2003 Fiscal Year Annual Research Report

欠損蛋白補充による新しい表皮水疱症治療法の開発

Research Project

Project/Area Number	13357008
Research Institution	HOKKAIDO UNIVERSITY
Principal Investigator	清水宏北海道大学, 大学院・医学研究科, 教授 (00146672)
Co-Investigator(Kenkyū-buntansha)	古市泰宏 (株)ジーンケア研究所, 所長(研究職) 清水忠道北海道大学, 医学部・歯学部附属病院, 講師 (70260396) 澤村大輔北海道大学, 大学院・医学研究科, 助教授 (60196334) 増永卓司株式会社コーセー研究本部, 主任研究員
Keywords	創傷治癒 / 表皮水疱症 / 栄養障害型 / 重症 / VII型コラーゲン / 蛋白補充 / 遺伝子導入 / 表皮移植
Research Abstract	今回の研究の目的は、重篤で患者数が多い劣性栄養障害型(VII型コラーゲン遺伝子の変異による)表皮水疱症を対象として、欠損あるいは,きわめて減少している蛋白を体外から補充する新しい治療法を開発することであった。1)第一の研究実績として、実際に表皮水疱症患者への培養表皮シートの移植臨床研究を行った。12歳男児の重症型表皮水疱症患者から皮膚を採取し培養表皮シートを作成した後、患者の数カ所の潰瘍部に移植した。一部のシートはそのまま正着し著効した部分もあったが、やはり患者自身の皮膚を使用しているため、欠損するVII型コラーゲン蛋白や遺伝子を補う必要性が再び明らかになった。2)次に、実際に用いるVII型コラーゲンの合成と単離を行った。遺伝子導入細胞を蛍光抗体法で確認したところ、蛍光が認められVII型コラーゲンが産生されていることがわかった。合成されたVII型コラーゲンはウエスタンブロットで290kDのバンドとして検出され、VII型コラーゲン全長が合成されているのが明らかになった。培養HaCaT細胞、3T3細胞、293細胞では、293細胞が一番産生が多かった。また、VII型コラーゲンを認識するモノクローナル抗体であるLH7.2のカラムを使用して、アフィニテイークロマトグラファイを行い、VII型コラ-ゲンをsmall scaleにて単離した。3)さらに、その合成VII型コラーゲンの創傷治癒促進作用を確認した。VII型コラーゲン産生細胞の情勢を濃縮して、ラットに作成した皮膚潰瘍に外用したところ、潰瘍の治癒が促進された。この結果は、VII型コラーゲン自体に創傷治癒促進作用があることを示し、表皮水疱患者への補充療法への期待がさらに高まった。研究開始時点での目標のほとんどを達成できた。今回の研究である程度の有効性を示すことができた蛋白補充療法を表皮水疱症患者の治療に臨床応用可能であると結論した。

Research Products
(20 results)

All Other

All Publications (20 results)

