<30332EBFF8C0FA2DB1E8BFB5C7D12E687770>

Size: px

Start display at page:

Download "<30332EBFF8C0FA2DB1E8BFB5C7D12E687770>"

보라 영
5 years ago
Views:

1 Korean Journal of Obstetrics and Gynecology Vol. 51 No. 11 November 2008 정상산모와중증전자간증산모의태반에서 Matrix Metalloproteinase (MMP) -2, -9 의발현양상 연세대학교의과대학산부인과학교실 문여정 황한성 김영한 권자영 박용원 Differential expression of Matrix Metalloproteinase (MMP)-2, -9 in normal and severe preeclamptic human placentas Yeo Jung Moon, M.D., Han-Sung Hwang, M.D., Young-Han Kim, M.D., Ja-Young Kwon, M.D., Yong-Won Park, M.D. Department of Obstetrics and Gynecology, Yonsei University Health System, Seoul, Korea Objective: The aim of the study was to investigate the differential expression of Matrix Metalloproteinase (MMP) -2,-9 in the normal and severe preeclamptic placenta. Methods: Placentas were obtained from women undergoing cesarean section with normal (n=10) and severe preeclamptic (n=10) pregnancies. Semi-quantitative RT-PCR was done to detect the placental gene expression of MMP-2,-9. Western blot analysis was performed to identify MMP-2,-9 protein expression in each placenta. Immunohistochemical staining were employed to localize MMP-2,-9 in placental tissues. Results: MMP-2,-9 genes were expressed in both normal and severe preeclamptic placenta. There were lower expressions MMP-2,-9 in severe preeclamptic placentas than in normal. MMP-2,-9 proteins were all present in each placental tissue. The expression for MMP-2,-9 was weaker in severe preeclamptic placenta than in normal. MMP-2,-9 were localized only to the trophoblast, and were also weakly positive in severe preeclamptic placenta compared with normal. Conclusions: MMP-2,-9 expressions were decreased in severe preeclampsia placenta compared to those from normal placenta. This study suggests that decreased expression of MMP-2,-9 may have a role in the development of severe preeclampsia. Key Words: MMP-2, MMP-9, Severe preeclampsia, Placenta 접수일 : 채택일 : 교신저자 : 김영한 yhkim522@yuhs.ac 서 론 임신중고혈압과단백뇨가특징인전자간증은다양한 기관에영향을미쳐태아에게는자궁내태아발육제한, 자궁내사망, 조산및미숙아와관련된합병증을유발하며, 임신부에게자간증, 신부전, 폐부종, 뇌졸중및사망등으로이어질수있다. 1 여러가설들 -염증성질환, 혈관관련물질, 태반허혈, 유전적요인, 비정상면역반응등- 이전자간증의병태생리로제시되고있으나, 2,3 최근에는많은연구들이비정상적인태반형성및태반허혈 (abnormal

