Mineral trioxide aggregate 가인간치수세포에서유전자발현에미치는영향 김용범 손원준 이우철 금기연 백승호 배광식 * 서울대학교치의학대학원치과보존학교실, 치의학연구소, BK21 Program ABSTRACT GENE EXPRESSION PROFILING IN HUMAN DENTAL PULP CELLS TREATED WITH MINERAL TRIOXIDE AGGREGATE Yong-Beom Kim, Won-Jun Shon, WooCheol Lee, Kee-Yeon Kum, Seung-Ho Baek, Kwang-Shik Bae* Department of Conservative Dentistry, Dental Research Institute, BK21 Program, School of Dentistry, Seoul National University, Seoul, Korea This study investigated the changes in gene expression when mineral trioxide aggregate (MTA) was applied in vitro to human dental pulp cells (HDPCs). MTA in a teflon tube (diameter 10 mm, height 2 mm) was applied to HDPCs. Empty tube-applied HDPCs were used as negative control. For microarray analysis, total RNA was extracted at 6, 24, and 72 hrs after MTA application. The results were confirmed selectively by performing reverse transcriptase polymerase chain reaction for genes that showed changes of more than two-fold or less than half. Of the 24,546 genes, 109 genes were up-regulated greater than twofold (e.g., FOSB, THBS1, BHLHB2, EDN1, IL11, FN1, COL10A1, and TUFT1) and 69 genes were downregulated below 50% (e.g., SMAD6 and DCN). These results suggest that MTA, rather than being a bioinert material, may have potential to affect the proliferation and differentiation of pulp cells in various ways. [J Kor Acad Cons Dent 35(3):152-163, 2010] Key words: Microarray, Mineral trioxide aggregate, Dental pulp capping, Human dental pulp cell, Differentiation, Proliferation -Received 2010.3.20., revised 2010.4.6., accepted 2010.4.12.- Ⅰ. 서론 직접치수복조 (direct pulp capping) 는생활치수가노출된경우치수조직에외부자극을차단하고수복상아질 (reparative dentin) 의형성을촉진하여치수의생활력을유지하기위해시행된다. 1) 수산화칼슘이직접치수복조술에수십년동안가장널리사용되어왔음에도불구하고형성된수복상아질이균일하지않으며내부에 tunnel 구조를가지 *Corresponding Author: Kwang-Shik Bae Department of Conservative Dentistry, School of Dentistry, Seoul National University 275-1 Yeongeon-Dong, Jongno-Gu, Seoul, 110-768, Korea Tel: 82-2-2072-2651 Fax: 82-2-2072-3859 E-mail: baeks@snu.ac.kr 며남은치수내의과도한석회화가일어나는등의단점이보고되고있다. 2-4) 비교적최근에소개된재료인 mineral trioxide aggregate (MTA) 는 perforation repair 나치근단역충전에주로사용되었으며여러연구에서생체친화성이좋은것으로보고되었다. 5,6) 이후 MTA 를이용한치수복조술을사용한경우에서도우수한결과가보고되었다. 원숭이와개의치아에 MTA 와수산화칼슘을이용한치수복조술을시행한경우 MTA 가수산화칼슘과비교하여유사하거나더좋은결과를보여주었다. 7-10) 이런연구들이조직학적소견에의해 MTA 를치수복조제재로써평가했던것과달리최근에는 mineralized matrix marker 의유전자발현을관찰하는연구들이보고되고있다. 11-13) 152
Mineral trioxide aggregate 가인간치수세포에서유전자발현에미치는영향 치수복조용제재에대한치수조직의반응에대해서많은연구가진행되어왔지만치수복조용제재의상아질형성능력이치수복조제재에의해유도되는고유한능력인지외상을입은치수자체의치유과정인지도아직확실히규명되지못했다. Microarray를이용한실험방법은다양한재료에대해수만개의유전자발현을동시에분석할수있는방법이다. 이는세포의발생, 세포내의신호전달, apoptosis, 질병의진단, 치료효과, 약물개발, 그리고약물안전평가에사용될수있다. 14) 최근인간치수의줄기세포 (human dental pulp stem cell) 와골수기질의줄기세포 (bone marrow stromal stem cell) 의유전자차이를분석한연구, 우식증이있는치아와없는치아의치수조직에서의유전자차이를분석한연구, 레진계열봉함제가조골세포에미치는영향을분석한연구그리고치수세포와치주인대세포의유전자차이를비교한연구에서 microarray가사용되었다. 