KR101089591B1 - 인간 배아 줄기세포 유래의 섬세포 - Google Patents
인간 배아 줄기세포 유래의 섬세포 Download PDFInfo
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- KR101089591B1 KR101089591B1 KR1020047008713A KR20047008713A KR101089591B1 KR 101089591 B1 KR101089591 B1 KR 101089591B1 KR 1020047008713 A KR1020047008713 A KR 1020047008713A KR 20047008713 A KR20047008713 A KR 20047008713A KR 101089591 B1 KR101089591 B1 KR 101089591B1
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Abstract
Description
표 1: pPS 세포로부터 섬세포로의 분화를 위한 인자 | |||
인자 | 화합물 유형 또는 패밀리 | 제안된 기능 | 초기의 작용농도 |
시클로파민 | 스테로이드성 알칼로이드 | 헤지호그 신호전달의 억제자; 또한 콜레스테롤 생합성의 억제자로서도 작용할 수 있음 | 10μM |
베타셀룰린 | EGF 패밀리 일원 | 베타세포분화의 미토겐 또는 촉진제. 상기 두 가지 기능은 단백질의 상이한 영역에 기인함. | 4nM |
액티빈 A | TGF-β패밀리 일원 | 분화인자, 관유사 세포주의 내분비 췌장세포로 분화를 야기함 | 4nM |
엑센딘-4 | 글루카곤-유사 펩티드 1 작용제 | GLP-1(하기 참조)의 더욱 안정한 형태 | 20nM |
글루카곤-유사 펩티드 1(GLP1) | 펩티드호르몬, 글루카곤 유전자의 단백질 산물 중 하나; G-단백질 커플드 수용체 리간드 | 글루코스 생산, 인슐린 분비를 유도함, 베타 세포 신생을 유도함, 베타 세포 증식을 유도함, | 20nM |
간세포 성장인자(HGF) |
c-Met 수용체의 리간드 | HGF를 과발현하는 트렌스제닉 동물의 베타 세포 질량을 증가 시킴. 관유사 세포주로부터 베타세포 형성을 유도함. |
10ng/ml |
니아신아미드(니코틴아미드) | 비타민 B 패밀리의 일원 | 세포의 산화상태 및/또는 ADP-리보실화에 영향을 미칠 수 있음; 베타 세포 분화를 촉진함 | 10mM |
인슐린-유사 성장 인자I(IGF-1) | 펩티드 호르몬 | 베타 세포 미토겐 | 10nM |
n-부티레이트 | 히스톤 데아세틸라아제 억제제(간세포의 생산을 위해 사용됨 | 0.5mM 내지 2.5mM | |
레티노산(all-trans) | 레티노이드 | 분화인자 | 10μM |
성장 호르몬 (인간 뇌하수체 소마토트로핀) |
소마토트로핀/프로락틴 패밀리 | 베타 세포 미토겐 | 100ng/ml |
태반 락토겐 | 소마토트로핀/프로락틴 패밀리 | PL을 과발현하는 트렌스제닉 동물의 베타 세포 질량, 임신동안의 베타 세포 미토겐을 증가시킴. | 50ng/ml |
혈관내피성장인자(VEGF) | Flt-1 및 Flk-1은 수용체임. PDGF/VEGF 패밀리 |
내피 섬전구세포에 대한 가능한 미토겐 | 50ng/ml |
인슐린-유사 성 장 인자 II(IGF-II) |
인슐린 패밀리 | IGF-1,미토겐과 유사, 췌장 발생 및 기능에 있어서는 강력하게 연관된 것은 아님. | 10nM |
표 1: pPS 세포로부터 섬세포로의 분화를 위한 인자 | |||
인자 | 화합물 유형 또는 패밀리 | 제안된 기능 | 초기작용 농도 |
3-이소부틸-1-메틸쟌틴(IBMX) | 포스포디에스터라아제 억제제, cAMP 수준 증가시킴. | 인슐린 분비 증가시킴. | 100μM |
워트마닌 | PI-3 키나아제 억제제 | 인슐린 분비를 증가시킬 수 있음 | 30nM |
가스트린 | 펩티드 호르몬 | 베타 세포 신생을 촉진할 수 있음 | 10ng/ml |
콜레시스토키닌 | 창자 호르몬 | 섬의 성숙을 보조할 수 있음 | 5μM |
신경성장인자 (NGF) |
NGF 패밀리 | 인슐린 분비 증가시킴 | 50ng/ml |
표피 성장인자(EGF) | EGF 패밀리(상기 베타셀룰린 참조) | 트렌스제닉 과발현이 베타세포의 증식으로 이어짐 | 4nM |
각질세포 성장인자 (KGF), 아카 FGF7 | FGF 패밀리 | 트렌스제닉 과발현은 베타세포의 증식으로 이어짐 | 10ng/ml |
혈소판-유래 성장인자(PDGF) | PDGF 패밀리(상기 VEGF 참조) | 잠재적 전구체 미토겐 | 50ng/ml |
재생유전자(Reg) | Reg 패밀리 | 췌장 손상 후 섬 재생에서 기능함 | 100ng/ml |