[Publications] Arita K, et al.: "Squamous cell carcinoma in a patien with non-bullous congenital ichthyosiform erythroderma"Br J Dermatol. 148. 363-384 (2003)
[Publications] Shimizu T, et al.: "Cetirizine, an H1-receptor antagonist, suppresses the expression of macrophage migration inhibitory factor (MIF) : Its potential anti-inflammatory action"Clin Exp Allergy. 34. 103-109 (2003)
[Publications] Matsuzaki Y, et al.: "Keratinocyte responsive element 3 : analysis of a keratinocyte-specific regulatory sequence in the 230 kDa bullous pemhigoid antigen promoter"J Invest Dermatol. 120. 308-312 (2003)
[Publications] Sawamura D, et al.: "Identification of COL7A1 alternative splicing inserting 9 amino acid residues into the fibronectin type III linker domain"J Invest Dermatol. 120. 942-948 (2003)
[Publications] Sawamura D, et al.: "Epidermolysis bullosa : directions for future research and new challenges for treatment"Arch Dermatol Res. 295 Suppl 1. S34-S42 (2003)
[Publications] Kawasaki H, et al.: "Squamous cell carcinoma developing in a 12-year-old boy with nonHallopeau-Siemens recessive dystrophic epidermolysis bullosa"Br J Dermatol. 148. 1047-1050 (2003)
[Publications] Liu Y, et al.: "Immunofluorescence studies using skin sections of recessive dystrophic epidermolysis bullosa patients indicated that the antigen of anti-p200 pemphigoid is not fragment of type VII collagen"J Dermatol Sci. 32. 125-129 (2003)
[Publications] McMillan JR, et al.: "Epidermal basement membrane zone components : ultrastructural distribution and molecular interactions"J Dermatol Sci. 31. 169-177 (2003)
[Publications] McMillan JR, et al.: "Ultarastructural orientation of laminin 5 in the epidermal basement membrane : an undated model for basement membrane organization"J Histochem Cytochem. 51. 1299-1306 (2003)
[Publications] McMillan JR, et al.: "Alterations in desmosome size and number coincide with the loss of keratinocyte cohesion in skin with homozygous and heterozygous defects in the desmosomal protein plakophilin 1"J Invest Dermatol. 121. 96-103 (2003)
[Publications] McMillan JR, et al.: "Immunomapping of EBA sera to multiple epitopes on collagen VII further evidence that anchoring fibrils originate and terminate in the lamina densa"Exp Dermatol. 12. 261-267 (2003)
[Publications] Siegel DH, et al.: "Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin-extracellular-matrix linker protein UNC-112, causes Kindler syndrome"Am J Hum Genet. 73. 174-187 (2003)
[Publications] Tomita Y, et al.: "Simultaneous occurrence of three squamous cell carcinomas in a recessive dystrophic epidermolysis bullosa patient"Acta Derm Venereol. 83. 225-226 (2003)
[Publications] Tsuji Y, et al.: "Changing pattern of deiminated proteins in developing human epidermis"J Invest Dermatol. 120. 817-822 (2003)
[Publications] Matsuyama N, et al.: "The dinucleotide microsatellite polymorphism of the IFNARI gene promoter correlates with responsiveness of hepatitis C patients to interferon"Hepatol Res. 25. 221-225 (2003)
[Publications] Satoh M, et al.: "Innate apoptosis of human B lymphoblasts transformed by Epstein-Barr virus : modulation by cellular immortalization and senescence"Cell Struct Funct. 28. 61-70 (2003)
[Publications] Akiyama M, et al.: "Truncation of CGI-58 Protein Causes Malformation of Lamellar Granules Resulting in Ichthyosis in Dorfman-Chanarin Syndrome"J Invest Dermatol. 121. 1029-1034 (2003)
[Publications] Arita K, et al.: "Severely hyperkeratotic erythroderma associated with Hogkin's diseas-Dose high serum G-CSFcontribute to the formation of skin lesions?"J Am Acad Dermatol. 49. 772-773 (2003)
[Publications] Sasaki H, et al.: "A novel Sp 1-family-related cis-acting element for transcription of type VII collagen gene (COL7A1)"J Dermaol Sci. 32. 239-242 (2003)
[Publications] Sato-Matsumura KC, et al.: "A novel insertion mutation in COL7A1 identified in Hallopeau-Siemens recessive dystrophic epidermolysis bullosa"Acta Dermato-Venereol. 83. 137-138 (2003)

2003 Fiscal Year Annual Research Report

欠損蛋白補充による新しい表皮水疱症治療法の開発

Principal Investigator

清水 宏 北海道大学, 大学院・医学研究科, 教授 (00146672)

Research Products

[Publications] Arita K, et al.: "Squamous cell carcinoma in a patien with non-bullous congenital ichthyosiform erythroderma"Br J Dermatol. 148. 363-384 (2003)

[Publications] Shimizu T, et al.: "Cetirizine, an H1-receptor antagonist, suppresses the expression of macrophage migration inhibitory factor (MIF) : Its potential anti-inflammatory action"Clin Exp Allergy. 34. 103-109 (2003)

