2000 Fiscal Year Annual Research Report

HNF-1β変異体発現マウスを用いた「優性阻害作用」による糖尿病発症機構の解析

Research Project

Project/Area Number	12671099
Research Institution	Gunma University
Principal Investigator	戸村秀明群馬大学, 生体調節研究所, 助手 (70217553)
Co-Investigator(Kenkyū-buntansha)	正公枝群馬大学, 生体調節研究所, 教務員 (40201561) 武田純群馬大学, 生体調節研究所, 教授 (40270855)
Keywords	MODY / HNF-1β / 変異体発現マウス
Research Abstract	これまでの研究結果から我々は、「HNF-1βは、優性阻害に働く変異体のみが糖尿病の成因となる」という仮説を立てた。本研究ではその仮説を検証するため、HNF-1β変異体発現マウスを作成しその解析を行う。本年度はまずHNF-1β変異体発現マウスの作成ため、β細胞で特異的な発現が期待されるインスリンプロモーターまたは内在性HNF-1βと同様の発現が期待されるオリジナルHNF-1β遺伝子プロモーター下流に、C端にHA-tagを付加した変異HNF-1βcDNAを組み込み、それぞれ発現ベクターを構築した。次に変異体発現マウス作成の前に、これら構築した発現ベクターが実際に変異体を発現するかどうかを、マウスβ細胞由来株であるMIN6細胞など、培養細胞系を用い検討した。発現の検出には、HA-tagに対する特異抗体を使用し、免疫染色法により発現を確認した。現在、発現が確認された変異HNF-1β発現ベクターよりDNAを調整し、マウス受精卵へのマイクロインジェクションを行い、変異体発現マウスの誕生を待っている。一方並行して本年度は、変異体発現マウスのスクリーニング法の検討を行った。HNF-1β変異体由来のRNAの発現は、Taqman system(パーキンエルマー社)を用い、迅速に定量する予定である。そのための適当なプローブ配列やPCR primer配列または反応条件など、変異体を発現させたMIN6細胞由来のRNAを用い検討した。このように若干の遅れはあるが、現在まで着実に計画は進行している。次年度は、上記スクリーニング法を用い変異体発現マウスを選別し、その組織学的、生化学的解析を行い上記仮説の検証を目指す。

Research Products

(13 results)

All Other

All Publications (13 results)

[Publications] Yamada,S., et al.: "Cloning of cDNA and the gene encoding human hepatocyte nuclear factor (HNF)-3β and mutation screening in Japanese subjects with MODY."Diabetologia. 43. 121-124 (2000)
[Publications] Yamazaki,Y., et al: "Edg-6 as a putative sphingosine 1-phosphate receptor coupling to Ca^<2+> signaling pathway."Biochem.Biophys.Res.Commun.. 268. 583-589 (2000)
[Publications] Aihara,Y., et al.: "Molecular cloning of a novel brain-type Na^+-dependent inorganic phosphate cotransporter."J Neurochem.. 74. 2622-2625 (2000)
[Publications] Shimizu,H., et al.: "Sphigosine 1-phosphate stimulates insulin secretion in HIT-T 15 cells and mouse islets."Endocrine Journal. 47. 261-269 (2000)
[Publications] Kimura,T., et al.: "Sphingosine 1-phosphate stimulates proliferation and migration of human endothelial cells possibly through the lipid receptors, Edg-1 and Edg-3."Biochem.J.. 348. 71-76 (2000)
[Publications] Zhu,Q., et al.: "Identification of missense mutations in the hepatocyte nuclear factor-3β gene in Japanese subjects with late-onset type 2 diabetes mellitus."Diabetologia. 43. 1197-1200 (2000)
[Publications] Murata,N., et al.: "Quantitative measurement of sphingosine 1-phosphate by radioreceptor-binding assay."Anal Biochem.. 282. 115-120 (2000)
[Publications] Tanaka,S., et al.: "A novel dominant-nagative mutation of the hepatocyte nuclear factor-1 α gene in Japanese early-onset type 2 diabetes."Hormone and Metabolic Res.. 32. 373-377 (2000)
[Publications] Murata N, et al.: "Interaction of sphingosine 1-phosphate with plasma components, including lipoproteins, regulates the lipid receptor-mediated actions."Biochem J.. 352. 809-815 (2000)
[Publications] Tamama Ki, et al.: "Extracellular mechanism through the Edg family of receptors might be responsible for sphingosine-1-phosphate-induced regulation of DNA synthesis and migration of rat aortic smooth-muscle cells."Biochem J.. 353. 139-146 (2001)
[Publications] Nishigori,H., et al.: "Mutations in the small heterodimer partner gene are associated with mild obesity in Japanese."Proc.Natl.Acad.Sci.USA.. 98. 575-580 (2001)
[Publications] Yu,L., et al.: "Genetic variation in the hepatocyte nuclear factor (HNF)-3α gene does not contribute to maturity-onset diabetes of the young in Japanese."Hormone and Metabolic Res.. (In press). (2001)
[Publications] Aihara,Y., et al.: "Assignment of the human gene encoding brain/pancreatic islet-type Na^+-dependent inorganic phosphate cotransporter to chromosome 11p14.3"Cytogenet Cell Genet.. (In press). (2001)