15-18) 이런치수조직과치주조직에서시도된다양한 microarray 연구들이보고되었지만, 현재까지 MTA로치수복조술을시행하였을때치수세포에서일어나는유전자발현양상에대해서조사한연구는없었다. 이번연구의목적은 MTA를치수세포에적용하였을때변화하는치수세포의유전자들중에서치수세포의분화와증식에관여하는유전자가어떤변화를보이는지분석하기위함이다. 본연구는 MTA를일차세포배양한인간치수세포에적용하고시간대별로 (6, 24, 72시간 ) 표본을채취하여처리한후 microarray를이용하여유전자변화를분석하고선택적으로역전사중합효소연쇄반응 (reverse transcriptase polymerase chain reaction (RT-PCR)) 을이용하여이를확인하였다. 와 3 mg/ml collagenase (Sigma, St Louis, MO, USA) 가 1:1 로혼합된용액에넣고 37 에서 10 분간반응시킨후, 떨어져나온조직덩어리와세포들을모아서원심분리한뒤침전물 (pellet) 을얻었다. 이들을 10% fetal bovine serum (Sigma) 이포함된 Dulbecco s Modified Eagle s Medium (DMEM; Invitrogen, Carlsbad, CA, USA) 배지에재부유시켜배양시킨지 4 일후에 6 well culture plate (SPL life science, Pocheon, Gyeonggi, Korea) 에자라나온세포들이확인되었다. 세포수와생존도는 trypan blue 염색을통해확인하였고, 5-7 번째계대의세포들을본실험에사용하였다. Hemocytometry 를이용하여치수세포를 10% fetal bovine serum (Sigma), 100 U/mL penicillin 과 100 μm/ml streptomycin 의항생제가포함된 DMEM 에 1 10 5 /well 의농도로적용한후 37, 5% CO2, 95% air 상태에서배양하였다. Well 에세포를적용한후 24 시간배양하여실험에사용하였다. 2. 시편제작 내경 10 mm 의테플론튜브를 2 mm 의두께로절단하여 ethylene oxide gas 소독을시행하였다. 이후 white MTA (ProRoot; Dentsply, Tulsa, OK, USA) 를제조사의지시대로혼합하여준비된테플론튜브에삽입하였다. 혼합 4 시간후시편을 0.4 μm 의 pore size 를가지는 permeable transwell insertion (Millipore, Billerica, MA, USA) 위에올려서치수세포에직접접촉이되지않게 6 well culture plate (SPL life science) 에넣었다. 각 well 에 3 ml 의배양액을담가주었으며빈튜브를넣은 well 을대조군으로사용하였다. 시편의적용후 6, 24, 72 시간에같은군 (n=3) 의세포를 pooling 하였다. Ⅱ. 실험재료및방법 3. Microarray analysis 1. 인간치수세포의일차세포배양 인간치수세포의일차세포배양은다음과같은방법을사용하였다. 19) 서울대학교치과병원구강악안면외과에서 2009년 10월내원한성인의건전한제3대구치를환자의동의하에발치하였으며모든실험과정은서울대학교치과병원연구윤리심의위원회의승인하에진행하였다 (CRI09020). 발치즉시항생제가들어있는 phosphatebuffered saline에넣은상태로실험실로이동하여 #330 bur를사용하여백악법랑질경계하방 1 mm에서수평으로치아를절단하였다. 이후치수조직을조심스럽게꺼내어 blade로작게썰어서 4 mg/ml dispase II (Roche, San Francisco, CA, USA) Pooling 한세포에서 total RNA 는 Trizol (Invitrogen) 을이용하여추출하였으며 RNeasy columns (Qiagen, chatsworth, CA, USA) 를이용하여정제하였다. 추출된 total RNA 를정량화하여 freezer 에서 -80 로보관하였다. RNA 의 purity 와 integrity 를 denaturing gel electrophoresis 와 OD260/280 ratio 를이용하여확인하였으며, Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) 을이용하여분석하였다. RNA 합성과정제과정은 Illumina TotalPrep RNA Amplification Kit (Ambion, Austin, TX, USA) 을사용하여제조사가제시하는방법으로 biotin labeled crna 를제작하였다. 간단히기술하면 first strand cdna 합성은 550 ng 의 RNA 에 T7 oligo (dt) primer 를사용하여역전 153
사 (reverse transcription) 과정을거쳤다. Second strand cdna 합성은 DNA polymeraese 와 RNase H 의반응으로 single-strand cdna 를 double-strand DNA 로변환하였다. cdna purification 후 in vitro transcription crna 합성단계에서 biotin-ntp 로표지화된 crna 를증폭시킨다. crna purification 후 ND-1000 분광측광기 (NanoDrop, Wilmington, DE, USA) 를사용하여정량하였다. Microarray 실험은 Whole-genome gene expression with IntelliHyb Seal System (Illumina, Inc., San Diego, CA, USA) 을이용하여제조사가제시한방법에따라실시하였다. 1.5 μg 의 labeled crna 시료에 GEX- HYB buffer 20 μl 를첨가하여 Illumina HumanRef-8 v3 bead chip (Illumina Inc., San Diego, CA, USA) 에혼성화하여 16-18 시간동안 58 항온기에서반응시켰다. Beadchip 세척은 High-Temp Wash buffer 에서 10 분동안 55 항온기에서반응후 E1BC solution 에서 5 분간세척하였다. 