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1 DOI : /kdj ORIGINAL ARTICLES 장내분비 K- 세포에서인슐린 - 발현세포로의분화 가톨릭대학교의과대학내과학교실 이에스더 유준모 이민경 류경렬 고승현 안유배 문성대 송기호 Transdifferentiation of Enteroendocrine K-cells into Insulin-expressing Cells Esder Lee, Jun Mo Yu, Min Kyung Lee, Gyeong Ryul Ryu, Seung-Hyun Ko, Yu-Bae Ahn, Sung-Dae Moon, Ki-Ho Song Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea Abstract Background: Despite a recent breakthough in human islet transplantation for treating type 1 diabetes mellitus, the limited availability of donor pancreases remains a major obstacle. Endocrine cells within the gut epithelium (enteroendocrine cells) and pancreatic β cells share similar pathways of differentiation during embryonic development. In particular, K-cells that secrete glucose-dependent insulinotropic polypeptide (GIP) have been shown to express many of the key proteins found in β cells. Therefore, we hypothesize that K-cells can be transdifferentiated into β cells because both cells have remarkable similarities in their embryonic development and cellular phenotypes. Methods: K-cells were purified from heterogeneous STC-1 cells originating from an endocrine tumor of a mouse intestine. In addition, a K-cell subclone expressing stable Nkx6.1, called Kn4-cells, was successfully obtained. In vitro differentiation of K-cells or Kn4-cells into β cells was completed after exendin-4 treatment and serum deprivation. The expressions of insulin mrna and protein were examined by RT-PCR and immunocytochemistry. The interacellular insulin content was also measured. Results: K-cells were found to express glucokinase and GIP as assessed by RT-PCR and Western blot analysis. RT-PCR showed that K-cells also expressed Pdx-1, NeuroD1/Beta2, and MafA, but not Nkx6.1. After exendin-4 treatment and serum deprivation, insulin mrna and insulin or C-peptide were clearly detected in Kn4-cells. The intracellular insulin content was also increased significantly in these cells. Conclusion: K-cells are an attractive potential source of insulin-producing cells for treatment of type 1 diabetes mellitus. However, more experiments are necessary to optimize a strategy for converting K-cells into β cells. (Korean Diabetes J 33: , 2009) Key words: Differentiation, Enteroendocrine cells, K-cell, Nkx6.1 protein, Pancreatic beta-cell 서 론 제 1 형당뇨병은인슐린을분비하는췌도 (pancreatic islet) 내베타세포가자가면역기전에의하여파괴됨에따라인슐린이절대적으로결핍되어고혈당이발생하고결국에는많은합병증이유발되는만성질환이다 1-3). 제1형당뇨병은 접수일자 : 2009 년 10 월 9 일, 통과일자 : 2009 년 11 월 24 일교신저자 : 송기호, 가톨릭대학교의과대학내과학교실, kihos@catholic.ac.kr * 이논문은 2009 년도정부 ( 교육과학기술부 ) 의재원으로한국연구재단의지원을받아수행된기초연구사업임 (No ). 475

