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당뇨병환자와 정상인의 간, 근육, 위장, 공장, 지방조직에서의CEACAM-1 CD1및SIRT1 단백 발현에 관한 연구 (CEACAM-1 CD1 and SIRT1 expression in liver, muscle, stomach, jejunum, abdominal fat of diabetics and non-diabetics )
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Project Number E00006
Year(selected) 2008 Year
the present condition of Project 종료
State of proposition 재단승인
Completion Date 2009년 08월 29일
Year type 결과보고
Year(final report) 2009년
Research Summary
  • Korean
  • 연구 배경 및 목적: CEACAM-1과 PC-1 단백은 당뇨병의 발생기전 및 치료와 관련하여 최근 매우 주목받고 있는 단백질로서 당뇨병 환자와 정상인의 각 장기에서의 본 단백 발현에 관한 연구는 당뇨병의 발생과정을 이해하고 치료법을 개발하는데 큰 도움이 될 것으로 기대하여 본 연구를 계획하였다.CEACAM1 (Carcinoembryonic antigen-related cell adhesion molecule 1) 은 carcinoembryonic antigen (CEA) gene family에 속하며 CD66a로도 불린다. CEACAM1 단백은 간의 담도에 발현하는 biliary glycoprotein 으로 처음 보고 되었으나 현재는 백혈구, 상피세포, 내피세포 등에 발현하는 cell-cell adhesion molecule으로 알려져 있다. CEACAM1 단백의 세포내에서 역할은 분화, 혈관형성, 세포자멸사, 종양 억제, 전이, 면역반응의 조절 등이 있다. 특히 CEACAM1 단백은 간내에서 인슐린 수용체의 기질로 활동하며 인슐린 청소를 담당함으로서 인슐린 활동을 조절한다. 유전자이전 (遺傳子移轉) 생쥐에서의 CEACAM1의 불활성은 인슐린 청소의 장해를 초래하여 고인슐린 혈증, 인슐린 저항, 내부장기 지방증을 초래한다. PC-1은 인슐린 리셉터의 inhibitor로서 인슐린 저항을 가진환자에서 증가되어 잇다고 보고되어 있다.
    연구 재료 및 방법: 당뇨병환자와 정상인의 간, 근육, 위장, 공장, 복부 지방을 Wakefield GE research institute Diabetes group의 tissue bank 파일로부터 각각 20예를 무작위 선택하여 연구대상으로 하였다. 본 조직표본 모두 뉴질랜드의 Central Regional Ethics Committee (CEN/05/02/2004)에 승인된 조직표본이다. 냉동 보관 되었던 조직을 바로 95% 포르말린에 이틀 동안 충분히 고정시킨 후 각 환자의 간, 근육, 위장, 공장, 복부 지방 조직을 하나의 파라핀 블록에 포매하였다. 즉 하나의 파라핀 블록에 한 환자의 간, 근육, 위장, 공장, 복부 지방 조직이 모두 포매되게 하였다. 파라핀 블록을 5um으로 박절하여 헤마톡실린-에오진 염색을 한 후 각 현미경적으로 조직을 확인하였다.파라핀 포매 조직을 4마이크론으로 박절 후 자일렌, 에탄올을 거쳐 탈파라핀 하였다. 항원복원을 위하여 10mM sodium citrate pH 7.0 용액에 슬라이드를 담근 후 전자레인지에서 10분간 가열한 후 세척하였다. Peroxidase blocking을 위하여 30분간 3% H2O2에 슬라이드를 담근 후 흐르는 물로 세척하였다. 일차항체는 mouse monoclonal CEACAM1 (clone 29H2, Abcam, Cambridge,UK)과 goat polyclonal ENPP1(Abnova)를 이용하였으며 각각 1:50 과 1:250으로 희석하여 사용하였다. 이차항체는 Immpess (Invitrogen) universal kit를 사용하였고 DAB로 발색하였고 Meyer's hematoxylin으로 염색하였다.
    결과: CAECAM1 및 PC-1 단백은 간조직에만 염색이 되었고 다른 조직 즉, 근육, 위장, 공장, 복부 지방 조직에는 염색이 되지 않았다. CEACAM1은 간세포의 세관을 따라서 염색이 되었고 PC-1은 간세포의 세포막을 따라서 염색이 되었다. CEACAM1 단백이 전체 간조직 중 1/3 이하에서 염색이 될 때를 CEACAM 저발현으로 판독하였다. PC-1 단백이 전체 간조직 중 2/3 이하에서 염색이 될 때를 PC-1 과발현으로 판독하였다. 20명의 당뇨병 간 조직 중 7명 (35%)에서 20명의 정상 간조직의 5명 (5%)에서 CEACAM 단백의 저발현이 관찰되었다. 20명의 당뇨병 간 조직 중 7명 (35%)에서 20명의 정상 간조직의 9명 (45%)에서 PC-1 단백의 과발현이 관찰되었다. 당뇨병 유무와 CEACAM 단백 및 PC-1 단백 발현과는 통계학적으로 상관 관계가 없었다. CEACAM 단백의 저발현은 간 지방이 없거나 저등도로 있는 환자의 0%에서 간 지방이 중등도 이상으로 있는 환자의 38.7%에서 CEACAM 단백의 저발현이 관찰되었다. 즉 간 지방이 중등도 이상으로 있는 환자에서 CEACAM 단백의 저발현이 유의성있게 관찰되었다 (p=0.026).