2 대한산부회지제 51 권제 11 호, 2008 placentation and placental vasculature insufficiency) 에기인한저산소증에의해어떤특정물질이분비되어임산부의혈관조직에작용함으로써전자간증의다양한임상양상이발생된다는이론이주로받아들여지고있다. 4,5 세포외기질분자, 단백분해효소 (extracellular matrix molecule, degrading enzyme) 는유착분자와세포외기질을분해하여영양막의분화및침습을도움으로써태반의형성과정에서중요하게작용하며대표적인물질로 matrix metalloproteinase (MMP) 가있으며이중 MMP-2 와 MMP-9 은세포외기질의주된물질인 IV형콜라젠과젤라틴을분해한다. MMP-2 는임신 6주정도부터분비되며점차감소하여임신 11주에는임신 6주에비하여 1/6 정도로감소하며임신말기까지지속적으로분비되는반면 MMP-9 는임신 6 주에는측정이안되다임신 7주부터증가하여임신 11주에는임신 7주때의 10배이상측정되나임신말기로갈수록감소한다. 6 MMP-2 는임신초기부터말기에걸쳐융모외영양막세포 (extravillious trophoblast) 에서나타나고, MMP-9 는임신초기융모영양막세포 (villous cytotrophoblast) 에서나타난다. 7 MMP-2,-9 은배란, 임신초기배아의착상및발생과정, 자궁경부숙화, 양막파수및태반분리등의정상임신과정뿐만아니라, 8-14 임신성융모질환, 조기양막파수및조기진통, 유착태반, 임신성당뇨, 전자간증등의이상임신의병태생리와밀접한관련을보인다 MMP-2,-9 의발현이적게이루어져세포외기질의적절한분해가이루어지지않아초기태반형성이부족하게되고, 이에따라서전자간증이일어날것으로생각된다. 그러나, 실제발표된논문들은전자간증에서 MMP-2,-9 의농도및태반발현을다르게보고하고있다. Galewska 등은전자간증의제대혈에서 MMP-2 는감소하고 MMP-9 은증가한다고보고하였다. 19 Narumiya 등은정상산모에비해전자간증산모혈장내 MMP-2 가임신기간중높게유지된다고보고하였다. 20 Huisman 등은전자간증, 자궁내태아발육지연등의이상임신과정상임신의임신초기태반내 MMP-2,-9 의발현이차이가없다고보고하였으나, 21 Merchant 등은자궁내태아발육지연임신에서는태반내 MMP-2,-9 의발현이감소되어있음을보고하였 다. 22 이상에서 MMP-2,-9 은임신과정에서중요한역할을하는것을알수있으며, 특히전자간증의병태생리를이해하는데도움을줄것으로사료된다. 이에본연구에서는중증전자간증 ( 이하전자간증 ) 임신부와정상임신부에서태반조직내의 MMP-2 와 MMP-9 의발현차이를살펴보고자하였다. 연구대상및방법 1. 연구대상본연구는 2005 년 6월 1일부터 12월 31일까지 6개월간본원에서정기적인산전진찰후분만한산모를대상으로하였다. 실험에참여한산모는총 20명으로정상산모 10 명과전자간증산모 10명을대상으로하였다. 참여산모들은모두제왕절개분만을시행하였고, 그때분만된태반을대상으로하였다. 제왕절개술의적응증으로정상산모중기왕제왕절개술력 8명, 기왕자궁근종절제술력 1명, 둔위태위 1명이었으며전자간증산모중기왕제왕절개술력 1명, 태아절박가사 2명, 자간증전구증상발현 7명이었다. 임신주수의산정은각산모의월경력과임신제1삼분기의태아초음파소견을고려하여적절하다고판단되는지표를기준으로삼았다. 중증전자간증의정의는임신 20 주가지나서고혈압과단백뇨가있는산모로정의하였고, 6시간이상간격을두고측정한혈압이수축기가 160 mmhg, 이완기가 110 mmhg 이상인경우를고혈압으로하였고, 4시간이상간격을두고측정한단백뇨가 dipstick 검사상적어도 3+ 이상이거나 24시간소변에서 5,000 mg 이상인경우로정의하였다. 23 그리고각각의정의는적어도두번이상의연속검사로반복확인하였다. 저출생체중아가동반된전자간증은실험대상에서제외하였으며, 24 그이외다태임신, 태아의구조적및유전적이상, 산모의기왕고혈압병력이있는경우및산모가기왕에신장질환, 간질환, 당뇨병, 결체조직질환등내과적, 외과적질환을가지고있는경우실험에서제외하였다. 임신과관련된기타의질병이나약물복용의기왕력이있거나조기분만의위험요소를가지고있는산모는실험에서제외되었다