2 일생동안인슐린주사와함께철저한자가관리가필요한질환으로일상생활에심한제약을유발하여삶의질을현격히저하시키고, 많은합병증으로인한사회적, 경제적손실이지대하다. 현재까지시행되는치료법으로는다회인슐린주사, 인슐린펌프, 인공췌장기등이있으나혈당조절이효과적으로장기간유지되는경우는거의불가능한실정이며, 인슐린주사에따른저혈당의발생이치료에큰걸림돌이다. 제1형당뇨병완치를위한치료법으로는췌도이식 (islet transplantation) 과췌장이식 (pancreas transplantation) 이매우제한적으로시행되고있으나췌장공여자가절대적으로부족하고, 이식후면역학적및비면역학적기전으로인해이식췌도의손상이문제가되고있다 3-7). 따라서이문제점을극복하고자새로운완치법으로줄기세포등을이용한세포치료법, 유전자치료법등이시도되고있다. 세포치료법은줄기세포를포함한비베타세포를베타세포로분화유도하는것이다 7,8). 현재까지췌관세포 (pancreatic duct cell), 췌장선포세포 (pancreatic acinar cell), 골수세포 (bone marrow cell), 간세포 (liver cell) 등이연구되어왔다. 그러나, 대부분의연구결과를살펴보면비베타세포로부터분화된 인슐린 -분비세포 는원래의베타세포에비해인슐린양이나인슐린 mrna가현저히낮았으므로이에대한많은개선과함께추가연구가필요한실정이다 8-12). 발생학적으로췌장은전장 (forgut) 으로부터유래하며전장의내배엽에서기원한세포들은증식, 분화하여췌관을형성하고이췌관세포는다시외부의환경인자와수많은전사인자들과의복합적이고도순차적인발현을거쳐성체의췌장을구성하는 3가지종류의췌관세포, 소화효소를분비하는선포세포, 주로인슐린을분비하는췌도세포 ( 베타세포 ) 등으로분화된다. 베타세포의발생과분화에관여하는필수전사인자로는 Pdx1, Ngn3, NeuroD1/ Beta2, Nkx6.1 등이잘알려져있다 10,11,13,14). 한편, 장내분비세포는장상피세포 (intestinal epithelial cell) 의 1% 를차지하지만양으로보면신체내에서가장많은내분비조직이다. 내배엽에서기원하며 MATH1, Ngn3, Nkx6.1 등이발현되면서여러종류의장내분비세포로의분화가이루어지며베타세포및장내분비세포는발생에서기원, 전사인자의발현등이매우유사하다. 특히, glucose -dependent insulinotrophic polypeptide (GIP) 호르몬을분비하는 K-세포는 glucokinase, proconvertase 1/3 (PC1/3), proconvertase 2 (PC2), Pdx-1 발현등의베타세포와유사한독특한특성을갖고있다 15-18). 따라서저자들은 K-세포의생물학적인특성이베타세포 와매우유사하다는점에착안하여이를베타세포로분화시키고자본연구를시행하였다. 대상및방법 1. K-세포분리및배양 K-세포의순수분리는이전의연구에서저자들이보고한방법대로시행하였다 19). 간단히요약하면, 생쥐의소장종양세포에서분리한복합장내분비세포주인 STC-1을 Dr. Hanahan (University of California, San Francisco) 으로부터기증받았으며이세포를 Epstein-Barr virus 유래벡터를이용하여 GIP promoter와 green fluorescent protein (GFP) 유전자를삽입함으로써 K-세포를순수하게분리하였다. K-세포는 Dulbecco's Modified Eagle Medium (DMEM; GIBCO, Grand Island, NY, USA) 배양액에배양하였다. DMEM 배양액에는 4.5 g/l D-Glucose, 584 mg/l L-glutamine, 25 mm HEPES (1M HEPES 25 ml/l), 10% fetal bovine serum (FBS) 가포함되었으며, 모든배양은 5% CO 2 와 37 의조건에서이루어졌다. 2. Nkx6.1을안정적으로발현하는 K-세포클론의확보덴마크의 Hagedorn Research Institute의 Dr. Serup으로부터쥐 Nkx6.1 full length cdna (1.9kb) 플라즈미드를기증받았다. 이플라즈미드벡터 (pcmv4 - Nkx6.1) 에서 Hind III와 Xba I으로자른 Nkx6.1 cdna를다시 pcdna 3.1 플라즈미드벡터에삽입하여새로운벡터 (pcdna Nkx6.1) 를제작하였다 (Fig. 1). K-세포를미리분주하여 60~70% 배양되면 pcdna 3.1-Nkx6.1 플라즈미드를주입 (transfection) 시키고 48시간후에 G418 (400 ug/ml) 이포함된배지로옮겨 G418내성콜로니 (colony) 를계대클로닝 (subcloning) 하여여러종류의 K-세포의클론을확보하였다. 이중에 Nkx6.1의 mrna 발현을조사하여 Nkx6.1의발현이높은클론을 Kn4-세포 로명명하여실험에이용하였다. 대조군으로서는 Nkx6.1 cdna 가없는빈 pcdna 3.1 플라즈미드벡터를주입하였으며이클론은 K_pcDNA-세포 로명명하였다. 3. 유전자발현조사 (RT-PCR) 1) RNA 분리및 cdna 합성 RNA 분리를위해생쥐에서분리한췌도나배양한세포를 Trizol reagent kit (Molecular Research Center, Cincinnati, OH, USA) 를이용하여총 RNA를분리하였다. 즉, Trizol 476

이에스더외 7 인 : 장내분비 K- 세포에서인슐린 - 발현세포로의분화 Fig. 1. Design of Nkx6.1-expressing vector. The Nkx6.1-expressing vector (pcdna3.1 + Nkx6.1) was made as shown in this cartoon.