2000 Fiscal Year Annual Research Report

HNF-1β変異体発現マウスを用いた「優性阻害作用」による糖尿病発症機構の解析

Principal Investigator

戸村 秀明 群馬大学, 生体調節研究所, 助手 (70217553)

Research Products

[Publications] Yamada,S., et al.: "Cloning of cDNA and the gene encoding human hepatocyte nuclear factor (HNF)-3β and mutation screening in Japanese subjects with MODY."Diabetologia. 43. 121-124 (2000)

[Publications] Yamazaki,Y., et al: "Edg-6 as a putative sphingosine 1-phosphate receptor coupling to Ca^<2+> signaling pathway."Biochem.Biophys.Res.Commun.. 268. 583-589 (2000)

[Publications] Aihara,Y., et al.: "Molecular cloning of a novel brain-type Na^+-dependent inorganic phosphate cotransporter."J Neurochem.. 74. 2622-2625 (2000)

[Publications] Shimizu,H., et al.: "Sphigosine 1-phosphate stimulates insulin secretion in HIT-T 15 cells and mouse islets."Endocrine Journal. 47. 261-269 (2000)

[Publications] Kimura,T., et al.: "Sphingosine 1-phosphate stimulates proliferation and migration of human endothelial cells possibly through the lipid receptors, Edg-1 and Edg-3."Biochem.J.. 348. 71-76 (2000)

[Publications] Zhu,Q., et al.: "Identification of missense mutations in the hepatocyte nuclear factor-3β gene in Japanese subjects with late-onset type 2 diabetes mellitus."Diabetologia. 43. 1197-1200 (2000)

[Publications] Murata,N., et al.: "Quantitative measurement of sphingosine 1-phosphate by radioreceptor-binding assay."Anal Biochem.. 282. 115-120 (2000)

[Publications] Tanaka,S., et al.: "A novel dominant-nagative mutation of the hepatocyte nuclear factor-1 α gene in Japanese early-onset type 2 diabetes."Hormone and Metabolic Res.. 32. 373-377 (2000)

[Publications] Murata N, et al.: "Interaction of sphingosine 1-phosphate with plasma components, including lipoproteins, regulates the lipid receptor-mediated actions."Biochem J.. 352. 809-815 (2000)

[Publications] Tamama Ki, et al.: "Extracellular mechanism through the Edg family of receptors might be responsible for sphingosine-1-phosphate-induced regulation of DNA synthesis and migration of rat aortic smooth-muscle cells."Biochem J.. 353. 139-146 (2001)

[Publications] Nishigori,H., et al.: "Mutations in the small heterodimer partner gene are associated with mild obesity in Japanese."Proc.Natl.Acad.Sci.USA.. 98. 575-580 (2001)

[Publications] Yu,L., et al.: "Genetic variation in the hepatocyte nuclear factor (HNF)-3α gene does not contribute to maturity-onset diabetes of the young in Japanese."Hormone and Metabolic Res.. (In press). (2001)

[Publications] Aihara,Y., et al.: "Assignment of the human gene encoding brain/pancreatic islet-type Na^+-dependent inorganic phosphate cotransporter to chromosome 11p14.3"Cytogenet Cell Genet.. (In press). (2001)

戸村秀明群馬大学, 生体調節研究所, 助手 (70217553)