3 문여정외 4 인. Differential expression of MMP-2/-9 in normal and preeclamptic human placentas 2. 연구방법 1) 검체수집각각의태반조직은제왕절개분만이이루어진후나온태반을대상으로하였다. 분만후 15분이내에태반조직을제대혈관부위중전체태반의중앙부분에서가로, 세로각각 1 cm 3 의크기로절제하여분석전까지 -70 의질소탱크에보관하였고, 나머지태반조직은 4% formal saline 에담근후 48시간이내에 paraffin wax 를처치하여면역조직화학염색법을위한슬라이드를만들때까지보관하였다. 2) Reverse transcriptase-polymerase chain reaction (RT-PCR) 실험을위한전체 RNA는 easy-spin total RNA extraction kit (Intron, Biotechnology, Sung Nam, Korea) 를사용하여태반으로부터분리하였다. 분리된 RNA 는 oligo (dt) primer 와 M-MLV reverse transcriptase (Fomentas life science, USA) 를사용하여역전사 (reverse transcription) 를실시하여 cdna 를합성하였다. 사용된 primer 들은 Table 1에서보여진것과같다. 이때의상태 (condition) 는 5분간 70, 60분간 37, 10분간 70 로하였다. PCR 과정은 35회 (cycles) 를시행하였고, 검체는 94 에서 1분간변성 (denature) 된후다시 1분간 β-actin 은 65, MMP-2,-9 은 55 에서각각의 primer 에불림 (annealing) 과정, 이후 30초간 72 에서확장 (extension) 과정을거쳤다. 합성된 cdna 는 -20 에사용전까지보관하였다. PCR 을통해얻어진산물은 1.5% agarose gel 상에전기영동을통하여분획시킨후분석하였다. 3) Western blotting 분쇄한태반조직샘플에 lysis buffer (Cell Signaling Technology, Beverly, Mass., USA) 를넣고 10분동안 incubation한후 4 에서 14,000 rpm으로원심분리하여가라앉힌후단백질 extract 를얻었다. 이렇게얻은단백질은환원제를포함하는 SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) 를통해분리시켰다. 전기영동후 gel 을상온에서 PVDF (polyvinylidene difluoride) membranes (Millipore, Bedford, MA) 에 blotting 하였다. 이후 5% non-fat dry milk/tbst 로 2시간동안상온에서 blocking 하였다. 상온에서 monoclonal antibody 인 MMP-2 Ab, MMP-9 Ab (MMP-2, MMP-9: 1:100, Calbiochem, SanDiego, CA) 와 β-actin (Sigma, MO, USA) 을 2시간동안처리하고, TBST washing buffer 로 10분씩 3번씻었다. 이후 secondary antibody (anti-rabbit polyclonal IgG, Horseradish peroxidase conjugated anti-goat polyclonal IgG: Santa Cruz Biotechnology, CA, USA. Anti-mouse monoclonal IgG: Amersham, Buckinghamshire, England) 를노출시켰다. 그후 ECL reagents (Amersham, Buckinghamshire, England) 를반응시키고, Hyper X-ray film 에 30초, 1분, 5분노출시켜영상을얻었다. 4) Densitometer RT-PCR 과 Western blotting 을시행하여얻은영상을 densitometer (IMAGE READER LAS-1000 lite, Fuji Photo Film Co., Ltd., Japan) 를사용하여 scanning 을하였고, digital analysis software (Fuji Photo Film Co., Ltd., Japan) 를사용하여분석하였다. Table 1. Primer sequence specific to the target genes Gene Dir sequence Size (bp) MMP-2 S 5 -AGGACATTGTATTTGATGGC A 5 -CTTCTTGTTTTTGCTCCAGT-3 MMP-9 S 5 -AGCTTTTCTTCTTCTCTGGG A 5 -ACTGCAGGATGTCATAGGTC-3 S: sense, A: antisense, bp: base pair, Dir: direction