    결론: CEACAM1 단백과 PC-1 단백은 당뇨병 간조직과 정상인의 간조직에서 면역조직학적으로 발현의 차이가 없었다. 본 연구의 결과는 CEACAM의 저발현이 당뇨병 환자 뿐만아니라 전반적인 지방간 환자의 인슐린 청소 저하와 관련성이 있을 것이라는 가정을 하게 한다.
  • English
  • Background: Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) also known as CD66a , is a human gene. The encoded protein was originally described in bile ducts of liver as biliary glycoprotein. Multiple cellular activities have been attributed to the encoded protein, including roles in the differentiation and arrangement of tissue three-dimensional structure, angiogenesis, apoptosis, tumor suppression, metastasis, and the modulation of innate and adaptive immune responses. CEACAM1, a substrate of insulin receptor in liver, regulates insulin action by promoting insulin clearance. Inactivation of CEACAM1 impairs insulin clearance and causes hyperinsulinemia, insulin resistance, dyslipidemia and visceral adiposity in transgenic mice. Thus, there is a strong association between visceral obesity, insulin resistance and reduced hepatic CEACAM1 level. PC-1 (plasma cell membrane glycoprotein-1), or ENPP1 (ectonucleotide pyrophosphatase/phosphodieterase) was originally described as a marker of terminally differentiated B cells within lymphoid system. It is an insulin receptor (IR) inhibitor that is elevated in cells and tissues of insulin-resistant humans. The suppression of PC-1/ENPP-1 expression improves insulin sensitivity in vitro and in vivo. The aim of this study is to determine whether the difference of CEACAM-1 and PC1 expression in liver, muscle, stomach, jejunum, abdominal fat between diabetics or non-diabetics.
    Material and Methods: Liver, muscle, stomach, jejunum, abdominal fat tissue of 20 diabetics and 20 non-diabetics will be selected from the files of Wakefield GE research institute Diabetes group. Any project involving experimentation with human subjects or animals requires approval from appropriate hospital or animal ethical committees. Approval has been given by the Central Regional Ethics Committee (CEN/05/02/2004) for the collection and use of all tissue for this study.
    The expression of CEACAM1 and PC-1 protein was determined by Immunohistochemical staining of formalin-fixed paraffin-embedded sections of each sample. The primary antibodies used were mouse monoclonal CEACAM1 (clone 29H2, Abcam, Cambridge,UK) and goat polyclonal ENPP1 (Abnova). Decreased expression of CEACAM1 was defined as negative staining in more than one third of hepatocytes. Overexpression of PC-1 was defined as positive staining in more than two third of hepatocytes. We statistically analyzed to compare the expression of CEACAM-1 and PC-1 in diabetics and non-diabetics groups.
    Results: CEACAM1 and PC-1 were expressed in liver tissue but not expressed in muscle, stomach, jejunum and abdominal fat tissue. CEACAM1 was expressed in cannalicular pattern of hepatocytes. PC-1 was expressed in membranous pattern of hepatocytes. Decreased expression of CEACAM1 was noted in 7 (35%) of 20 diabetes and 5 (24%) of 20 non-diabetes. Overexpression of PC-1 was noted in 7 (35%) of 20 diabetes and 9 (45%) of 20 non-diabetes. There was no significant correlation between decreased expression of CEACAM1 or overexpression of PC-1 and presence of diabetes. Decreased expression of CEACAM1 was noted in 12 (38.7%) of 31 moderate or severe fatty liver and 0 (0%) of 8 non- or mild fatty liver. Overexpression of PC-1 was noted in 12 (38.7%) of 31 moderate or severe fatty liver and 4 (44.4%) of 9 non- or mild fatty liver. There was significant correlation between decreased expression of CEACAM1 and presence of moderate to severe fatty liver (p=0.026). There was no significant correlation between overexpression of PC-1 and presence of moderate to severe fatty liver.
