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1 ORIGINAL ARTICLE J Korean Thyroid Assoc Vol. 4, No. 1, May 2011 형질전환마우스를이용한갑상선유두암모델의확립 가톨릭대학교대전성모병원병리과 1, 충남대학교의학전문대학원내과학교실 2 이정의 1 *, 정경혜 2 *, 조영석 2, 송민호 2 Model Development of Papillary Thyroid Carcinoma by Transgenic Mice Jung Uee Lee, MD, PhD 1, Kyong Hye Joung, MD 2, Young Suk Jo, MD, PhD 2 and Minho Shong, MD, PhD 2 Department of Pathology, Daejeon St. Mary's Hospital, The Catholic University of Korea 1 ; and Research Center for Endocrine and Metabolic Diseases, Chungnam National University School of Medicine 2, Daejeon, Korea Background and Objectives: The mutation has been regarded as the leading cause of papillary thyroid cancer (PTC). However, the multi-step carcinogenic process induced by has been remained to be elucidated in thyroid gland. In this study, to investigate staged development of papillary thyroid carcinoma, we observed the histo-pathological findings of thyroid gland from transgenic mice with a period of 60 weeks. Materials and Methods: We histologically inspected 3, 9, 20, 27, 39, 44, 48 and 60 week old transgenic mice derived from FVB/N background mice with a bovine thyroglobulin promoter which are providing thyroid specific expression. Results: Thyroid glands from 3 and 9 week old transgenic mice were enlarged and showed abnormal histologic feature such as distorted follicular architectures. The 20 and 27 week old transgenic mice showed irregularly enlarged thyroid gland sprouting out above the carotid arteries. Thyroid gland derived from 39 week old mice showed reduced formation of intact follicular structure and increased solid area. Thyroid glands were entirely replaced by firm tumor mass composed of poorly differentiated cell at 44 weeks. Interestingly, we could observe tracheal invasion, surrounding muscle invasion in thyroid gland from 48 week old mice and detect lung metastasis in 60 week old mice. Conclusion: Thyroid specific expression of induced staged development of thyroid cancer. This finding may support that have a role in entire carcinogenic process such as tumor initiation, development and progression. Key Words: Disease model, Proto-Oncogene Proteins B-raf, Thyroid neoplasm, Transgenic mice 서론 최근갑상선암의발생이증가하면서갑상선암의원인유전자에대한연구가활발하게진행되고있다. 또한, 이러한연구에힘입어전통적으로잘알려진 RET/ PTC나 TRK/T rearrangements 이외에 돌연변이가갑상선암의주요한원인유전자로제시되고, 그분자생물학적역할이밝혀지고있다. 1-4) RAF kinase는 A-, B-, C-RAF 세가지의 paralogue가알려져있는데, 이들은 RAS-MEK-MAP/ERK 신호경로의중요한인자로서세포의성장과분화에관여하는 논문접수일 : 2011 년 4 월 21 일 / 심사완료일 : 2011 년 5 월 19 일교신저자 : 송민호, 대전시중구대사동 640, , 충남대학교병원내분비대사내과 Tel: , Fax: , minhos@cnu.ac.kr *The first two authors equally contributed to this work. 이논문은 2009 년도충남대학교병원대덕특구연구과제연구비에의하여수행되었음. 30