대한산부회지제 51 권제 11 호, 2008 5) 면역조직화학염색법 (immunohistochemistry) 10% 중성포르말린용액에고정하고, 파라핀조직을 4 μm의두께로절편 (section) 을만들어 xylene 으로 15분간탈파라핀화하고 10%, 90%, 70% 의알코올을각 3분씩거쳐증류수로함수 (rehydration) 시켰다.

4 대한산부회지제 51 권제 11 호, ) 면역조직화학염색법 (immunohistochemistry) 10% 중성포르말린용액에고정하고, 파라핀조직을 4 μm의두께로절편 (section) 을만들어 xylene 으로 15분간탈파라핀화하고 10%, 90%, 70% 의알코올을각 3분씩거쳐증류수로함수 (rehydration) 시켰다. 내인성 peroxidase 의활성을억제하기위하여 3% 과산화수소수로 10분간처리한후항원의노출을위하여극초단파를이용하여 30분간끓이고, 다시 30분간실온에서식힌후 PBS buffer 로 10분간처리하였다. 일차항체인 MMP-2,-9 항체 (Santa Cruz Biotechnology, CA, USA) 를각각 1:100 으로희석하여 4 에서밤샘처리한후 anti goat -IgG (Santa Cruz Biotechnology, CA, USA ) 를 20분간처리하여비특이적결합을억제하였다. 다시 PBS buffer 에서 10분간처리한후 Streptavidin-HRP (DAKO, Carpinteria, CA, USA) 를 20분간처리하였다. PBS buffer 에 10 분간처리한후 DAB chromogen kit (ScyTek, Utah, USA) 로발색한다음증류수로씻어냈다. 6) 통계학적분석실험에참여한대상환자들에대한비교는평균과표준편차를이용하여표현하였고, densitometry 를이용한얻은결과는중위수와최대및최소값으로표현하였다. 두군사이의비교에사용한통계법은 Mann-Whitney U test 를사용하였다. P value 가 0.05 미만이경우만을통계적으로의미가있는것으로하였다. 통계에상용된프로그램은 SPSS 로 version 12.0 을사용하였다. 결과실험에참여한산모들의특징은 Table 2와같았고, 산모 Table 2. Clinical characteristics of normal and preeclamptic pregnant women Variable Normal group (n=10) Preeclampsia group (n=10) P value Maternal age (years) 30±2.9 31±3.5 NS Gestational age at delivery (weeks) 37±1.6 35± Birth weight (g) 3,049±409 2,674± Smoker (%) - - NA Systolic blood pressure (mmhg) 112±9 169± Proteinuria none 10/10* Data: Mean±SD. * 2+ on dipstick in ten patients. Mann-Whitney U test, P<0.05; statistically significant, NS: not significant, NA: not applicable. Fig. 1. Expression of Matrix Metalloproteinase (MMP) -2, -9 in human placenta. Differential expressions of MMP-2,-9 were analyzed in normal and preeclamptic placentas by (A) RT-PCR and (B) Western blot. N: normal pregnancy, P: preeclamptic pregnancy