이 beadchip 은다시 100% ethanol 에서 10 분그리고 E1BC solution 에서 2 분간세척하였다. Beadchip 은 wash tray 로옮겨 E1 buffer 4 ml 을가하여 10 분동안반응후 1 mg 의 streptavidin-cy3 (GE Healthcare Bio- Sciences, Little Chalfont, UK) 를첨가한 E1 buffer 에서 10 분동안반응시켰다. 최종적으로 E1BC solution 에서세척후원심분리기를이용하여 beadchip 을건조시켰다. Beadchip scan 은 Illumina BeadArray Reader Confocal Scanner (Illumina Inc.) 를사용하여제조사가제시한방법으로진행하였다. Scanner 로도출된 beadchip image 를 Illumina BeadStudio Gene Expression Module v3.3.8 (Illumina Inc.) 로분석하여 raw data 를추출하였다 Raw data 에서얻어지는 detection pvalue 값을이용하여, 50% 샘플에서 p-value < 0.05 를만족하는리스트를선별하여, Log2, Quantile normalization 을수행하였다. 샘플간의비교로서, Fold-change 가사용되었으며, 유의한차이를지니는 ( Fold > 2) 유전자들대상으로데이터를도식화하였다. 데이터분석과도식화에 ArrayAssist (Stratagene, La Jolla, CA, USA) 를사용하였으며, Panther database (http://www.pantherdb.org) 를이용하여해당유전자들에대해 ontology 를나타내었다. 4. RT-PCR analysis RT-PCR 분석에사용된 total RNA 는위에 microarray analysis 에사용하고남은것을사용하였다 Maxime RT- PreMix (Intron, Suwon, Gyeonggi, Korea) 를제조사의지시대로사용하여 RNA 를 cdna 로바꾸고, 분석하고자하는유전자의 cdna 는 Table 1 에명시된각각의특이 primer 와 Maxime PCR Premix (Intron) 를이용하여증폭하였다. 각유전자들의증폭산물은 denaturation (94 3 분 ), 30cycle 의 amplification (94, 30 초 ; 57, 30 초 ; 72, 30 초 ) 단계로반응시킨뒤, 72 에서 7 분간반응비활성화의단계를거친뒤얻었다. 각증폭산물은 1.5% agarose gel 에서전기영동되었고, 10 분간 ethidium bromide 용액으로염색시켜 Gel-Doc (Bio-rad, Hercules, CA, USA) 으로관찰된뒤, 이들에대한 band intensity 를측정하였다. 측정된각유전자의 band intensity 는우선각 GAPDH 의 band intensity 로보정된다음, MTA 를적용한그룹의 band intensity 를대조군의 band intensity 로나누어비율로표시하였다. Ⅲ. 결과 6, 24, 72 시간중어느시간에서라도 fold 값이 2 배이상차이가나는유전자를확인하였다. BeadChip 에표현된 24,546 개의유전자중총 178 개의유전자가대조군과비 Table 1. Primer sequence list in RT-PCR Target gene Primer Molecular Forward Reverse size(bp) GAPDH GAA GTG GAA GGT CGG AGT C GAA GAT GGT GAT GGG ATT TC 313 FOSB GGC TCA ACA AGG AAA AAT GC GAG GCC AGA AAT TCC AAT CA 297 BHLHB2 TGC TAT ACC CAG GCC TCA AC TGT CTT GCT CCA AGC TCT GA 153 TUFT1 GCA GAA AGA GGC AGA AGT CG CCA AGC AGA ACG AGA AAA GG 152 DCN GGA CCG TTT CAA CAG AGA GG AAT GCC ATC TTC GAG TGG TC 147 THBS1 CCT GTG CTG CAG ACA TTG AT GAA CAC AAT CCG GAT CAG CT 148 FN1 TGG ATG CTC CCA CTA ACC TC GCA GTA CAA TGT GGG TCC CT 150 COL10A1 CCA GGA GTC CCT GGT ATG AA AGA GGT GAA AAT GGG GTT CC 149 154
Mineral trioxide aggregate 가인간치수세포에서유전자발현에미치는영향 교하여 2 배이상의차이를보였으며이중 109 개의유전자가대조군보다 up-regulation 되었으며 69 개의유전자가 down-regulation 되었다. 이중대조군과비교하여 3 배이 상의차이를보이는유전자들과 2 배이상변화한유전자들중에서세포의증식과분화에관여하는유전자들을 Table 2 에표시하였다. Table 2. Biological processes classification of genes in MTA-treated cells. The genes that showed a difference of more than three-fold, and those considered to be related to differentiation and proliferation out of genes that showed a difference of more than two-fold are listed. SYMBOL DEFINITION ACCESSION FOLD BIOLOGICAL PROCESS 6hrs 24hrs 72hrs ACTA2 actin, alpha 2, smooth muscle, NM_001613.1-1.08-1.22 3.25 Intracellular protein traffic, aorta (ACTA2), Transport, Cell