2 형질전환마우스표현형분석 것으로알려져있다. 5,6) 흥미롭게도암을일으키는돌연변이는이들세가지 paralogue 중에 B-RAF에서만관찰되는데현재까지알려진돌연변이만도 40여개가넘는다. 특히, B-RAF의 1799번째염기인 Thymine (T) 가 Adenine (A) 로치환된 missense mutation (BRAF T1799A, ) 는현재까지알려진 B-RAF 돌연변이의 90% 가량을차지하는것으로보고되고있다. 는 B-RAF의키나아제활성을증가시키는 Thr599나 Ser602이인산화된 wild type BRAF과유사한구조적변화를가지며, 이를통해 Ras에의한활성화없이도 MEK-MAP/ERK 신호경로를활성화시키게된다. 7) 결국, 는궁극적으로다양한 ERK 의존적전사인자의작용을통해세포의성장을지속적으로촉진시키고암을일으키는것으로생각되고있다. 이러한가설은세포수준의연구뿐만아니라, 갑상선특이적 형질전환마우스에서도증명되고있다. 1,5) 그러나, 2007년에보고된 Dankort 등의 폐암모델을보면, 6주령이하의마우스에서는 hyperplasia 발생부위에 Ki67와같은 proliferation marker 발현만이확인되지만, 8주령마우스부터는 proliferation marker 발현은감소하고 p19 ARF 와같은 senescence marker 발현이증가하였다. 또한 돌연변이만발생시킨경우에는 3 5% 에서만폐선암이발생하였으나 Ink4a/ Arf와 TP53 결손이함께발생한경우에는마우스대부분에서폐선암이발생하였다. 8) 이연구결과들을통해서, 폐암모델에서 는세포의성장을촉진하나 growth arrest와 oncogene-induced senescence를야기하며, Ink4a/Arf와 TP53에의존적으로폐암을일으키는것을알수있다. 또한, 2009년에보고된 Goel 등의연구에서도, melanocyte를타겟으로한 의발현이 benign melanocytic hyperplasia와동반된 senescence를야기하며, CDKN2A의소실과동반될때흑색종을일으키는것이확인되었다. 9) 결국, 폐암이나흑색종동물모델에서 는세포의성장과동반된 senescence를야기하여세포의사멸을가져오는것으로판단되며, 종양억제유전자의기능소실이동반될때비로소악성화과정을유도하는것으로판단된다. 8-10) 그러나, 갑상선암의형성에있어 와종양억제유전자의기능소실과의관련성은많이연구되어있지않은실정이다. 본연구에서는 3 60주령의 형질전환마우스의주령별조직학적특성을분석하여 변이에의한갑상선암의발생과진행과정을알아보고, 폐암및흑색종동물모델의종양형성및 발달단계 (staged development) 를 갑상선암동물모델과비교해보고자하였다. 대상및방법 실험동물 형질전환마우스 20마리와 FVB/N 마우스 20마리를각각실험군과대조군으로이용하였다. 형질전환마우스와 FVB/N 마우스는 Memorial Sloan Kettering Cancer Center의 James A. Fagin 박사로부터제공받았다. 실험동물은충남대학교의학전문대학원실험동물사육실에서사육되었으며동물들은온도 22±2 o C, 습도 45 55%, 조도 300 lux ( 단상 cm 2 ) 및명암주기 12시간의환경에서마우스용고형사료 ( 대한바이오링크, Korea) 및무균음용수를무제한으로공급받았다. 동물실험은충남대학교의학연구소의실험동물윤리위원회 (Institutional Animal Care and Use Committee) 방침및법규를준수하였다. 돌연변이확인 형질전환마우스는 bovine thyroglobulin promoter, β-globin intron 2, myc-tagged BRAF T1799A 를사용하여제작되었으며 (Fig. 1A), 마우스의꼬리에서추출한 DNA를 PCR로분석하여형질전환여부를판정하였다. 사용한 Primer의염기서열은순방향 5'- TGGTAGAAACAACTACATCCTGGTC-3' 와역방향 5'-GGGATTAGACCTCCATCATAG-3' 이었고, HiPi PCR PreMix (ELPis Biotech, Korea) 를사용하여 PCR을수행하였다. 부검, 조직표본준비및조직학적관찰 3, 9, 20, 27, 39, 44, 48, 60주령의실험동물을이산화탄소로희생시킨후, 목부위의피부를절개하여갑상선을포함한주변조직및장기를적출하였고흉골을절개하여폐를적출하였다 (Fig. 1B). 적출된조직은육안검사를거친후 10% 중성포르말린으로고정하여파라핀에포매하였다 (Fig. 1C, D). 조직의파라핀블럭을 4μm 두께로절편한후, Hematoxylin & Eosin (H&E) 염색을시행하였다. 면역염색법 형질전환마우스에서적출한갑상선조직의파라핀블럭을 4μm 두께로절편하여면역염색용슬라이드에부착시키고 xylene에 5분씩 2회담구어파 31 J Korean Thyroid Assoc

이정의외 Fig. 1. (A) Map of the Tg-BRAF transgene.

for injection. Upper arrow, transcription start site; blue boxes, 5 V untranslated regions (Adopted from Cancer Res 2005;65(10):4238-4245).

Removal of the central compartment of neck including skin and strap muscle exposes the trachea and thyroid gland. Thyroid glands are located on each side of trachea (arrows).

