유전자 조작 근육모세포에서의 포도당 자극에 의한 인슐린 분비 = Glucose Regulated Production of Human Insulin in Genetically Modified Myoblast Cell Line (C2C12)

다국어 초록 (Multilingual Abstract)

Background: To develop somatic gene therapy model for diabetes mellitus, it is the most important to control it by glucose concentration. In order to develop the myoblasts that produce insulin by glucose concentration, the transfection of genes of human insulin, rat glucokinase and rat GLUT2 was conducted using C2C12, the murine myoblast cell line.Methods: pMLC-hINSmut plasmid vector to which human insulin cDNA was inserted in C2C12 cell line, pCB7/GLUT2 and pCB7/GK to which GLUT2 and glucokinase were inserted. Based on the inserted gene, C2C12/INS-GLUT2, C2C12/INS-GK and C2C12/INS-GK-GLUT2 were prepared. In each cell line, its mRNA and protein expression were measured. Also, the capability of producing insulin in low glucose (2.7 mM) and high glucose (25 mM) were compared.

국문 초록 (Abstract)

연구 배경: 당뇨병을 치료하기 위한 세포이식을 하는데 있어 가장 중요한 것은 포도당에 의해 조절되는 것이다. 포도당에 반응하는 유전자는 현재 여러 종류가 있지만, 췌도세포에서는 glucokinase와 GLUT2가 그 역할을 하고 있다. 저자는 포도당에 의해 인슐린을 생산하는 근육모세포를 개발하기 위하여 생쥐 근육모세포 세포주인 C2C12를 이용하여 사람 인슐린, 쥐 glucokinase, 쥐 GLUT2 유전자를 transfection 시켰다.방법: C2C12 세포주에 사람 인슐린 cDNA가 삽입된 pMLC-hINSmut 플라스미드와 쥐 GLUT2와 glucokinase가 삽입된 pCB7/GLUT2와 pCB7/GK를 transfection시켜 안정된 세포주를 얻었다. 각각의 삽입된 유전자에 따라 C2C12/INS-GLUT2, C2C12/INS-GK, C2C12/INS-GK-GLUT2 세포주를 만들었다. 이들 세포주에 대하여 각 유전자의 mRNA 및 단백질 발현을 관찰하였다. 그리고 저농도 포도당(2.7 mM)과 고농도 포도당(25 mM)에서의 인슐린 생산능을 비교하였다. C3H 생쥐에 streptozotocin으로 당뇨병을 유발시킨 뒤 C2C12/INS-GK-GLUT2 세포주를 이식한 후 혈당의 변화를 관찰하였다.

참고문헌 (Reference)

1 Panza JL, "Treatment of rat pancreatic islets with reactive PEG" 21 : 1155-1164, 2000

2 Liu EH, "Transplantation of the Islets of Langerhans.New Hope for Treatment of Type 1 Diabetes Mellitus" 11 : 379-382, 2000

3 Wilson MO, "Tetracycline-regulated secretion of human(pro)insulin following plasmid-mediated transfection of human muscle" 34 : 391-403, 2005

4 Scougall KT, "Tetracycline-regulated secretion of human insulin in transfected primary myoblasts" 304 : 167-175, 2003

5 Barr E, "Systemic delivery of recombinant proteins by genetically modified myoblasts" 254 : 1507-1509, 1991

6 Dhawan J, "Systemic delivery of human growth hormone by injection of genetically engineered myoblasts" 254 : 1509-1512, 1991

7 Simonson GD, "Synthesis and processing of genetically modified human proinsulin by rat myoblast primary cultures" 7 : 71-78, 1996

8 Yaffe D, "Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle" 270 : 725-727, 1977

9 Shaw JA, "Secretion of bioactive human insulin following plasmid-mediated gene transfer to non- neuroendocrine cell lines,primary cultures and rat skeletal muscle in vivo" 172 : 653-672, 2002

10 Vollenweider F, "Processing of proinsulin by transfected hepatoma(FAO)cells" 267 : 14629-14636, 1992

11 Arcelloni C, "Processing and release of human proinsulin-cleavage products into culture media by different engineered non-endocrine cells:a specific assessment by capillary electrophoresis" 166 : 437-445, 2000

12 Sutherland DE, "Pancreas transplantation:a review" 30 : 1940-1943, 1998

13 "Pancreas transplantation for patients with type 1 diabetes: American Diabetes Association" 23 : 85-, 2000

14 Clark SA, "Novel insulinoma cell lines produced by iterative engineering of GLUT2,glucokinase,and human insulin expression" 46 : 958-967, 1997

15 Gros L, "Insulin production by engineered muscle cells" 10 : 1207-1217, 1999

16 Mitanchez D, "Glucose- stimulated genes and prospects of gene therapy for type I diabetes" 18 : 520-540, 1997

17 Aomatsu Y,, "Efficacy of agarose/polystyrene sulfonic acid microencapsulation for islet xenotransplantation" 32 : 1071-1072, 2000

18 Yamasaki K, "Differentiation-induced insulin secretion from nonendocrine cells with engineered human proinsulin cDNA" 265 : 361-365, 1999

19 Zwillich T, "Diabetes research.Islet transplants not yet ready for prime time" 289 : 531-533, 2000