[Publications] Matsuzaki Y, et al.: "Keratinocyte responsive element 3 : analysis of a keratinocyte-specific regulatory sequence in the 230 kDa bullous pemhigoid antigen promoter"J Invest Dermatol. 120. 308-312 (2003)

[Publications] Sawamura D, et al.: "Identification of COL7A1 alternative splicing inserting 9 amino acid residues into the fibronectin type III linker domain"J Invest Dermatol. 120. 942-948 (2003)

[Publications] Sawamura D, et al.: "Epidermolysis bullosa : directions for future research and new challenges for treatment"Arch Dermatol Res. 295 Suppl 1. S34-S42 (2003)

[Publications] Kawasaki H, et al.: "Squamous cell carcinoma developing in a 12-year-old boy with nonHallopeau-Siemens recessive dystrophic epidermolysis bullosa"Br J Dermatol. 148. 1047-1050 (2003)

[Publications] Liu Y, et al.: "Immunofluorescence studies using skin sections of recessive dystrophic epidermolysis bullosa patients indicated that the antigen of anti-p200 pemphigoid is not fragment of type VII collagen"J Dermatol Sci. 32. 125-129 (2003)

[Publications] McMillan JR, et al.: "Epidermal basement membrane zone components : ultrastructural distribution and molecular interactions"J Dermatol Sci. 31. 169-177 (2003)

[Publications] McMillan JR, et al.: "Ultarastructural orientation of laminin 5 in the epidermal basement membrane : an undated model for basement membrane organization"J Histochem Cytochem. 51. 1299-1306 (2003)

[Publications] McMillan JR, et al.: "Alterations in desmosome size and number coincide with the loss of keratinocyte cohesion in skin with homozygous and heterozygous defects in the desmosomal protein plakophilin 1"J Invest Dermatol. 121. 96-103 (2003)

[Publications] McMillan JR, et al.: "Immunomapping of EBA sera to multiple epitopes on collagen VII further evidence that anchoring fibrils originate and terminate in the lamina densa"Exp Dermatol. 12. 261-267 (2003)

[Publications] Siegel DH, et al.: "Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin-extracellular-matrix linker protein UNC-112, causes Kindler syndrome"Am J Hum Genet. 73. 174-187 (2003)

[Publications] Tomita Y, et al.: "Simultaneous occurrence of three squamous cell carcinomas in a recessive dystrophic epidermolysis bullosa patient"Acta Derm Venereol. 83. 225-226 (2003)

[Publications] Tsuji Y, et al.: "Changing pattern of deiminated proteins in developing human epidermis"J Invest Dermatol. 120. 817-822 (2003)

[Publications] Matsuyama N, et al.: "The dinucleotide microsatellite polymorphism of the IFNARI gene promoter correlates with responsiveness of hepatitis C patients to interferon"Hepatol Res. 25. 221-225 (2003)

[Publications] Satoh M, et al.: "Innate apoptosis of human B lymphoblasts transformed by Epstein-Barr virus : modulation by cellular immortalization and senescence"Cell Struct Funct. 28. 61-70 (2003)

[Publications] Akiyama M, et al.: "Truncation of CGI-58 Protein Causes Malformation of Lamellar Granules Resulting in Ichthyosis in Dorfman-Chanarin Syndrome"J Invest Dermatol. 121. 1029-1034 (2003)

[Publications] Arita K, et al.: "Severely hyperkeratotic erythroderma associated with Hogkin's diseas-Dose high serum G-CSFcontribute to the formation of skin lesions?"J Am Acad Dermatol. 49. 772-773 (2003)

[Publications] Sasaki H, et al.: "A novel Sp 1-family-related cis-acting element for transcription of type VII collagen gene (COL7A1)"J Dermaol Sci. 32. 239-242 (2003)

[Publications] Sato-Matsumura KC, et al.: "A novel insertion mutation in COL7A1 identified in Hallopeau-Siemens recessive dystrophic epidermolysis bullosa"Acta Dermato-Venereol. 83. 137-138 (2003)

清水宏北海道大学, 大学院・医学研究科, 教授 (00146672)