3 이에스더외 7 인 : 장내분비 K- 세포에서인슐린 - 발현세포로의분화 Fig. 1. Design of Nkx6.1-expressing vector. The Nkx6.1-expressing vector (pcdna3.1 + Nkx6.1) was made as shown in this cartoon. reagent에녹여세포를깨고, bromochloropropane을첨가하여원심분리후 RNA를침전시키기위해상층액과 isopropanol 을혼합후에탄올을사용하여불순물을세척하고 diethyl pyrocarbonate 처리한증류수에녹여총 RNA를분리하였다. 분리후 Spectrometry (UV/VIS Spectrophotometer ND-1000; Nanpdrop, Silmington, DE, USA) 로정량하였다. 0.5~1 ug 의 RNA를 85 에서 3분반응하고 5X First-Strand buffer (Invitrogen, Carlsbad, CA, USA), 0.1 M dtt (Invitrogen), 50 ug/ul Random hexamer (Invitrogen), 10 mm dntp (Invitrogen), RNaseOUT TM (40 U/uL, Invitrogen), SuperScrip TM ⅡReverse Transcriptase (200 U/uL, Invitrogen) 를차례로넣어서잘섞은후 25 에서 10분, 4 2 에서 60분, 95 에서 10분으로역전사시켜 cdna를합성하였다. 2) PCR 합성된 cdna로부터 TEMPase HOT Start Master Mix (Ampligon) 등을이용한 PCR 방법으로써여러가지유전자를증폭하였다. 대조유전자로는 cyclophilin 등을이용하였다 (Table 1). PCR 산물은 1~2% agarose gel에서전기영동함으로써특정유전자의발현을반정량하여분석하였다. 4. 시험관내분화유도 K-세포또는 Kn4-세포가 60~70% 자라면분화를유도하기위해서 FBS가없는배양액으로바꾸었다. 이배양액에는 584 mg/l L-glutamine, g/l sodium bicarbonate, 0.1% bovine serum albumin (Sigma, St. Louis, MO, USA), 10 mm nicotinamide (Sigma), 500 ul ITS (5 ug/ml insulin, 5 ug/ml transferrin, 5 ng/ml [ M] sodium selenite; ITS, Roche, Germany) 가포함되었다. 또한, 추가로 exendin-4, betacellulin, activin A 또는 hepatocyte growth factor 등을각각처리하여분화유도를시도하였다. 5. 면역세포화학염색 (Immunocytochemistry) 면역세포화학염색을하기위해세포를 12 well 배양용기 (NUNC TM ; Roskilde, Denmark) 에코팅된커버슬립 (cover slip) 을넣고분주배양하였다. 부유배양하는세포는수거하여 4% (para)formaldehyde (PFA) 로상온에서 10분동안고정하고세척한후 Phosphate Buffered Saline (PBS) 에 0.2% TritonX-100 희석한것을상온에서 20분동안처리한후세척하였다. 블로킹전에 C-펩타이드면역염색은 10 mm citrate buffer로 37 에서 1~1시간 30분동안처리하 477

