod Immunol 1995; 34: Moffett-King A. Natural killer cells and pregnancy. Nat Rev Immunol 2002; 2: Chantakru S, Miller C, Roach LE, Kuziel WA, Maeda N, Wang WC, et al. Contributions from self-renewal and trafficking to the uterine NK cell population of early pregnancy. J Immunol 2002; 168: = 국문초록 = 목적 : 말초혈액자연살해세포가증가된반복유산환자군과증가되지않은반복유산환자군에서다음번유산시탈락막의자연살해세포의발현양상을관찰하여말초혈액자연살해세포와상관관계가있는지보고자하였다. 연구방법 : 말초혈액자연살해세포가 15% 이상증가된반복유산환자군 14명을실험군으로, 15% 이하인군 12명을대조군으로하여연구를진행하였다. 이환자들에게서다음번유산시착상부위를포함한자궁탈락막세포를확인하여면역조직화학염색을통해 CD56 + 와 CD16 + 자연살해세포의수를세어실험군과대조군을비교하였다. 결과 : 실험군과대조군은탈락막내 CD56 + 자연살해세포의수간에통계적으로유의한차이 (170.1±132.1 vs ±66.1, p=0.02) 를보였으나둘간의상관관계 (r=0.229, p=0.261) 는없었다. 또한두군간에 CD16 + 자연살해세포는통계적으로유의한차이 (25.70±11.72 vs ±22.67) 를보이지않았다. 결론 : 본연구는말초혈액 CD56 + 자연살해세포가증가된반복유산환자군에서말초혈액자연살해세포가증가되지않은군에비하여탈락막의 CD56 + 자연살해세포가증가되어있음을알수있었다. 이는탈락막자연살해세포가반복유산의면역학적기전에서중요한역할을할것이라는가능성을제시해주는연구결과이며또한말초혈액자연살해세포의측정이반복유산환자에서다음번유산을예측할수있는유용한지표임을알수있다. 중심단어 : 말초혈액 CD56 + 자연살해세포, 탈락막 CD56 + 자연살해세포, 반복유산

Microsoft Word - 04-양광문.DOC

Microsoft Word - 04-양광문.DOC 大韓不妊學會誌 : 第 32 卷第 3 號 2005 Kor. J. Fertil. Steril., Vol. 32, No. 3, 2005, 9 습관성유산환자에서저용량면역글로불린치료와말초혈액내 Natural Killer (NK) 세포의임계치에관한연구 성균관대학교의과대학, 삼성제일병원산부인과불임및생식내분비 차선화 김해숙 김혜옥 송인옥 유근재 궁미경 강인수 양광문 The