cycle, Cell structure and motility ADAMTS1 ADAM metallopeptidase NM_006988.3 1.16 1.7 3.15 Protein metabolism and with thrombospondin type 1 modification, Signal transduction, motif, 1 (ADAMTS1), Developmental processes ANGPTL4 angiopoietin-like 4 (ANGPTL4), NM_139314.1 1.99 1.53 3.3 Immunity and defense transcript variant 1, ANKRD1 ankyrin repeat domain 1 NM_014391.2 1.44 2.1 3.19 Biological process unclassified (cardiac muscle) (ANKRD1), BHLHB2 basic helix-loop-helix domain NM_003670.1 2.47 3.55 2.54 Nucleoside, nucleotide and containing, class B, 2 (BHLHB2), nucleic acid metabolism, Developmental processes, Cell proliferation and differentiation C5orf13 chromosome 5 open reading frame NM_004772.1 1.16 2.81 4.04 Biological process unclassified 13 (C5orf13), C5orf46 chromosome 5 open reading NM_206966.2 1.25 1.01 6.69 Biological process unclassified frame 46 (C5orf46), COL10A1 collagen, type X, NM_000493.3-1.13 1.16 2.88 Signal transduction, Cell adhesion, alpha 1 (COL10A1), Developmental processes, Cell structure and motility CTGF connective tissue growth NM_001901.2 1.03 1.39 2.06 Signal transduction factor (CTGF), CYFIP2 cytoplasmic FMR1 NM_014376.2 1.23 1.03 3.18 Signal transduction, interacting protein 2 (CYFIP2), Developmental processes transcript variant 3, DACT1 dapper, antagonist of beta-catenin, NM_016651.4 1.53 3.16 2.62 Biological process unclassified homolog 1 (Xenopus laevis) (DACT1), DACT1 dapper, antagonist of beta-catenin, NM_016651.5 1.4 3.43 2.88 Biological process unclassified homolog 1 (Xenopus laevis) (DACT1), transcript variant 1, DCN decorin (DCN), NM_001920.3-1.16-1.16-3.69 Signal transduction, transcript variant A1, Developmental processes 155
DCN decorin (DCN), NM_133503.2-1.15-1.22-3.19 Signal transduction, transcript variant A2, Developmental processes DHRS3 dehydrogenase/reductase (SDR family) NM_004753.4-1 -1.35-3.31 Other metabolism member 3 (DHRS3), EDN1 endothelin 1 (EDN1), NM_001955.2 2.83 3.11 1.71 Signal transduction, Blood circulation and gas exchange, Cell proliferation and differentiation, Homeostasis FN1 fibronectin 1 (FN1), transcript NM_054034.2-1.03 1.58 2.25 Signal transduction, Cell adhesion, variant 7, Intracellular protein traffic FN1 fibronectin 1 (FN1), transcript NM_212474.1-1.03 1.6 2.1 Signal transduction, Cell adhesion, variant 6, Intracellular protein traffic, Cell structure and motility FOSB FBJ murine osteosarcoma viral NM_006732.1 2.17 1.49 1.24 Nucleoside, nucleotide and oncogene homolog B (FOSB), nucleic acid metabolism, Developmental processes, Cell cycle FZD8 frizzled homolog 8 (Drosophila) NM_031866.1 1.47 1.87 3.63 Biological process unclassified (FZD8), GDF15 growth differentiation factor NM_004864.1 1.11 1.67 2.9 Developmental processes 15 (GDF15), HMOX1 heme oxygenase (decycling) NM_002133.1 3.56 2.05 1.48 Coenzyme and prosthetic group 1 (HMOX1), metabolism HSPA1A heat