3 이정의외 Fig. 1. (A) Map of the Tg-BRAF transgene. A Spe I/Sal I fragment containing the bovine thyroglobulin promoter, rabbit h-globin intron 2, and the myc-tagged BRAFT1799A cdna containing the endogenous human BRAF polyadenylation signal was used for injection. Upper arrow, transcription start site; blue boxes, 5 V untranslated regions (Adopted from Cancer Res 2005;65(10): ). (B) Gross inspection of thyroid glands derived from 27 week old thyroid specific transgenic mouse. Removal of the central compartment of neck including skin and strap muscle exposes the trachea and thyroid gland. Thyroid glands are located on each side of trachea (arrows). (C) Gross inspection of thyroid gland extracted from a wild type FVB/N mouse. Thyroid glands consist of two obvious lobes without an isthmus (arrows). (D) Gross inspection of thyroid gland extracted from 39 week old thyroid specific transgenic mouse. Thyroid glands are noticeably lager than wile type FVB/N mouse. Both lobes are joined on the trachea. 라핀을제거한후, 농도차가있는 alcohol (100% 부터 80% 까지 ) 에 3분씩처리하여함수화하였다. 항원성회복을위하여 10 mm 구연산완충액 (ph 6.0) 에조직절편을담가 autoclave (CHS-ACCE-860, Choong Wae Medical Corporation, Korea) 에서 25분간끓인다음실온에서식혔다. 내인성 peroxidase의활성을제거하기위해조직슬라이드를 3% hydrogen peroxide에서 5분간둔다음 normal horse serum (DakoCytomation LSAB2 System-HRP kit; DakoCytomation, Denmark) 에서 10분간처리하였다. 일차항체인 Galectin-3 rabbit polyclonal (1:50, ab53082, Abcam Inc., Cambridge, MA) 와실온에서 30분간반응시킨뒤, 수세후조직슬라이드를 biotin 결합이차항체와 10분동안반응시키고 Streptavidin-HRP & 3,3'-diaminobenzidine (DAB) solution 에서다시 10분동안반응시켰다. 조직슬라이드는 Harris hematoxylin으로대조염색을한후봉입제로봉입하였다. 결과 FVB/N 마우스갑상선의조직학적소견대조군 FVB/N 마우스의정상갑상선은후두의아래쪽기도양옆에위치하고연결구조없이우엽과좌엽으로나누어져있었다. 양쪽엽에각각한개의부갑상선 (parathyroid gland) 과한개의림프절 (lymph node) 이붙어있었으며, 갑상선을둘러싼막 (membrane) 은존재하지않았으며갈색지방조직과백색지방조직으로둘러싸여있었다. 정상갑상선은다양한크기를가진둥근모양의여포 (follicle) 로이루어져있었는데, 여포는평평또는입방형의여포세포 (follicular cell) 가단층으로배열되어있고여포강 (follicular lumen) 은분홍색으로균질하게염색된콜로이드로채워져있었다 (Fig. 2A C). 여포사이간질조직 (interfollicular area) 에는소포곁세포 (parafollicular C-cell), 모세혈관, 갈색지방세포 (brown adipose tissue) 가관찰되었다 (Fig. 2D). Vol. 4, No. 1,

형질전환마우스표현형분석 Fig. 2. Representative figures of thyroid glands from wild type FVB/N mice. (A, B) Thyroid glands are composed of relatively round uniform follicles.

(D) Brown adipose tissues are scattered in the interfollicular space.

실험군과대조군간의갑상선전체크기를비교한결과, 형질전환마우스의갑상선은같은주령의 FVB/N 마우스에비해서크기가커져있었다 (Fig. 1C, D). 3주령부터 60주령까지 형질전환마우스의갑상선조직을 H&E 염색으로관찰하였다. 형질전환마우스 20마리전체에서갑상선암의발생을확인할수있었다.

여포세포는 Galectin-3 면역염색에양성을보여서형질전환이일어났음을확인할수있었다 (data not shown). 9주령 형질전환마우스의갑상선 : 육안적으로갑상선의좌엽과우엽은서로연결되어있지않은상태로존재하였다. 양엽의크기는비슷하였고한엽의크기는평균장경이 0.