    Conclusion: Immunohistochemically, CEACAM1 or PC-1 expression between in diabetes and non-diabetes liver was not different. However, CEACAM1 expression was inversely correlated with severity of fatty liver. The result suggests the defect of insulin clearance by decreased expression of CEACAM1 might be associated with diabetic fatty liver as well as non-diabetic fatty liver
Research result report
  • Abstract
  • Background: Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) also known as CD66a , is a human gene. The encoded protein was originally described in bile ducts of liver as biliary glycoprotein. Multiple cellular activities have been attributed to the encoded protein, including roles in the differentiation and arrangement of tissue three-dimensional structure, angiogenesis, apoptosis, tumor suppression, metastasis, and the modulation of innate and adaptive immune responses. CEACAM1, a substrate of insulin receptor in liver, regulates insulin action by promoting insulin clearance. Inactivation of CEACAM1 impairs insulin clearance and causes hyperinsulinemia, insulin resistance, dyslipidemia and visceral adiposity in transgenic mice. Thus, there is a strong association between visceral obesity, insulin resistance and reduced hepatic CEACAM1 level. PC-1 (plasma cell membrane glycoprotein-1), or ENPP1 (ectonucleotide pyrophosphatase/phosphodieterase) was originally described as a marker of terminally differentiated B cells within lymphoid system. It is an insulin receptor (IR) inhibitor that is elevated in cells and tissues of insulin-resistant humans. The suppression of PC-1/ENPP-1 expression improves insulin sensitivity in vitro and in vivo. The aim of this study is to determine whether the difference of CEACAM-1 and PC1 expression in liver, muscle, stomach, jejunum, abdominal fat between diabetics or non-diabetics.
    Material and Methods: Liver, muscle, stomach, jejunum, abdominal fat tissue of 20 diabetics and 20 non-diabetics will be selected from the files of Wakefield GE research institute Diabetes group. Any project involving experimentation with human subjects or animals requires approval from appropriate hospital or animal ethical committees. Approval has been given by the Central Regional Ethics Committee (CEN/05/02/2004) for the collection and use of all tissue for this study.
    The expression of CEACAM1 and PC-1 protein was determined by Immunohistochemical staining of formalin-fixed paraffin-embedded sections of each sample. The primary antibodies used were mouse monoclonal CEACAM1 (clone 29H2, Abcam, Cambridge,UK) and goat polyclonal ENPP1 (Abnova). Decreased expression of CEACAM1 was defined as negative staining in more than one third of hepatocytes. Overexpression of PC-1 was defined as positive staining in more than two third of hepatocytes. We statistically analyzed to compare the expression of CEACAM-1 and PC-1 in diabetics and non-diabetics groups.
    Results: CEACAM1 and PC-1 were expressed in liver tissue but not expressed in muscle, stomach, jejunum and abdominal fat tissue. CEACAM1 was expressed in cannalicular pattern of hepatocytes. PC-1 was expressed in membranous pattern of hepatocytes. Decreased expression of CEACAM1 was noted in 7 (35%) of 20 diabetes and 5 (24%) of 20 non-diabetes. Overexpression of PC-1 was noted in 7 (35%) of 20 diabetes and 9 (45%) of 20 non-diabetes. There was no significant correlation between decreased expression of CEACAM1 or overexpression of PC-1 and presence of diabetes. Decreased expression of CEACAM1 was noted in 12 (38.7%) of 31 moderate or severe fatty liver and 0 (0%) of 8 non- or mild fatty liver. Overexpression of PC-1 was noted in 12 (38.7%) of 31 moderate or severe fatty liver and 4 (44.4%) of 9 non- or mild fatty liver. There was significant correlation between decreased expression of CEACAM1 and presence of moderate to severe fatty liver (p=0.026). There was no significant correlation between overexpression of PC-1 and presence of moderate to severe fatty liver.
    Conclusion: Immunohistochemically, CEACAM1 or PC-1 expression between in diabetes and non-diabetes liver was not different. However, CEACAM1 expression was inversely correlated with severity of fatty liver. The result suggests the defect of insulin clearance by decreased expression of CEACAM1 might be associated with diabetic fatty liver as well as non-diabetic fatty liver
  • Research result and Utilization method
  • 최근 제 2형 당뇨병의 치료방법을 물색하기 위한 방법으로 CEACAM1과 PC-1/ENPP1 단백에 대한 연구가 활발히 진행되고 있으나 면역조직화학 염색결과에 대한 데이터가 미흡하다. 본 연구 결과는 제 2형 당뇨병에 있어서의 CEACAM1과 PC-1/ENPP1 단백에 대한 연구의 기초 자료로 활용될 수 있을 것으로 기대한다.
  • Index terms
  • 당뇨병, 간, 근육, 위장, 공장, 지방, CEACAM-1 , PC-1, 면역조직화학염색
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