5 문여정외 4 인. Differential expression of MMP-2/-9 in normal and preeclamptic human placentas Fig. 2. Densitometric values normalized to GAPDH levels for MMP-2,-9 gene expression, and β-actin levels for MMP-2,-9 protein expression. Gene expressions of MMP-2,-9 were significantly higher in normal pregnancies compared to preeclampsia (A, B). Protein expressions of MMP-2,-9 were significantly higher in normal pregnancies compared to preeclampsia (C, D). P<0.001 by Mann-Whitney U test. 의나이를제외한분만주수, 출생체중, 수축기및이완기혈압, 단백뇨모두두군사이에유의한차이를보였다. MMP-2,-9 에대한유전자발현차이를비교하기위하여반정량적 RT-PCR 을시행한결과두유전자모두전자간증산모에서하향조절 (down regulation) 되었다 (Fig. 1A). MMP-2,-9 단백질의발현차이를확인하기위해 western blotting 을시행한결과 MMP-2,-9 단백질모두전자간증에서단백질발현이감소하여나타났다 (Fig. 1B). RT-PCR 과 western blot 결과를바탕으로 densitometry 를시행한결과 MMP-2,-9 의유전자및단백질발현모두전자간증에서통계학적으로유의하게적었다 (P< 0.001) (Fig. 2). 중위수 ( 최대값- 최소값 ) 로표현한결과 MMP-2 유전자는정상에서 ( ), 전자간증에서 59.2 ( ), MMP-9 유전자는정상에서 ( ), 전자간증에서 ( ) 로측정되었다. MMP-2 단백질은정상에서 ( ), 전자간증에서 86.8 ( ), MMP-9 단백질은정상에서 ( ), 전자간증에서 ( ) 로나타났다. 정상산모에비해서전자간증산모의태반에서감소되어있는 MMP-2 (Fig. 3) 와 MMP-9 (Fig. 4) 단백질에대한면역학적위치선정 (immunolocalization) 을위하여면역조직화학염색을시행하였다 (Fig. 3, Fig. 4). MMP-2 과 MMP-9 은모두태반내융모막에서만발현하였으며, 혈관내피세포나태반기질세포등에서는발현되지않았다. 고찰전자간증은아직까지그원인이불확실하며, 많은병인론이제기되고있는질환으로그중하나로알려져있는것은임신초기착상시태반영양막침습의결함으로인한자궁나선동맥의부적절한재형성 (remodeling) 이다

대한산부회지제 51 권제 11 호, 2008 Fig. 3. Immunohistochemical staining of MMP-2 in normal and preeclamptic placentas. MMP-2 was expressed in normal and preeclamptic placental tissues.

A, C: placentas from normal pregnancies, B, D: placentas from preeclamptic pregnancies. A, B: 100, C, D: 400. Fig. 4. Immunohistochemical staining of MMP-9 in normal and preeclamptic placentas.