4 Table 1. Sequences of primers and PCR conditions Primer Sequences (5'-3') Product size Annealing Cycles (bp) temperature ( ) (n) Cyclophilin Sense AAC CCC ACC GTG TTC TTC Antisense TGC CTT CTT TCA CCT TCC C Insulin 1 Sense TAG TGA CCA GCT ATA ACC AGA G Antisense ACG CCA AGG TCT GAA GGT CC Insulin 2 Sense AGC CCT AAG TGA TCC GCT ACA A Antisense AGT TGC AGT AGT TCT CCA GCT G Isl1 Sense CAC TAT TTG CCA CCT AGC CAC Antisense AAA TAC TGA TTA CAC TCC GCA C MafA Sense CAC CAC GTG CGC TTG G Antisense CAG AAA GAA GTC GGG TG NeuroD1/Beta2 Sense ACT CCA AGA CCC AGA AAC TGT C Antisense ACT GGT AGG AGT AGG GAT GCA C Nkx 2.2 Sense GTC CGG AAC CAT GTC GCT GA Antisense GAC TTG GAG CTC GAG TCT TG Nkx6.1 (mouse) Sense ACT TGG CAG GAC CAG AGA GA Antisense AGA GTT CGG GTC CAG AGG TT Nkx6.1 (rat) Sense TCT TCT GGC CTG GGG TGA TG Antisense GGC TGC GTG CTT CTT TCT CCA Pax6 Sense AAC AAC CTG CCT ATG CAA CC Antisense ACT TGG ACG GGA ACT GAC AC Pdx-1 Sense ATT CTT TGC CAA CAG GTC TA Antisense AAT GAA ATG GAA ACA TCG AC 고상온에서 20분동안블로킹을시행하였다. 베타세포의표지자로 polyclonal guinea pig anti-insulin (1:200; Zymed, San Francisco, CA, USA), rabbit anti-c-peptide (1:100; Cell Signaling, Danvers, MA, USA) 에대한 1차항체를 4 에서하루동안처리하였다. 2차형광항체로는 anti-guinea pig rhodamine (1:100, Jackson ImmunoResearch Laboratries, West Grove, PA, USA), anti-rabbit Texas Red (1:50, Jackson ImmunoResearch Laboratries) 을사용하였다. 핵은 4',6'-diamindino-e-phenylindole (DAPI) 로염색하였다. 면역염색이끝나고면역형광현미경또는공초점현미경 (Confocal microscope; BIORAC MRC 1024; BIORAC, MO, UK) 을이용하여관찰하였다. 6. 세포내인슐린양측정세포내의인슐린양을측정하기위해서배양액을제거하고 Krebs-Ringer bicarbonate 버퍼 (25 mm HEPES, 115 mm NaCl, 24 mm NaHCO 3, 5 mm KCl, 2.5 mm CaCl 2, 1 mm MgCl 2, 0.5% BSA, 5 mm glucose) 로세포를세척한후 acid ethanol을첨가하여 sonication으로세포를깨고 4 에서하루가지난후에 Rat/Mouse Insulin ELISA kit (Linco Research, St. Charles, MO, USA) 를이용하여인슐린농도를측정하였다. 동시에단백질의양도 Coomassie Billiant Blue G-250 (Thermo Fisher Scientific, Rockford, IL, USA) 를이용한 Bradford 분석방법으로측정하여세포내인슐린양을보정하였다. 7. 통계분석두군간의연속변수를비교하기위해서 Student's T-test 및 Wilcoxon's rank sum test를시행하였고, P값이 0.05 이하를유의하다고판정하였다. 결과 1. 췌도-특이적인전사인자들의발현 RT-PCR을이용하여췌도- 특이적인전사인자들의발현을분석하였다. 대조군인생쥐췌도와비교하였을때 K-세포는 Pdx-1, NeuroD1/Beta2, MafA, Pax6, Isl1, Nkx 2.2가모두발현하였다. 그러나, Nkx6.1는 K-세포에서는발현되지않았다 (Fig. 2). 따라서이미기술한대로 Nkx6.1을안정적으로발현하는 K-세포의클론 (Kn4-세포) 을추가로얻어 478

이에스더외 7 인 : 장내분비 K- 세포에서인슐린 - 발현세포로의분화 서실험에사용하였다. 2. 베타세포로의분화유도 먼저 K- 세포가 60~70% 자라면 7 일동안 FBS 가없는배 양액으로바꾸고 exendin-4, nicotinamide를첨가하여베타세포로의분화를유도하였다.

K-cells were found to express transcription factors which were all present in mouse islets except Nkx6.1. Fig. 3. RT-PCR of insulin 1 mrna.