4 형질전환마우스표현형분석 Fig. 2. Representative figures of thyroid glands from wild type FVB/N mice. (A, B) Thyroid glands are composed of relatively round uniform follicles. Parathyroid gland (arrows) and lymph node are noted on thyroid gland. (C) Follicular cells are flat or cuboidal. Follicular lumen contains a homogeneously eosinophilic stained colloid. (D) Brown adipose tissues are scattered in the interfollicular space. 주령별 형질전환마우스갑상선의조직학적소견 형질전환마우스를번식시키고, 이들마우스에서 형질전환유전자의일부인 β- globin intron을확인함으로써갑상선특이적으로 의발현을증명하였으며, 형질전환마우스에서갑상선기능저하가나타날수있어서 8) 머리에서꼬리까지의길이를측정하여대조군인 FVB/N 마우스와비교하였으나유의한차이는없었다 (data not shown). 실험군과대조군간의갑상선전체크기를비교한결과, 형질전환마우스의갑상선은같은주령의 FVB/N 마우스에비해서크기가커져있었다 (Fig. 1C, D). 3주령부터 60주령까지 형질전환마우스의갑상선조직을 H&E 염색으로관찰하였다. 형질전환마우스 20마리전체에서갑상선암의발생을확인할수있었다. 3주령 형질전환마우스의갑상선 : 육안적으로기관을중심으로좌엽과우엽이서로연결되어있지않은상태로존재하였고, 크기는대조군과비슷하였다. H&E 염색상에서는다양한크기의비교적원형을유지하는여포를관찰할수있었다. 대조군과비교했을때실험군여포의크기가더다양하고여포세포의크기가더크게보여정상과다름을확인할수있었다. 대조군과마찬가지로여포사이간질조직에서갈색지방세포를관찰할수있었다. 여포세포는 Galectin-3 면역염색에양성을보여서형질전환이일어났음을확인할수있었다 (data not shown). 9주령 형질전환마우스의갑상선 : 육안적으로갑상선의좌엽과우엽은서로연결되어있지않은상태로존재하였다. 양엽의크기는비슷하였고한엽의크기는평균장경이 0.2 cm로대조군에비해상당히커져있었으며, 기도와경동맥 (carotid artery) 사이에위치하였다 (Fig. 3A). 대부분의여포는비교적원형을유지하였으나일부는다각형을띠었다. 여포들사이사이에얇은섬유조직이발달하여구획 (nest) 을형성하였다 (Fig. 3B). 여포세포는입방형으로대조군에비해서핵이더컸으며호산성 (amphophilic) 의세포질을보였다 (Fig. 3C). 대조군과비교했을때여포사이간질조직에서존재하던갈색지방세포는관찰되지않았으며, 여포강은탁한분홍색을띠는콜로이드와대식세포, dust-like material로채워져있었다 (Fig. 3D). 20주령 형질전환마우스의갑상선 : 갑상선의양엽은경동맥 (carotid artery) 의바깥부분에위치하고크기는각각장경 0.25 cm이었다 (Fig. 3E, F). 갑상선은다양한크기의불규칙한모양을지닌여포로구성되어있었고, 입방형의여포세포는약간커진핵이가운데위치하고있었으며, 일부에서는섬모가관찰되었다 (Fig. 3G, H). 여포세포에서유사분열은관찰되지않았다. 여포강의콜로이드는탁한분홍색이거나푸른빛이도는분홍색을띠었고대식세포나 dust-like material이관찰되기도하였다 (Fig. 3G). 여포사이간질조직에서갈색지방세포는관찰되지않았다. 27주령 형질전환마우스의갑상선 : 갑상선이경동맥을감싸면서성장한것을확인할수있었다 (Fig. 3I, J). 여포사이간질조직의섬유화가더심해져서갑상선조직의구획형성은더뚜렷해졌다. 갑상선은다양한크기와좀더불규칙한모양을가진여포로구성되어있고간혹유두상 (papillary structure) 이관찰되었다. 일부에서는여포를구성하지못한여포세포들이고형성분 (solid portion) 을형성하였는데이것은 33 J Korean Thyroid Assoc

mice. (A D) Thyroid glands of 9 weeks old mice.

side of trachea. (B) Thyroid gland is composed of relatively round follicles.

(C) Follicle consists of a cuboidal cell with a slightly enlarged, uniform nucleus.

(E H) Thyroid glands of 20 weeks old mice.

(F) Both lobes of thyroid gland have grown outside carotid arteries which are ascended

(G) Thyroid gland consists of variable sized and relatively irregular shaped follicles.

Colloid shows a turbid grayish-pink or bluish-pink color and contains macrophages or

(H) Some of follicular cells have a cillia-like sturecture (arrows).

(I, J) Carotid artery is enclosed with thyroid gland.

Fibrous septa surrounding follicles are more prominent.

Mitoses are occationally found in follicular cell.