shock 70kDa protein NM_005345.4 3.1-1.08 1.18 Protein metabolism and modification, 1A (HSPA1A), Immunity and defense IGFBP3 insulin-like growth factor NM_000598.4-1.12 1.21 4.59 Signal transduction, Homeostasis binding protein 3 (IGFBP3), transcript variant 2, IGFBP3 insulin-like growth factor NM_001013398.1 1.03 1.23 4.91 Signal transduction, Homeostasis binding protein 3 (IGFBP3), transcript variant 1, IL11 interleukin 11 (IL11), NM_000641.2 2.31 2.6 1.96 Biological process unclassified ITIH3 inter-alpha (globulin) inhibitor NM_002217.3 1.17-1 -3.67 Protein metabolism and modification H3 (ITIH3), KIAA1199 KIAA1199 (KIAA1199), NM_018689.1-1.47-3.17-5.34 Biological process unclassified KYNU kynureninase (L-kynurenine NM_003937.2-1.06-1.56-3.01 Amino acid metabolism hydrolase) (KYNU), transcript variant 1, LMCD1 LIM and cysteine-rich domains 1 NM_014583.2 2.54 4.31 2.95 Biological process unclassified (LMCD1), LRRC15 leucine rich repeat containing NM_130830.2-1.13 1.46 3.46 Signal transduction, Cell adhesion, 15 (LRRC15), Immunity and defense LRRC17 leucine rich repeat containing 17 NM_005824.1-1.03 1.52 4.58 Signal transduction, (LRRC17), transcript variant 2, Developmental processes NOX4 NADPH oxidase 4 (NOX4), NM_016931.2 1.19 3.44 3.99 Electron transport PDE5A phosphodiesterase 5A, NM_001083.3-1.94-2.63-3.12 Nucleoside, nucleotide and cgmp-specific (PDE5A), nucleic acid metabolism, transcript variant 1, Signal transduction 156
Mineral trioxide aggregate 가인간치수세포에서유전자발현에미치는영향 PDGFC platelet derived growth NM_016205.1-1.26 1.46 2.17 Signal transduction, factor C (PDGFC), Developmental processes, Cell proliferation and differentiation PENK proenkephalin (PENK), NM_006211.2 1.09-1.29-6.18 Signal transduction, Neuronal activities PMEPA1 prostate transmembrane protein, NM_199169.1 2.3 6.12 5.46 Biological process unclassified androgen induced 1 (PMEPA1), transcript variant 2, PMEPA1 prostate transmembrane protein, NM_199169.1 2.12 5.98 3.97 Biological process unclassified androgen induced 1 (PMEPA1), transcript variant 2, PTHLH parathyroid hormone-like hormone NM_198964.1 2.01 1.44 1.29 Developmental processes (PTHLH), transcript variant 3, PTHLH parathyroid hormone-like hormone NM_198965.1 2.11 1.83 1.37 Developmental processes (PTHLH), transcript variant 1, SERPINE2 serpin peptidase inhibitor, clade E NM_006216.2-1.06 2.29 3.08 Protein metabolism and modification (nexin, plasminogen activator inhibitor type 1),member 2 (SERPINE2), SGK serum/glucocorticoid regulated NM_005627.2 1.25 1.84 3.38 Protein metabolism and modification, kinase (SGK), Signal transduction, Apoptosis SMAD6 SMAD family member 6 NM_005585.3-1.92-1.99-2.24 Nucleoside, nucleotide and (SMAD6), nucleic acid metabolism, Signal transduction, Immunity and defense, Developmental processes, Oncogenesis SMYD3 SET and MYND domain containing NM_022743.1-1.01 1.14 3.41 