1, Mouse islets; 1-1, K-cells; 2, K-cells, 7 days in serum-free medium; 3, K-cells, 7 days in serum-free medium with 10 mm nicotinamide; 4, K-cells, 7 days in serum-free medium with 10 mm

5 이에스더외 7 인 : 장내분비 K- 세포에서인슐린 - 발현세포로의분화 서실험에사용하였다. 2. 베타세포로의분화유도 먼저 K- 세포가 60~70% 자라면 7 일동안 FBS 가없는배 양액으로바꾸고 exendin-4, nicotinamide를첨가하여베타세포로의분화를유도하였다. RT-PCR 결과에서 insulin 1 mrna는 30 cycle에서는보이지않았지만, 40 cycle에서는뚜렷한 insulin 1 mrna Fig. 2. RT-PCR of transcription factors mrna. K-cells were found to express transcription factors which were all present in mouse islets except Nkx6.1. Fig. 3. RT-PCR of insulin 1 mrna. Insulin1 band was weakly detected in K-cells after 7 day-treatment with 10 mm nicotinamide and 10 pm exendin-4 in serum-free medium. 1, Mouse islets; 1-1, K-cells; 2, K-cells, 7 days in serum-free medium; 3, K-cells, 7 days in serum-free medium with 10 mm nicotinamide; 4, K-cells, 7 days in serum-free medium with 10 mm nicotinamide and 10 pm exendin-4. Fig. 4. RT-PCR of insulin 1 and Nkx6.1 mrna. Insulin1 band was clearly detected in Kn4-cells after 7 day-treatment with 10 mm nicotinamide and 10 pm exendin-4 in serum-free medium. 1, Rat islets; 1-1, Mouse islets; 2, K-cells, 7 days in serum-free medium; 3, K_pcDNA-cells, 7 days in serum-free medium; 4, Kn4-cells, 7 days in serum-free medium; 5, K-cells, 7 days in serum-free medium with 10 mm nicotinamide and 10 pm exendin-4; 6, K_pcDNA-cells, 7 days in serum-free medium with 10 mm nicotinamide and 10 pm exendin-4; 7, Kn-4 cells, 7 days in serum-free medium with 10 mm nicotinamide and 10 pm exendin

또한, betacellulin, activin A, 또는 hepatocyte growth

다음으로, K-세포, K_pcDNA-세포와 Kn4-세포에서각각베타세포로의분화를유도하고

Kn4-세포에서는 K-세포또는 K_pcDNA-세포와달리 A B C D Fig. 5.

in K-cells and transdifferentiated Kn4-cells.

insulin-positive (A, Insulin staining in Ins-1 cells,

staining in differentiated Kn4-cells, 400; D, GFP

6 의발현을확인할수있었다. 그러나, 이상의결과는 FBS가없는상태에서 nicotinamide, exendin-4를모두처치했을경우에만관찰되었다 (Fig. 3). Insulin 2 mrna의발현은관찰되지않았다. 또한, betacellulin, activin A, 또는 hepatocyte growth factor는뚜렷한효과가없었다 ( 결과제시안함 ). 다음으로, K-세포, K_pcDNA-세포와 Kn4-세포에서각각베타세포로의분화를유도하고 RT-PCR을시행하여비교하였다. Kn4-세포에서는 K-세포또는 K_pcDNA-세포와달리 A B C D Fig. 5. Fluorescent microscopy for insulin immunocytochemistry in K-cells and transdifferentiated Kn4-cells. Some of differentiated Kn4-cells were found to be insulin-positive (A, Insulin staining in Ins-1 cells, 200; B, Insulin staining in K-cells, 200; C, Insulin staining in differentiated Kn4-cells, 400; D, GFP expression, 400). A B C D Fig. 6. Confocal microscopy for C-peptide immunocytochemistry in transdifferentiated Kn4-cells. Some of differentiated Kn4-cells were found to be C-peptide-positive (A, Nuclei staining with DAPI; B, GFP expression; C, C-peptide staining D, Merged image, 400). 480

이에스더외 7 인 : 장내분비 K- 세포에서인슐린 - 발현세포로의분화 Nkx6.1의뚜렷한발현을확인할수있었다. 또한, insulin 1 은 30 cylce의 PCR 조건에서 Kn4-세포에서만뚜렷한발현이관찰되었으며 K-세포, K_pcDNA-세포에서는거의발현되지않았다 (Fig. 4). Fig. 5는인슐린면역세포화학염색의면역형광현미경소견이다.