섬 신생관련 단백질(INGAP) | Reg 패밀리 | 섬 재생에서 기능할 수 있음 | 100ng/ml |
표2: 세포 규명을 위한 표현형 마커 | |
SSEA-4 | 배아줄기 및 생식 세포 |
Oct-4 | 비분화된 배아 다능성 세포 |
텔로머라아제 역전사효소(TERT) | 무한정 복제가 가능한 세포(예를 들면, 비분화된 pPS 세포) |
Pdx1 | 췌장으로 되는 십이지장부위에 있는 췌장성 돌기 형성전에 발현됨. 성숙한 베타세포에서도 또한 발현됨. |
뉴로게닌 3(Ngn3) | 섬전구세포의 마커 |
글루카곤 | 알파 세포 |
Nkx6.1 | 베타 세포마커 |
글루코키나아제 | 베타 세포마커 |
인슐린 또는 프로인슐린 | 성숙한 베타 세포 |
소마토스타틴 | 델타 세포 |
췌장 폴리펩티드 | PP 세포 |
섬 아밀로이드 폴리펩티드(IAPP) | 섬 마커 |
Islet-1 | 섬마커, 또한 신경 발현 |
베타-2/뉴로D | 범 섬세포 마커 |
HNF3b | 내배엽 마커 |
HNF4a | 내배엽 마커 |
Sox17 | 확정성 내배엽 마커 |
아밀라아제 | 외분비 세포 |
HES | 외분비 췌장 마커 |
네스틴 | 잠재적 전구세포 마커 |
소닉 헤지호그 | 신호전달 분자, 췌장 형성에 부재가 요구됨 |
CK19 | 췌장관 마커(가능한 췌장 전구체) |
Glut-2 | 글루코스 운반체 |
패치드(patched) 동족체 | 헤지호그 수용체 |
스무든드(smoothened) 동족체 | 헤지호그 수용체 |
표 3: 간세포 분화 프로토콜 | |||
단계 I 비분화 세포 (컨플루언트 할 때까지) |
단계 II 분화전 (4 일) |
단계 III 간세포 분화 (6 일) |
단계 IV 간세포 성숙 (7-9 군 만; 4 일) |
무영양세포 조건 | 20% SR 배지 + 1% DMSO |
20% SR 배지 + 1% DMSO + 2.5mM 부티레이트 |
HCM 30ng/ml hEGF + 10ng/ml TGF-α+ 30ng/ml HGF + 2.5mM 부티레이트 |
Claims (24)
- 내인성 c-펩티드를 발현하고 영장류 다능성 줄기(pPS)세포의 확립된 세포주와 동일한 게놈을 갖는 세포 무리를 포함하는, pPS 세포를 분화시켜 수득되는 분리된 영장류 세포 집단.
- 제 1 항에 있어서, 세포의 5% 이상이 두 가지 이상의 하기 마커를 발현하는 분화된 세포 집단: Pdx1, Ngn3, 인슐린, IAPP 및 Nkx6.1.
- 제 1 항에 있어서, 고혈당 비-비만 당뇨(NOD; non-obese diabetic) 마우스로 이식된 경우 공복 혈당 수준이 200mg/dL 아래로 떨어지는 것을 야기하는 분화된 세포 집단.
- 제 1 및 제 2 세포 집단을 포함하고, 상기 제 1 세포 집단은 제 1 항 내지 제 3 항 중 어느 한 항의 분화된 세포 집단을 포함하고, 제 2 세포 집단은 이들이 분화되는 비분화된 pPS 세포주를 포함하는, 췌장 호르몬 분비 세포를 생성하는 시스템.
- pPS 세포 또는 이들의 자손을 적어도 섬세포 분화인자의 혼합물 중에서 배양하고, 이에 의해 c-펩티드를 발현하는 세포를 포함하는 세포 집단을 수득하는 것을 포함하는 c-펩티드 발현 세포를 수득하는 방법.
- 제 5 항 또는 제 6 항에 있어서, 뉴로게닌3 과 같은 췌장 전사인자의 발현을 야기하도록 세포를 유전적으로 변경하는 것을 추가로 포함하는 방법.
- 하기를 포함하는 인슐린 c-펩티드의 생산을 위한 장치:a) 제 1 항 내지 제 3 항 중 어느 한 항에 따른 분화된 세포 집단, 및b) 세포 집단의 통과는 막지만, 상기 세포 집단에 의해 분비되는 인슐린, 글루카곤, 또는 소마토스타틴은 통과시키는 반투막.
- 인슐린, 글루카곤, 또는 소마토스타틴의 결핍, 또는 제 1 형 당뇨와 연관된 상태의 치료용 의약의 제조에서 제 1 항 내지 제 3 항 중 어느 한 항에 따른 분화된 세포 집단을 사용하는 방법.
- 제 1 항 내지 제 3 항 중 어느 한 항에 있어서, pPS 세포가 인간 배아 줄기세포이거나, 또는 인간 포배로부터 수득된 세포의 자손인 분리된 세포 집단.
- 제 4 항에 있어서, pPS 세포가 인간 배아 줄기세포이거나, 또는 인간 포배로부터 수득된 세포의 자손인 시스템.
- 제 5 항에 있어서, pPS 세포가 인간 배아 줄기세포이거나, 또는 인간 포배로부터 수득된 세포의 자손인 방법.
- 제 8 항에 있어서, pPS 세포가 인간 배아 줄기세포이거나, 또는 인간 포배로부터 수득된 세포의 자손인 장치.
- 제 9 항에 있어서, pPS 세포가 인간 배아 줄기세포이거나, 또는 인간 포배로부터 수득된 세포의 자손인 방법.
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