Nucleoside, nucleotide and 3 (SMYD3), nucleic acid metabolism SPOCK1 sparc/osteonectin, cwcv and NM_004598.3-1.2 1.1 3.31 Biological process unclassified kazal-like domains proteoglycan (testican) 1 (SPOCK1), THBS1 thrombospondin 1 (THBS1), NM_003246.2-1.21 1.69 2.3 Immunity and defense, Developmental processes TMEM45A transmembrane protein 45A NM_018004.1 1.07 1.77 3.52 Biological process unclassified (TMEM45A), TRIB1 tribbles homolog 1 (Drosophila) NM_025195.2 2.7 3.6 2.4 Protein metabolism and modification, (TRIB1), Signal transduction TUFT1 tuftelin 1 (TUFT1), NM_020127.1 1.4 2.78 1.95 Developmental processes 여기서 fold 값이란실험군이대조군의값에대해서실제로몇배의차이가있는지를 ( 실험군의값 / 대조군의값 ) 으로살펴본수치이다. 예를들어, 2.8 fold 라면대조군에비해실험군에서 2.8 배더많이발현된것이고 -5.5 fold 라면, 대조군에비해실험군에서 5.5 배가덜발현됨을의미한다. 178 개의유전자중에서 up-regulation 된유전자와 down-regulation 된유전자를 biological process 에따라분류하여각각 Figure 1a, 1b 에표현하였다. Up-regulation 된유전자들중에서는신호의전달 (signal transduction), 발생과정 (development process), 단백질대사 157
a b Figure 1. Differentially expressed genes (178 genes) up- or down-regulated by at least two-fold following a cellular treatment with MTA at every time point. (a) The biological processes of up-regulated 109 genes, (b) The biological processes of downregulated 69 genes. Table 3. The expression level of the messenger RNA determined by RT-PCR. RT-PCR was conducted three times. The relative level of gene expression was normalized against GAPDH messenger RNA, and the control was set as 1.0. Optical density values represent the mean. 6hrs 24hrs 72hrs FOSB BHLHB2 TUFT1 DCN THBS1 FN1 COL10A1 Control 1.00 1.00 1.00 1.00 1.00 1.00 1.00 MTA 1.53 1.91 1.09 0.46 1.55 1.92 1.12 Figure 2. Optical density of messenger RNA measured by densitometry. (protein metabolism), 세포의구조와운동성 (cell structure and motility), 세포의부착 (cell adhesion), 세포의증식과분화 (cell proliferation and differentiation) 에관여하는유전자가많았으며 down-regulation 된유전자들중에서는신호의전달 (signal transduction), 면역반응과방어기전 (immunity and defense), 발생과정 (development process), 단백질대사 (protein metabolism) 에관여하는유전자가많았다. RT-PCR 은 FBJ murine osteosarcoma viral oncogene homolog B (FOSB), thrombospondin 1 (THBS1), basic helix-loop-helix domain containing, class B, 2 (BHLHB2), fibronectin 1 (FN1), decorin (DCN), collagen, type X, alpha 1 (COL10A1), tuftelin 1 (TUFT1) 에대하여시행하였으며그결과는 Table 3, Figure 2 에표현하였다. 158
Mineral trioxide aggregate 가인간치수세포에서유전자발현에미치는영향 Ⅳ. 총괄및고찰 최근들어생체적합성이뛰어난 MTA가치수복조제재로사용되었을때우수한연구결과들을보이고있지만, 유전자수준에서 MTA가치수세포에어떤영향을주는지에대한전반적인정보는많이부족한상태이다. 이번연구는 MTA 를적용한치수세포에서발생하는일련의유전자변화를 microarray로보여준최초의연구이다. 이번연구의한계내에서, 본연구의결과는치수복조용제재로서 MTA가치수세포의분화와증식을촉진시키고상아질형성능력을유도하는유전자의변화를유도할가능성을시사하고있다. 손상받은치수의상부에서수복상아질이생성되기까지는몇단계의과정을거친다. 20) 먼저, 치수에존재하는줄기세포나전구세포 (progenitor cell) 가손상받은부위로모이게된다. 이세포들이증식한후 odontoblast-like cell이나 osteoblast-like cell로분화한다. 