6 대한산부회지제 51 권제 11 호, 2008 Fig. 3. Immunohistochemical staining of MMP-2 in normal and preeclamptic placentas. MMP-2 was expressed in normal and preeclamptic placental tissues. MMP- 2 immunoreactivity was localized only to the trophoblast. Note that the expression of MMP-2 decreased in the preeclamptic placentas compared to the normal. A, C: placentas from normal pregnancies, B, D: placentas from preeclamptic pregnancies. A, B: 100, C, D: 400. Fig. 4. Immunohistochemical staining of MMP-9 in normal and preeclamptic placentas. MMP-9 was expressed in normal and preeclamptic placental tissues. MMP- 9 immunoreactivity was localized only to the trophoblast. Note that the expression of MMP-9 decreased in the preeclamptic placentas compared to the normal. A, C: placentas from normal pregnancies, B, D placentas from preeclamptic pregnancies. A, B: 100, C, D: 400. 정상임신초기바닥탈락막에있는나선동맥은이후태반에혈액을공급하게되는데, 영양막세포의침습이임신 6주경에탈락막내의나선동맥에서시작하여임신 18주경에는자궁평활근안쪽 1/3 에위치한나선동맥까지확장되는것이보통이다. 나선동맥은영양막침습에의하여본래의혈관구조가파괴되고평활근이나내피세포가없는골격 만남게되어모체혈관운동신경의조절을받지않아혈액을안정적으로태반에공급하는비탄력적인확장된관의형태인자궁태반혈관으로변화하게된다. 전자간증에서는나선동맥의약 30~50% 가혈관의재형성 (remodeling) 이적절하게이루어지지않아자궁태반혈관의저항이감소되지않고높은상태로지속된다. 그결과자궁태반순환이

7 문여정외 4 인. Differential expression of MMP-2/-9 in normal and preeclamptic human placentas 감소하며임신이진행할수록태반허혈이악화되어 25 tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1 α) 등의싸이토카인 (cytokine) 분비를증가시켜내피세포기능부전을일으키게된다. 26 영양막침습과정중유착분자와세포외기질을분해하여영양배엽세포의분화및침습을도와주는 MMP 는이런태반형성과정에중요하게작용하는물질이다. MMP 는혈관내피세포, 영양막등의여러세포에서분비되며 zinc 에의존하여작용하는다유전체군 (multi gene family) 단백분해효소로, 27 정상및비정상적인조직의개조에관여하며, 이들은기질특이성에따라크게 4개의군으로분류할수있다. 28 제1군 Collagenase 는 I형, II형, III 형콜라겐을분해하며 interstitial collagenase (MMP-1), neutrophil collagenase (MMP-8), collagenase 3 (MMP- 13) 가이에속한다. 제2군 Gelatinase 는젤라틴, IV형콜라겐을분해하며 gelatinase A (MMP-2), gelatinase B (MMP-9) 가이에속한다. 제3군 stromelysins 는화이브로넥틴, 라미닌, III형, IV형, V형콜라겐, 엘라스틴을분해하며, stromelysin-1 (MMP-3), stromelysin-2 (MMP- 10), stromelysin-3 (MMP-11), metalloelastase (MMP- 12) 등이이에속한다. 제4군 membrane-type MMP 는최근에밝혀진것으로이중 MT1-MMP, MT2-MMP, MT3-MMP, MT5-MMP 는 MMP-2 활성화에관여하는것으로추정된다. MMP 는 N-terminal 프로펩타이드의분할 (cleavage) 이나재접합 (reconfiguration) 을통해여러단계로활성화되며, 전사 (transcription), 분비, 활성화또는 tissue inhibitors of metalloproteinase (TIMPs) 같은억제제에의해활성도가조절된다. 29 따라서 MMP 의 mrna 나 protein 의발현이활성화된 MMP 와비례한다고단정지을수는없다. MMP 와 TIMPs 의균형은태반의형성에매우중요한역할을한다. 30 전자간증의태반조직에서 TIMP-1 과 TIMP-2 발현이증가되어있는것으로알려져있으며, 31 본연구에서는전자간증의태반조직에서 MMP-2,-9 의발현이감소되어있음을확인하였다. 이렇게균형이깨질경우영양막의침투가미약하여태반형성이적절치못하게될것이라고추측할수있다. 또한정상임신에서임신말기까지 MMP-2 가지속적으로높게유지되며본연구결과전자간 증에서 MMP-9 보다 MMP-2 의감소가더뚜렷하게나타나는것으로보아전자간증의병태생리에 MMP-2 가더깊게관여하는것으로보인다. MMP-2 는태반형성에관여할뿐아니라혈관의긴장도를조절하는인자중강력한혈관수축인자인 big endothelin 의분할을일으켜혈관반응성즉혈관수축을증가시키며, 32 calcitonin gene-related peptide 를분해하여혈관이완을억제시킴으로, 34 전자간증에서영양막침습을통한나선동맥의재구성장애와혈관작용인자들에대한혈관반응도증가모두에서중요한역할을하는것으로생각된다. Narumiya 등은전자간증임산부에서혈관신생, 혈관재구성및혈관투과도의생리적조절인자인혈관내피세포성장인자 (VEGF) 에의해혈중 MMP-2 의생성이증가된다고보고하여이두가지병태생리가서로연관되어있음을시사하였다. 20 MMP-9 는초기착상과정에서의혈관내피세포증식및영양배엽세포의형태를변화시키는데 urokinase plasminogen activator (upa) 와함께중요한역할을하는것으로알려져있으며, 34 Kolben 등은전자간증산모의태반조직추출물에서 MMP-9 농도가정상산모의것보다낮다는보고를하였고, 35 감소된 MMP-9 으로인해초기태반형성이부족하게일어나전자간증을일으키는것으로생각된다고보고하였다. 본연구를통하여전자간증에서 MMP-2, MMP-9 의발현이감소되었음을확인하였으나어떤인자들이무슨기전으로 MMP 의발현을조절하는지명확히밝혀지지않았다. 사람의태반에서 Nitric oxide 에의해 MMP 가활성화되며, 36 쥐에서 Nitric oxide 생성이억제될경우자궁내 MMP-2,-9 의유전자발현이억제되고, MMP-9 의활성또한억제한다는연구가있었다. 37 또다른조절인자로아데노신 A1수용체가 MMP-2 의분비를촉진하며, 38 아데노신 A2a 수용체는 MMP-9 의분비를억제한다는보고가있었다. 39 또한저산소태에서다양한영양막양세포주의침습정도를비교한결과대부분저산소상태에서침습이억제되는것으로보고하였다. 40 하지만, 임신과관련하여그조절기전에관여하는물질및기전에대해서는앞으로많은연구가이루어져야할것으로여겨진다. 본연구에서는전자간증및대조군산모의태반조직에서 MMP-2 와 MMP-9 을측정하여병태생리에서의 MMP