5 이정의 외 V600E Fig. 3. Representative figures of thyroid glands from 9, 20 and 27 week old BRAF transgenic mice. (A D) Thyroid glands of 9 weeks old mice. (A) Small sized thyroid glands, about 2 mm in length of one lobe, are located on each side of trachea. (B) Thyroid gland is composed of relatively round follicles. There is no brown adipose tissue in interfollicular spaces. (C) Follicle consists of a cuboidal cell with a slightly enlarged, uniform nucleus. (D) Colloid shows a turbid pink color and contains macrophages or dust-like materials. (E H) Thyroid glands of 20 weeks old mice. (E) One lobe of thyroid gland measures about 2.5 mm in length. (F) Both lobes of thyroid gland have grown outside carotid arteries which are ascended on the both side, anterolateral to the trachea. (G) Thyroid gland consists of variable sized and relatively irregular shaped follicles. Follicle consists of cuboidal cells with a slightly enlarged, uniform nucleus. Colloid shows a turbid grayish-pink or bluish-pink color and contains macrophages or dust-like materials. Mitotic figures are rarely founded in follicular cells. (H) Some of follicular cells have a cillia-like sturecture (arrows). (I L) Thyroid glands of 27 weeks old mice. (I, J) Carotid artery is enclosed with thyroid gland. (K) Thyroid gland consists of variable sized and more irregular shaped follicles. Fibrous septa surrounding follicles are more prominent. Follicular cell consists of a slightly polygonal flat or cuboidal cell. Mitoses are occationally found in follicular cell. (L) Cillia-like structures of follicular cell are prominent (arrows). 분화도가 나쁜 부분으로 이해할 수 있다(Fig. 3K). 여포 39주령 BRAF V600E 형질전환 마우스의 갑상선: 육 세포는 평평한 모양부터 다각형(polygonal)까지 다양한 안상 갑상선의 양엽이 커지면서 가운데에서 만나 고리 형태를 보이며, 좀 더 많은 세포에서 섬모가 관찰되었 모양으로 기관지를 둘러싸고 있었다. 한 엽의 길이는 다(Fig. 3L). 여포세포의 핵은 커지고 불규칙한 핵막을 0.35 cm이었다(fig. 4A). H&E 염색상, 갑상선은 주변 가졌으며 유사분열이 간혹 관찰되었다. 여포강은 20주 골격근을 밀어내면서 성장하지만 골격근, 부갑상선, 림 령과 유사하게 탁한 콜로이드와 대식세포, dust-like 프절 등의 주변 조직으로의 침윤은 관찰되지 않았다 material로 채워져 있다. (Fig. 4A, B). 갑상선은 다양한 크기와 모양을 가진 여 Vol. 4, No. 1,

형질전환마우스표현형분석 Fig. 4. Representative figures of thyroid glands from 39 and 44 week old transgenic mice.

Both lobes of thyroid gland fuse in the midline of trachea.

muscle. (C) Thyroid gland consists of variable sized and irregular shaped follicles.

(D) Follicular cell consists of a variable shaped and sized cell.

(F K) Thyroid glands of 44 weeks old mice. (F) Thyroid gland measures about 5 mm in the largest length.

Irregular shaped follicles are rarely founded among solid portion.

(J) Follicular cells with cilia-like structures are noted. 포로구성되며유두상이나고형성분이차지하는비율이좀더많아졌다 (Fig. 4C, D).

호중구등의염증세포가침윤되어있었다 (Fig. 4G I). 여포를형성하는여포세포들에서섬모가관찰되었다 (Fig. 4J).

H&E 염색상, 갑상선은다양한크기와모양의여포로구성되어있는데비교적크기가큰여포가갑상선의외곽에존재하고중앙에는고형성분이분포하였다 (Fig. 5A).