마지막으로세포외기질을생성하고석회화과정을거치는것이다. MTA를적용한치수세포에서 odontoblast-like cell 혹은 osteoblast-like cell로의분화를유도하는유전자들의변화가본연구의결과에서확인이되었다. Drosophila protein인 mothers against decapentaplegic (Mad) 와 C. elegans (Sma) 의 homolog인 SMAD 는세포의신호체계 (cell signaling) 에작용을하며특히 BMP와같은 TGF-βfamily를조절하는역할을한다. 21) 포유류에서확인된 SMAD 단백질은 8가지이며이중 SMAD6는 Co-Smad인 SMAD4와경쟁하는 inhibited Smad로작용하는것으로알려져있다. 22) 본실험에서 MTA를 6시간과 24시간적용한실험군에서 SMAD6가감소하였으며 72시간에가장많이감소하는경향을보였다 (-2.24 fold). TGF-βfamily가치수세포의분화에중요한영향을미친다고알려진것을고려할때 MTA에의한이러한변화는치수세포의분화와연관이있을것으로사료된다. FBJ murine osteosarcoma viral oncogene homolog B (FOSB) 는본실험에서 MTA를 6시간적용한실험군에서 2배이상발현되었다 (2.17 fold). 세포의분화와증식에관여하는 FOS family의특성상 FOSB가조상아세포와조골세포에미치는영향에관해서도여러가지실험결과가발표되었다. 기존연구에서체세포분열이끝나분화가완료된상태인조상아세포와분화가완료되지않은상태인외배엽성중배엽세포 (ectomesenchymal cell) 가포함된치수세포에서의 FosB의발현에는유의성있는차이가없다는관찰결과를근거로 FosB가치수세포의분화와증식에는영향을주지않는것같다고보고하였다. 23) 그러나최근의연구에서 FosB의 short splicing isoform인 deltafosb가골형성을촉진하는것이발견된후 deltafosb가조골세포형성을조 절하는가능성이제시되었다. 24) MTA에의한치수세포상의 FOSB의변화가가지는의미와치수세포에서조상아세포로의분화와증식과정에서 FOSB가어떤역할을하는지에대한계속적인연구가필요하리라사료된다. Basic helix-loop-helix transcription factor Dec1 (BHLHB2) 는세포의분화를조절하는유전자중하나이다. 본실험에서는 BHLHB2가 MTA를 6, 24, 72시간적용한모든실험군에서 2배이상증가하는양상을보였다 (2.47, 3.55, 2.54 fold). BHLHB2는중배엽성줄기세포 (mesenchymal stem cell) 의연골성분화 (chondogenic differentiation) 에영향을주는것으로알려져왔다. 25) 그러나최근의연구에서중배엽성줄기세포에골형성을유도하는물질을넣은경우 BHLHB2가많이발현되며지방형성을유도하는물질을넣은경우적게발현되는현상을보였다. 26) 또 BHLHB2을제거하였을때조골세포의표현형 (osteoblastic phenotype) 을억제하는양상을보였다. 이와같은사실에주목해보면 BHLHB2이 6, 24, 72시간에서전반적으로증가하는양상이 MTA에의한경조직형성의과정의일부분이라고사료된다. Thrombospondin 1 (THBS1) 은부착성당단백질 (adhesive glycoprotein) 의일종으로세포간상호작용이나세포와기질의상호작용을매개하는기능을한다. 27) 또한 TGF-β에결합하여이를활성화시키는기능을하는것으로알려져있다. 28) THBS1은치수조직이나치은조직에서는관찰되지않으며오직 predentin에분포하며특히조상아세포에서만관찰된다. 29) 이러한현상을종합해볼때 THBS1 이치수의석회화과정에서중요한역할을하는것으로생각된다. 본실험에서 MTA를 72시간적용한실험군에서 THBS1이 2배이상증가하였다 (2.30 fold). 이러한증가는 MTA가적용되었을때치수세포혹은줄기세포에서조상아세포가존재하는환경으로변화되는것이라추측할수있다. Endothelin 1 (EDN1) 은 vascular tone, 혈압, 그리고나트륨과물의항상성 (homeostasis) 등의기전을조절하는등다양한기능을가지는것으로알려진 21-amino acid peptide이다. 최근의연구에서 EDN1이치수의혈관에서관찰되었다. 30) 또다른연구에서 endothelin receptor가발육중인치아조직에다양하게분포하는것을확인한후 EDN1이치아의발생과정에다양한기능을할가능성을제시하였다. 31) EDN1은섬유아세포에작용하여세포외기질단백질을생성하며특히 fibronectin의생성을촉진시키는것으로알려져있다. 32-34) 최근의연구에서 EDN1을일차세포배양한인간치수세포에적용시킨경우치수세포가증식되고 mitogen 기전이활성화되며 type I collagen의합성이증가하는양상을관찰하였으며이를근거로 EDN1이치수의치유와치수세포의분화에중요한역할을할것이라 159
고추론하였다. 35) 본실험에서는 MTA를 6, 24시간적용한실험군에서 EDN1이 2배이상증가하는양상을보였다 (2.83, 3.11 fold). Interleukin 11 (IL11) 은골수의기질세포 (bone marrow stromal cell) 에서발견되며여러가지기능을가지는 cytokine의하나이다. IL11의중요한기능중한가지는조골세포생성을촉진시키는것이다. 36) IL11이조골세포의분화와골생성을촉진시키는현상은여러연구에서확인되었다. 37) 또한 IL11을정상보다많이발현하는 transgenic mice에서골괴 (bone mass) 가더많이형성된다는사실이발견되었다. 38) 본실험에서는 MTA를 6, 24시간적용한실험군에서 IL11이 2배이상발현되었다 (2.31, 2.60 fold). Fibronectin (FN) 은 integrins, collagen, fibrin 등과결합하는세포외기질당단백질중하나이다. 세포의부착, 성장, 이동, 그리고분화에영향을미치며창상치유와배아의발생 (embryonic development) 에중요한역할을한다. 39) 치아가생성되는동안 dental basement membrane 과관계되어관찰되는 FN은조상아세포의분화에중요한역할을한다. 40) 더욱이 FN은조상아세포의분극화 (polarization) 와이동 (migration) 에중요한역할을하여치아의형성과수복상아질의형성에기여한다. 