C-펩타이드가양성인세포의비율 (C-펩타이드양성인세포 /DAPI 양성인세포 ) 은 9~14% 이었다. 인슐린또는 C-펩타이드와 GFP 염색은대부분 colocalization이되지않았다. 세포내인슐린양을측정한결과, 분화가유도된 Kn4-세포의인슐린양은 K-세포에서보다현저하게높았다 (Fig. 7).

7 이에스더외 7 인 : 장내분비 K- 세포에서인슐린 - 발현세포로의분화 Nkx6.1의뚜렷한발현을확인할수있었다. 또한, insulin 1 은 30 cylce의 PCR 조건에서 Kn4-세포에서만뚜렷한발현이관찰되었으며 K-세포, K_pcDNA-세포에서는거의발현되지않았다 (Fig. 4). Fig. 5는인슐린면역세포화학염색의면역형광현미경소견이다. 양성대조군으로쥐베타세포주인 Ins-1 세포를이용하였다. K-세포에서는인슐린의발현은관찰되지않았으나, 분화가유도된 Kn4-세포에서인슐린이양성으로염색된세포들이다수발견되었으며대부분의세포는 GFP를발현하고있었다. 또한, 인슐린발현을재확인하고자 C-펩타이드면역세포화학염색을실시하고공초점현미경으로관찰한결과인슐린염색과비슷한결과를얻었다 (Fig. 6). C-펩타이드가양성인세포의비율 (C-펩타이드양성인세포 /DAPI 양성인세포 ) 은 9~14% 이었다. 인슐린또는 C-펩타이드와 GFP 염색은대부분 colocalization이되지않았다. 세포내인슐린양을측정한결과, 분화가유도된 Kn4-세포의인슐린양은 K-세포에서보다현저하게높았다 (Fig. 7). 육안관찰상분화유도과정에서세포사 (cell death) 가관찰되었으며 trypan blue를이용하여조사하였을때세포사는약 40% 이었다 (Fig. 8). 고찰본연구는장내분비세포의하나인 K-세포가시험관내조건에서인슐린을발현하는세포로분화가가능함을증명하였다. 인크레틴호르몬인 GIP를분비하는 K-세포는발생학적으로베타세포와비슷한전사인자의조절을받을뿐아니라, Fig. 7. Intracellular content of insulin. Insulin content was significantly higher in transdifferentiated Kn4-cells as compared with K-cells. * P < Before After Fig. 8. Cell morphology of Kn4-cells before and after 7 day-treatment with 10 mm nicotinamide and 10 pm exendin-4 in serum-free medium ( 400). 481