8 대한산부회지제 51 권제 11 호, 2008 의역할을알아보았고, 전자간증산모의태반에서정상산모에비해 MMP-2 와 MMP-9 의발현이감소하였음을확인하였다. 이런결과를기초로적절치못한태반형성에의해나타나는전자간증의병태생리기전에 MMP-2 와 MMP- 9이관여하는것으로추정할수있을것이다. 추후태반내 MMP-2 와 MMP-9 의발현을조절하는물질에대한연구나저산소상태에서발현차이등추가적인연구가필요할것으로사료된다. 1. Sibai BM, Anderson GD. Hypertension. In: Gabbe SG, Niebyl JR, Simpson JL, Anderson GD, editors. Obstetrics: Normal and problem pregnancies. 2nd ed. New York: Churchill Livingstone; p Brosens IA. Morphological changes in the uteroplacental bed in pregnancy hypertension. Clin Obstet Gynaecol 1977; 4: Brosens I, Dixon HG, Robertson WB. Fetal growth retardation and the arteries of the placental bed. Br J Obstet Gynaecol 1977; 84: Lam C, Lim KH, Karumanchi SA. Circulating angiogenic factors in the pathogenesis and predcition of preeclampsia. Hypertension 2005; 46: Roberts JM, Cooper DW. Pathogenesis and genetics of pre-eclampsia. Lancet 2001; 357: Huppertz B, Kertschanska S, Demir AY, Frank HG, Kaufmann P. Immunohistochemistry of matrix metalloproteinases (MMP), their substrates, and their inhibitors (TIMP) during trophoblast invasion in the human placenta. Cell Tissue Res 1998; 291: Isaka K, Usuda S, Ito H, Sagawa Y, Nakamura H, Nishi H, et al. Expression and activity of matrix metalloproteinase 2 and 9 in human trophoblasts. Placenta 2003; 24: Liu K, Wahlberg P, Ny T. Coordinated and cell-specific regulation of membrane type matrix metalloproteinase 1 (MT1-MMP) and its substrate matrix metalloproteinase 2 (MMP-2) by physiological signals during follicular development and ovulation. Endocrinology 1998; 139: Alexander CM, Hansell EJ, Behrendtsen O, Flannery ML, Kishnani NS, Hawkes SP, et al. Expression and function of matrix metalloproteinases and their inhibitors at the maternalembryonic boundary during mouse embryo implantation. Development 1996; 122: 참고문헌 10. Novaro V, Pustovrh C, Colman-Lerner A, Radisky D, Lo Nostro F, Paz D, et al. Nitric oxide induces gelatinase A (matrix metalloproteinase-2) during rat embryo implantation. Fertil Steril 2002; 78: Cañete Soler R, Gui YH, Linask KK, Muschel RJ. MMP-9 (gelatinase B) mrna is expressed during mouse neurogenesis and may be associated with vascularization. Brain Res Dev Brain Res 1995; 88: Chin JR, Werb Z. Matrix metalloproteinases regulate morphogenesis, migration and remodeling of epithelium, tongue skeletal muscle and cartilage in the mandibular arch. Development 1997; 124: Stygar D, Wang H, Vladic YS, Ekman G, Eriksson H, Sahlin L. Increased level of matrix metalloproteinases 2 and 9 in the ripening process of the human cervix. Biol Reprod 2002; 67: Xu P, Alfaidy N, Challis JR. Expression of matrix metalloproteinase (MMP)-2 and MMP-9 in human placenta and fetal membranes in relation to preterm and term labor. J Clin Endocrinol Metab 2002; 87: Okamoto T, Niu R, Yamada S, Osawa M. Reduced expression of tissue inhibitor of metalloproteinase (TIMP)-2 in gestational trophoblastic diseases. Mol Hum Reprod 2002; 8: Vadillo-Ortega F, Hernandez A, Gonzalez-Avila G, Bermejo L, Iwata K, Strauss JF 3rd. Increased matrix metalloproteinase activity and reduced tissue inhibitor of metalloproteinases-1 levels in amniotic fluids from pregnancies complicated by premature rupture of membranes. Am J Obstet Gynecol 1996; 174: Ke Y, Lu JH, Yang BL, Guo HQ, Ma QY, Zhu H, et al. Involvement of matrix metalloproteinase-2, -9, and tissue inhibitors of metalloproteinase-1, 2 in occurrence of the accrete placenta. Zhonghua Fu Chan Ke Za Zhi 2006; 41: Pustovrh MC, Jawerbaum A, Capobianco E, White V, López-Costa JJ, González E. Increased matrix metalloproteinases 2 and 9 in placenta of diabetic rats at midgestation. Placenta 2005; 26: Galewska Z, Bańkowski E, Romanowicz L, Jaworski S. Pre-eclampsia (EPH-gestosis)-induced decrease of MMP-s content in the umbilical cord artery. Clin Chim Acta 2003; 335: Narumiya H, Zhang Y, Fernandez-Patron C, Guilbert LJ, Davidge ST. Matrix metalloproteinase-2 is elevated in the plasma of women with preeclampsia. Hypertens Pregnancy 2001; 20: Huisman MA, Timmer A, Zeinstra M, Serlier EK, Hanemaaijer R, Goor H, et al. Matrixmetalloproteinase activity in first trimester placental bed biopsies in further complicated and uncomplicated pregnancies. Placenta 2004; 25: Merchant SJ, Crocker IP, Baker PN, Tansinda D, Davidge ST, Guilbert LJ. Matrix metalloproteinase release from placental explants of pregnancies complicated by intrauterine growth restriction. J Soc Gynecol Investig 2004; 11: ACOG Committee on Obstetric Practice. ACOG practice bulletin. Diagnosis and management of preeclampsia and eclampsia. Number 33, January American College of Obstetricians and Gynecologists. Int J Gynaecol Obstet 2002; 77: Mayhew TM, Wijesekara J, Baker PN, Ong SS. Morphometric Evidence that Villous Development and Fetoplacental Angiogenesis are Compromised by Intrauterine Growth Restriction but not by Pre-eclampsia. Placenta 2004; 25: Augest P, Lindheimer MD. Pathophysiology of pre-eclampsia. In: Laragh JH, Brenner BM, editors. Hypertension : pathophysiology, diagnosis, and management. 2nd ed. New York: Raven Press; p