41) 최근의연구에서 bovine plasma FN이사람치수세포의부착과이동에영향을주며조상아세포로의분화를조절하는역할을한다고주장하였다. 42) 본실험에서 FN은 72시간실험군에서 2배이상증가하였으며 (2.10 fold) 이러한현상이치수세포내의특정세포가조상아세포로분화하는과정중이라는것을증명한다고사료된다. 이런사실에근거하여치수세포에서 odontoblast-like cell이나 osteoblast-like cell로의분화에관여하는유전적흐름에 MTA가미치는영향이비교적이른시기에시작됨을알수있다. 수복상아질을형성함에있어서다른단계인석회화를조절하는단계와관련된유전자적변화역시본연구의결과에서확인할수있었다. 본실험에서는 collagen fibrillogenesis와성숙에관련이있는 decorin (DCN) 이 72시간에서급격히감소하였다 (-3.69 fold). Collagen 기질의석회화과정중 DCN의발현과석회화의반비례관계가여러연구에서보고되었다. 석회화가발생하기전 DCN이없어지거나변성되는것을발견한후 DCN이석회화를순간적으로조절할것이라는주장이제시되었다. 43) 최근의연구에서 DCN을정상보다많이발현하는조골세포 clone과정상보다적게발현하는 clone을비교하였을때적게발현하는 clone에서석회화가가속화되며석회화결절 (mineralized nodule) 의수가증가하는것을보고하였다. 44) 본실험에서 MTA를적용한후나타나는 DCN의급격한감소가 MTA가치수복조제재로 서지니는중요한상아질형성능력을유전자수준에서뚜렷하게반영하는결과라고생각한다. Collagen, type X, alpha 1 (COL10A1) 은연골내골화 (endochondral ossification) 이일어나는동안발생하는비후성연골세포 (hypertrophic chondrocyte) 의산물로알려져있다. 45) 최근의연구에서발생중인인간의법랑질 organ과법랑모세포에 Collagen type X이다양하게존재하는것을확인하였다. 46) 아직까지 Collagen type X이부족할때치아에서생기는변화에대한보고는없으나 Collagen type X이부족할때성장판연골 (growth plate cartilage) 과해면골 (trabecular bone) 에서는구조이상이관찰된다. 47) 본실험에서는 MTA를 72시간적용한실험군에서 COL10A1이 2배이상증가는양상을보였다 (2.88 fold). Tuftelin (TUFT) 는치아의법랑질에서발견되는인산화된당단백질 (phosphorylated glycoprotein) 이다. TUFT 는주로상아질이형성되는시기중짧은기간에만나타나기때문에법랑질의석회화를시작시키는물질로지목되었다. 48) TUFT는특히결정핵생성 (nucleation) 과성장 (crystal growth) 에영향을미친다. TUFT는법랑질형성의초기에분비성법랑모세포 (secretory ameloblast cell) 에서주로관찰되며특히 Tomes process에서주로발견된다. 또한법랑질이형성되는상아법랑질경계부위에서집중되는현상을보인다. 49) 본실험에서는 MTA를 24시간적용한실험군에서 TUFT1은 2배이상증가하였다 (2.78 fold). MTA를적용한치수세포에서 COL10A1과함께법랑질형성과관계되는 TUFT의유전자가발현되는것은재미있는현상이라고할수있다. 위에서언급한 10개의유전자는 microarray를사용한실험결과중에서세포의분화와증식에관여한다고알려져있는유전자들을찾아서기존의논문을고찰하였으며이중비교적변화가명확한유전자에대하여 RT-PCR을시행하였다. MTA는 bio-inert material이라기보다는치수세포에여러가지경로를통해영향을주는재료라고생각된다. 특히 MTA는치수세포의분화와증식에관여하는유전자의변화에영향을미치며석회화의과정에관여하는유전자의변화에영향을준다는사실이확인되었다. 앞으로더긴시간동안관찰할수있는실험과다른치수복조제재와의비교실험이필요할것으로사료된다. 참고문헌 1. American Association of Endodontists. Glossary of endodontic terms, 7th ed. pp. 40, 2003. 2. Cox CF, Subay RK, Ostro E, Suzuki S, Suzuki SH. Tunnel defects in dentin bridges: their formation fol- 160
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국문초록 Mineral Trioxide Aggregate 가인간치수세포에서유전자발현에미치는영향 김용범 손원준 이우철 금기연 백승호 배광식 * 서울대학교치의학대학원치과보존학교실, 치의학연구소, BK21 Program 이연구에서는 mineral trioxide aggregate (MTA) 를 in vitro 로인간치수세포에적용하였을때유전자들의변화를조사하였다. 실험군은 MTA 를 teflon tube ( 직경 10 mm 길이 2 mm) 에담아 4 시간경화시킨후 HDPCs 에적용하였고, 대조군은빈 tube 만을적용하였다. 6, 24, 72 시간후 total RNA 를추출하여 microarray 를이용하여분석하여, 2 배이상또는절반이하의변화를보이는유전자중선택적으로역전사중합효소연쇄반응 (reverse transcriptase polymerase chain reaction) 을사용하여발현을확인하였다. 24,546 개의유전자중에서 109 개의유전자가 2 배이상 up-regulation 되었으며 ( 예. FOSB, THBS1, BHLHB2, EDN1, IL11, FN1, COL10A1, TUFT1) 69 개의유전자가 50% 이하로 down-regulation 되었다 ( 예. SMAD6, DCN). MTA 는 bio-inert 한재료라기보다는치수세포에다양한경로로영향을주는재료로사료된다. 특히치수세포의분화와증식에관여하는유전자의변화에영향을주며석회화과정에관여하는유전자의변화에직접적인영향을주리라사료된다. 주요단어 : 치수복조, 치수세포, microarray, mineral trioxide aggregate, 세포분화, 세포증식 163