6 형질전환마우스표현형분석 Fig. 4. Representative figures of thyroid glands from 39 and 44 week old transgenic mice. (A E) Thyroid glands of 39 weeks old mice. (A) One lobe of thyroid gland measures about 3.5 mm in length. Both lobes of thyroid gland fuse in the midline of trachea. (B) There is no evidence of metastasis to the lymph node, parathyroid gland and surrounding skeletal muscle. (C) Thyroid gland consists of variable sized and irregular shaped follicles. Thyroid follicular cells focally grow as a solid mass of cells. (D) Follicular cell consists of a variable shaped and sized cell. (E) Cilia-like structures of follicular cell are more prominent (arrows). (F K) Thyroid glands of 44 weeks old mice. (F) Thyroid gland measures about 5 mm in the largest length. Thyroid gland is a large solid mass. (G I) Thyroid follicular cells nearly entirely grow as a solid mass. Irregular shaped follicles are rarely founded among solid portion. There is a infiltate of inflammatory cells in follicules. (J) Follicular cells with cilia-like structures are noted. 포로구성되며유두상이나고형성분이차지하는비율이좀더많아졌다 (Fig. 4C, D). 여포세포는 27 주령에서관찰되었던것과비슷하였다 (Fig. 4C E). 44주령 형질전환마우스의갑상선 : 육안상으로갑상선은기도를둘러싸는하나의단단한종괴를이루고있었고표면은비교적매끈하였다 (Fig. 4F). 갑상선의크기는길이 0.5 cm이었다. H&E 염색상, 갑상선의대부분은여포를형성하지않은여포세포들이군집을이루고있고이들사이에서비전형적인모양의여포를드물게관찰할수있었으며, 갑상선전반에걸쳐다수의림프구, 형질세포, 호중구등의염증세포가침윤되어있었다 (Fig. 4G I). 여포를형성하는여포세포들에서섬모가관찰되었다 (Fig. 4J). 48주령 형질전환마우스의갑상선 : 육안상에서갑상선의표면은약간울퉁불퉁하였고주변조직과분리가잘되지않았다. H&E 염색상, 갑상선은다양한크기와모양의여포로구성되어있는데비교적크기가큰여포가갑상선의외곽에존재하고중앙에는고형성분이분포하였다 (Fig. 5A). 갑상선은주변조직과의경계가명확하지않았고일부여포가주변골격근으로침윤성성장을하고있었다 (Fig. 5B, C). 60주령 형질전환마우스의갑상선 : 육안상과 H&E 염색상, 48주령 형질전환마우스의갑상선과비슷하였다 (Fig. 5D F). 그리고기관의점막하조직에갑상선여포가관찰되어갑상선암의기관지로침윤을확인할수있었다 (Fig. 5G). 적출된폐에서 cm 크기의둥근고립성폐결절이관찰되었고, H&E 염색상에서유두상구조를이루는갑상선여포세포를관찰할수있어서갑상선암의폐전이를확인하였다 (Fig. 5H, I). 갑상선암의폐전이병변은주변정상폐조직과경계가좋았고원발병소에비해서유두상구조가뚜렷하였다 (Fig. 5J). 사람의갑상선유두암진단에서핵내가성봉입체 (nuclear pseudoinclusion) 가진단에중요한역할을하나, 형질전환마우스의갑상선암에서는핵내가성봉입체가관찰되지않았다. 그러나, 흥미롭게도 60 주령형질전환마우스의폐전이병변에서는여포세포의핵내가성봉입체가관찰되었다 (Fig. 5K). 원발병소와폐전이병변에서여포세포는 Galectin-3 면역염색에양성임을확인하였다 (data not shown). 35 J Korean Thyroid Assoc

(A C) Thyroid glands of 48 weeks old mice. (A) Thyroid gland measures about 3.

Follicles are variable in size and irregular in shape.

(B, C) Tumor growths are evident in surrounding skeletal muslce.

(D K) Thyroid glands and metastatic lung lesion of 60 weeks old mice.

(G) There is a thyroid tumor follicle in submucosa of trachea.

Metastatic lesion is a bulging out mass, measuring 2.5x2.5 mm.

고찰 본연구에서 3주령 형질전환마우스에서전형적인갑상선유두암의조직학적특징을확인하지는못했지만 Galectin-3

11,12) 그리고, 형질전환마우스의주령이증가할수록둥글거나타원형이었던갑상선여포의모양이불규칙하게변화하였다.

44주령에는육안적으로하나의단단한덩어리로보이면서갑상선전체를분화가나쁜암세포가차지한것을관찰할수있어서종양이진행되면서분화도가나빠짐을확인할수있었다.

7 이정의외 Fig. 5. Representative figures of thyroid glands from 48 and 60 week old transgenic mice. (A C) Thyroid glands of 48 weeks old mice. (A) Thyroid gland measures about 3.5 mm in the largest length. Follicles are variable in size and irregular in shape. More larger follicles are located in periphery of gland. (B, C) Tumor growths are evident in surrounding skeletal muslce. But, there is no evidence of distant metastasis. (D K) Thyroid glands and metastatic lung lesion of 60 weeks old mice. (D, F) Thyroid glands are similar to thyroid glands of 48 weeks old mice. (G) There is a thyroid tumor follicle in submucosa of trachea. (H) Gross finding of metastatic lung lesion. Metastatic lesion is a bulging out mass, measuring 2.5x2.5 mm. (I, J) Metastatic lesion is a relatively well defined and consists of a well formed papillary structure. (K) Nuclear pseudoinclusion is frequently noted in metastatic lesion (arrows). 고찰 본연구에서 3주령 형질전환마우스에서전형적인갑상선유두암의조직학적특징을확인하지는못했지만 Galectin-3 면역화학염색에양성을보여세포의변형이동반되어있었음을알수있었다. 11,12) 그리고, 형질전환마우스의주령이증가할수록둥글거나타원형이었던갑상선여포의모양이불규칙하게변화하였다. 20주령부터갑상선은경동맥바깥쪽으로성장하였고, 39주령에는갑상선의양엽이기도의중앙부분에서만나고리형태를이루었다. 44주령에는육안적으로하나의단단한덩어리로보이면서갑상선전체를분화가나쁜암세포가차지한것을관찰할수있어서종양이진행되면서분화도가나빠짐을확인할수있었다. 13) 또한, 48주령의 형질전환마우스에서갑상선주변골격근으로의침윤을, 60주령마우스에서기도로의침윤및폐전이를보임으로써종양이더공격적으로변화함을알수있었다. 이런결과 들을종합해볼때 돌연변이가종양의발생과탈분화를유도하고종양의공격성에도영향을미쳐전이를야기함을확인할수있었다. 14) 본연구를통해확인한가장주목할만한사실은 에의한폐전이를확인한것으로, 이는 형질전환마우스를제작, 관찰한 Knauf 등 의연구에서도확인되지않았다. 5) 과거 RET/PTC3를과발현시킨형질전환마우스에서갑상선유두암의고형성변종 (solid variant of papillary thyroid carcinoma) 이발생하는것은확인이되나원격전이가관찰되지않았던것과도대별되는결과이다. 15) 이와더불어, RET/ PTC3 형질전환마우스와비교해보면, 12주령이상된 RET/PTC3 형질전환마우스의경우, 50 60% 에서갑상선유두암의고형성변종이발생하였으나, 분화도가나쁜암으로진행하지는않았다. 그에반해서 형질전환마우스에서는 12주령이상에서대부분의마우스에서전체갑상선세포가암화과정을밟는것으로확인되었으며, 27주령이상의마우스에서는 Vol. 4, No. 1,