8 베타세포에특이적으로발현하는 glucokinase, PC1/3, PC2, Pdx-1 등의발현함이알려져있다. 본연구에서도베타세포의분화, 기능에중요한전사인자들의발현을 RT-PCR로조사한결과, Pdx-1, NeuroD1/Beta2, MafA, Pax6, Isl1, Nkx2.2가 K-세포에서도발현됨을관찰하였다. 그러나, 태생후에는베타세포에서만발현하는것으로알려진 Nkx6.1 은발현되지않았다. 따라서저자들은 K-세포와베타세포가세포학적특성이매우유사하다는데에착안하여 K-세포가특정한조건에서는베타세포로분화또는전환될수있다는가설을검증하고자하였다. 베타세포로의분화를유도하기위해서무혈청상태에서 nicotinamide, exendin-4, betacellulin, activin A, 또는 hepatocyte growth factor를첨가하였다. 이상의조건은다른연구들에서베타세포의분화를유도내지는촉진시킴이알려져왔다 3,7,12,20-33). 또한, K-세포에서는발현되지않는 Nkx6.1 전사인자를안정적으로발현하도록하기위해 Nkx6.1 벡터를제작하고이를 transfection시킨후이미기술한바와같이 Nkx6.1을지속적으로발현하는 K-세포클론 (Kn4-세포) 을얻었다. 그결과, Kn4-세포에서 nicotinamide, exendin-4를처치했을경우에뚜렷한 insulin 1 mrna의발현이 RT-PCR에서확인되었다. Insulin 2 mrna의발현은관찰되지않았다. 인슐린또는 C-펩타이드면역염색에서도인슐린또는 C-펩타이드가양성으로염색된세포들이다수발견되었다. 따라서 K-세포가특정한시험관내조건에서는베타세포로분화가됨을확인할수있었다. 공초점현미경관찰상인슐린또는 C-펩타이드와 GFP 염색은대부분 colocalization이되지않았는데이는 GFP 발현은 GIP promoter의활성화에의존적이기때문에 Kn4-세포가인슐린또는 C-펩타이드를발현하는세포로분화가이루어지면아마도 GFP 발현이소실되기때문일것으로추측한다. 세포내인슐린양도분화가유도된 Kn4-세포에서증가됨을관찰하였다. 그러나, 세포내인슐린양은생쥐의췌도에서의인슐린양 ( 인슐린 / 총단백질양 = ~250 ug/mg) 34) 에비해서는현저히낮았다. 이와같이인슐린양이낮은사실로보아분화가유도된 Kn4-세포는아직은베타세포로의분화가완성되지않은미분화상태임을시사한다. 또한, 분화유도과정에서많은세포가사멸됨이관찰되었는데이때문에도세포내인슐린양이적게나왔을것이라고판단된다. 다만, 배양액에첨가한 ITS에포함된인슐린때문에인슐린측정값이실제값보다높았을수있다. 향후에베타세포의분화를좀더확인하기위해서전자현미경적관찰이나포도당및여러인슐린분비촉진물질등에의한인슐린분비능을자세하게조사 할계획이다. 결론적으로, 본연구의결과는 K-세포가특정한조건에서인슐린을발현하는세포로분화가가능하므로제1형당뇨병의세포치료법으로서유용할것임을시사한다. 그러나, 베타세포로의분화를극대화하기위한최적의분화프로토콜의개발이필요하다. 요약연구배경 : 최근제1형당뇨병치료법으로췌도이식의새로운발견에도불구하고췌장공여자가부족한것이여전히중요한장애물이다. 장상피에존재하는내분비세포 ( 장내분비세포 ) 와췌장베타세포는발생과정에서유사한분화경로를공유한다. 특히, GIP를분비하는 K-세포는베타세포에서관찰되는중요한단백질들이많이발현됨이알려져있다. 따라서저자들은 K-세포의생물학적인특성이베타세포와매우유사하다는점에착안하여이를베타세포로분화시키고자본연구를시행하였다. 방법 : 생쥐췌장의내분비종양으로부터기원한복합 STC-1세포에서 K-세포를순수분리하였다. 또한, 안정적으로 Nkx6.1을발현하는 K-세포클론 ( Kn4-세포 로명명 ) 을성공적으로확보하였다. 혈청이없는조건에서 exendin-4를처리함으로써 K-세포또는 Kn4-세포가시험관내에서베타세포로분화하도록유도하였다. 인슐린 mrna와인슐린단백질의발현은 RT-PCR과면역세포화학염색으로조사하였다. 세포내인슐린양도측정하였다. 결과 : K-세포에서 RT-PCR과 western blot 분석을통하여 glucokinase와 GIP가발현함을확인하였다. RT-PCR의결과에서 K-세포에서는 Pdx-1, NeuroD1/Beta2, MafA mrna가발현됨을확인하였으나 Nkx6.1은발현하지않았다. Kn4-세포를혈청이없는조건에서 exendin-4을처리한결과, 인슐린 mrna와인슐린또는 C-펩타이드단백질이뚜렷이발현됨을관찰하였다. 또한세포내인슐린양도유의하게증가하였다. 결론 : K-세포는특정한조건에서인슐린을발현하는세포로분화가되므로장차제1형당뇨병의세포치료법으로써활용될가능성이있다. 참고문헌 1. Ko SH, Lee WY, Lee JH, Kwon HS, Lee JM, Kim SR, Moon SD, Song KH, Han JH, Ahn YB, Yoo SJ, Son 482

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Æ¯ÇãÃ» '' - 1 - - 2 - - 3 - - 4 - - 5 - ' - 6 - - 7 - - 8 - - 9 - - 10 - - 11 - Super-Bio Co., Ltd. KWON, Suk-Tae Thermostable DNA Polymerase-encoding Gene from Thermus sp. X-1 an d Amino Acid Sequence