9 문여정외 4 인. Differential expression of MMP-2/-9 in normal and preeclamptic human placentas 26.Benyo DF, Miles TM, Conrad KP. Hypoxia stimulates cytokine production by villous explants from the human placenta. J Clin Endocrinol Metab 1997; 82: Birkedal-Hansen H, Moore WG, Bodden MK, Windsor LJ, Birkedal-Hansen B, DeCarlo A, et al. Matrix metalloproteinases: A review. Crit Rev Oral Biol Med 1993; 4: Bischof P, Meisser A, Campana A. Paracrine and autocrine regulators of trophoblast invasion a review. Placenta 2000; 21 Suppl A: S Murphy G, Willenbrock F, Crabbe T, O'Shea M, Ward R, Atkinson S, et al. Regulation of matrix metalloproteinase activity. Ann N Y Acad Sci 1994; 732: Niu R, Okamoto T, Iwase K, Nomura S, Mizutani S. Quantitative analysis of matrix metalloproteinase 2 and 9, tissue inhibitors -1,and -2 in human placenta throughout gestation. Life Sci 2000; 66: Pang ZJ, Xing FQ. Expression profile of trophoblast invasion-associated genes in the preeclamptic placenta. Br J Biomed Sci. 2003; 60: Fernandez-Patron C, Radomski MW, Davidge ST. Vascular matrix metalloproteinase-2 cleaves big endothelin-1 yielding a novel vasoconstrictor. Circ Res 1999; 85: Fernandez-Patron C, Stewart KG, Zhang Y, Koivunen E, Radomski MW, Davidge ST. Vascular matrix metalloproteinase-2 dependent cleavage of calcitonin gene-related peptide promotes vasoconstriction. Circ Res 2000; 87: de Jager CA, Linton EA, Spyropoulou I, Sargent IL, Redman CW. Matrix metalloprotease-9, placental syncytiotrophoblast and the endothelial dysfunction of preeclampsia. Placenta 2003; 24: Kolben M, Lopens A, Bläser J, Huber A, Frank M, Wilhelm O, et al. Measuring the concentration of various plasma and placenta extract proteolytic and vascular factors in pregnant patients with HELLP syndrome, pre-/eclampsia and highly pathologic Doppler flow values. Gynakol Geburtshilfliche Rundsch 1995; 35 Suppl 1: Pustovrh C, Jawerbaum A, Sinner D, Pesaresi M, Baier M, Micone P, et al. Membrane-type matrix metalloproteinase-9 activity in placental tissue from patients with pre-existing and gestational diabetes mellitus. Reprod Fertil Dev 2000; 12: Zhang X, Wang HM, Lin HY, Liu GY, Li QL, Zhu C. Regulation of Matrix Metalloproteinases (MMPs) and their Inhibitors (TIMPs) during Mouse Peri-implantation: Role of Nitric Oxide. Placenta 2004; 25: Shearer TW, Crosson CE. Adenosine A1 receptor modulation of MMP-2 secretion by trabecular meshwork cells. Invest Ophthalmol Vis Sci 2002; 43: Ernens I, Rouy D, Velot E, Devaux Y, Wagner DR. Adenosine inhibits matrix metalloproteinase-9 secretion by neutrophils: implication of A2a receptor and camp/pka/ca2+ pathway. Circ Res 2006; 99: Lash GE, Hornbuckle J, Brunt A, Kirkley M, Searle RF, Robson SC, et al. Effect of low oxygen concentrations on trophoblast-like cell line invasion. Placenta 2007; 28: = 국문초록 = 목적 : 본연구는정상태반및중증전자간증태반에서의 MMP-2 와 MMP-9 의발현차이를비교하고자하였다. 방법 : 실험에사용된정상및중증전자간증산모의태반은만삭에제왕절개술을시행한산모들을대상으로하였다. MMP-2,-9 의유전자발현정도는반정량적 RT-PCR을통하여측정되었다. 단백질의정량화를위하여 western blot을시행하였고, 면역학적위치선정을위하여 immunohistochemical staining을시행하였다. 결과 : RT-PCR 결과 MMP-2,-9 의유전자는정상및중증전자간증태반모두에서발현되었고, 중증전자간증에서발현이감소되었다. Western blot을시행한결과중증전자간증에서단백질발현이감소되었다. 면역조직화학염색결과 MMP-2,-9 은모두태반내융모막에서만발현하였으며, 중증전자간증태반에서발현이감소하였다결론 : 중증전자간증산모의태반에서 MMP-2,-9 의발현이감소되어있음을확인하였으며, 본연구를통하여 MMP-2,-9 이중증전자간증의병인에관여할것으로사료된다. 중심단어 : MMP-2, MMP-9, 중증전자간증, 태반

α α α α α

α α α α α α α α 太陰調胃湯加減方 dbdb 마우스 肝에 대한 아디포사이토카인 및 발현에 미치는 영향 SREBPs 와 섞어서 하고 마커 또한 하여 전기 영동을 하였다 전기영동을 한 후에 에 를 쪼여서 각 를 확인하였다 이 를 프로그램을 이용해 수치화하 여 분석하였다 肝 조직 동결 절편 분리한 조직은 로 시간 동안 고정 시킨 후 에 세척한 후 물기를