8 형질전환마우스표현형분석 분화가나쁜암으로의진행도확인되었다. 따라서, 형질전환마우스가문헌에보고된 RET/ PTC3 형질전환마우스의표현형에비해보다공격적인성향의갑상선암을형성함을알수있었다 ) Knauf 등의연구에서 형질전환마우스는갑상선특이적유전자발현의감소로인한갑상선호르몬의생성감소와이차적인 TSH 증가, 더불어갑상선종이발생하였고주변조직을침범하는공격적성향의갑상선암이발생하였다. 5) 이후, Franco 등의연구에서는 형질전환마우스에발생한갑상선종과갑상선암이 TSH 증가때문인지를확인하기위해서 TSH receptor knockout을일으킨 형질전환마우스를제작하였다. 이형질전환마우스는갑상선종이생기지않았고, 형질전환마우스에비해서갑상선암이느리게성장하였으며주변조직으로의침윤을일으키지않았다. 이런연구결과들을통해서 에의해암화과정이시작되고, TSH에의해갑상선종발생과갑상선암의빠른성장이일어남을알수있다. 19) 따라서, 본연구에서 TSH를측정하지는않았지만, 형질전환마우스에서갑상선종이발생과갑상선암의빠른성장은 TSH 증가때문일것으로추측할수있었다. 갑상선유두상암에서흔하게확인되는 돌연변이는흑색종 (70%), 난소암 (30%), 대장암 (15%), 비소세포성페암 (3%) 에서도확인되지만각조직에서의역할은다양할것으로생각된다. 예를들어 Dankort 등은폐에서 형질전환을일으킨마우스를개발하였으나 8) 본연구에서관찰한 형질전환마우스에서와는달리대부분의마우스에서선종만이발생하였고, 40주령이상된마우스의 3 5% 에서만폐선암이발생하였다. 또한, 는흑색종모델에서도갑상선특이적 형질전환마우스와같은공격적인성향의종양을형성하지는않았다. 9,20,21) 이는폐선암과흑색종의형성과정과갑상선유두상암의형성과정에서 의역할이다름을시사하는중요한자료로생각된다. 10) 요약하면, 는마우스갑상선에서갑상선암을일으키며, 사람에서관찰되는탈분화와주변조직으로침윤을야기시킨다. 또한, 형질전환마우스의종양형성과정은주령에따라특징적인조직학적변화를밟으며이루어지는것을알수있었다. 이는향후 에의한갑상선암의발생및진행단계를파악하는중요한모델을제공할것으로생각된다. 중심단어 : 질환모델, 원암유전자단백질 B-raf, 갑상선암, 형질전환마우스. References 1) Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, et al. Mutations of the BRAF gene in human cancer. Nature 2002;417: ) Tang KT, Lee CH. BRAF mutation in papillary thyroid carcinoma: pathogenic role and clinical implications. J Chin Med Assoc 2010;73(3): ) Kimura ET, Nikiforova MN, Zhu Z, Knauf JA, Nikiforov YE, Fagin JA. High prevalence of BRAF mutations in thyroid cancer: Genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma. Cancer Res 2003;63: ) Frattini M, Ferrario C, Bressan P, Balestra D, De Cecco L, Mondellini P, et al. Alternative mutations of BRAF, RET and NTRK1 are associated with similar but distinct gene expression patterns in papillary thyroid cancer. Oncogene 2004;23: ) Knauf JA, Ma X, Smith EP, Zhang L, Mitsutake N, Liao XH, et al. Targeted expression of in thyroid cells of transgenic mice results in papillary thyroid cancers that undergo dedifferentiation. Cancer Res 2005;65: ) Mitsutake N, Miyagishi M, Mitsutake S, Akeno N, Mesa C Jr, Knauf JA, et al. BRAF mediates RET/PTC-induced mitogen-activated protein kinase activation in thyroid cells: Functional support for requirement of the RET/PTC- RAS-BRAF pathway in papillary thyroid carcinogenesis. Endocrinology 2006;147: ) Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, Good VM, et al. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 2004;116: ) Dankort D, Filenova E, Collado M, Serrano M, Jones K, McMahon M. A new mouse model to explore the initiation, progression, and therapy of -induced lung tumors. Genes Dev 2007;21: ) Dhomen N, Reis-Filho JS, da Rocha Dias S, Hayward R, Savage K, Delmas V, et al. Oncogenic Braf induces melanocyte senescence and melanoma in mice. Cancer Cell 2009;15: ) Motti ML, De Marco C, Califano D, De Gisi S, Malanga D, Troncone G, et al. Loss of p27 expression through RAS BRAF MAP kinase-dependent pathway in human thyroid carcinomas. Cell Cycle 2007;6: ) Volante M, Bozzalla-Cassione F, DePompa R, Saggiorato E, Bartolazzi A, Orlandi F, et al. Galectin-3 and HBME-1 expression in oncocytic cell tumors of the thyroid. Virchows Arch 2004;445: ) Saggiorato E, Aversa S, Deandreis D, Arecco F, Mussa A, Puligheddu B, et al. Galectin-3: presurgical marker of thyroid follicular epithelial cell-derived carcinomas. J Endocrinol Invest 2004;27: ) Nikiforova MN, Kimura ET, Gandhi M, Biddinger PW, Knauf JA, Basolo F, et al. BRAF mutations in thyroid tumors 37 J Korean Thyroid Assoc

9 이정의외 are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas. J Clin Endocrinol Metab 2003;88: ) Cantwell-Dorris ER, O'Leary JJ, Sheils OM. : Implications for carcinogenesis and molecular therapy. Mol Cancer Ther 2011;10: ) Powell DJ Jr, Russell J, Nibu K, Li G, Rhee E, Liao M, et al. The RET/PTC3 oncogene: Metastatic solid-type papillary carcinomas in murine thyroids. Cancer Res 1998;58: ) Nucera C, Lawler J, Parangi S. and microenvironment in thyroid cancer: A functional link to drive cancer progression. Cancer Res 2011;71: ) Romei C, Ciampi R, Faviana P, Agate L, Molinaro E, Bottici V, et al. mutation, but not RET/PTC rearrangements, is correlated with a lower expression of both thyroperoxidase and sodium iodide symporter genes in papillary thyroid cancer. Endocr Relat Cancer 2008;15: ) Mesa C Jr, Mirza M, Mitsutake N, Sartor M, Medvedovic M, Tomlinson C, et al. Conditional activation of RET/PTC3 and in thyroid cells is associated with gene expression profiles that predict a preferential role of BRAF in extracellular matrix remodeling. Cancer Res 2006;66: ) Franco AT, Malaguarnera R, Refetoff S, Liao XH, Lundsmith E, Kimura S, et al. Thyrotrophin receptor signaling dependence of Braf-induced thyroid tumor initiation in mice. Proc Natl Acad Sci U S A 2011;108(4): ) Wajapeyee N, Serra RW, Zhu X, Mahalingam M, Green MR. Oncogenic BRAF induces senescence and apoptosis through pathways mediated by the secreted protein IGFBP7. Cell 2008;132: ) Decarlo K, Yang S, Emley A, Wajapeyee N, Green M, Mahalingam M. Oncogenic BRAF-positive dysplastic nevi and the tumor suppressor IGFBP7--challenging the concept of dysplastic nevi as precursor lesions? Hum Pathol 2010;41: Vol. 4, No. 1,

Kaes010.hwp

Kaes010.hwp 갑상선자극호르몬이갑상선암세포의 VEGF, 신생혈관형성, 성장, 침윤및전이에미치는영향 Effects of TSH on the Production of VEGF, Angiogenesis, Growth, Invasion and Metastasis in Thyroid Cancer Cells Euy Young Soh, M.D., Hee Boong Park, M.D.,