2003 Fiscal Year Annual Research Report

合成VII型コラーゲンを用いる新しい創傷治療戦略の開発

Research Project

Project/Area Number	15390336
Research Institution	Hokkaido University
Principal Investigator	清水宏北海道大学, 医学研究科, 教授 (00146672)
Co-Investigator(Kenkyū-buntansha)	秋山真志北海道大学, 医学部・歯学部付属病院, 講師 (60222551) 澤村大輔北海道大学, 大学院・医学研究科, 助教授 (60196334)
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 / 皮膚潰瘍
Research Abstract	VII型コラーゲンは、表皮真皮境界部に存在するアンカリングフィブリルの構成成分であり、表皮と真皮の接着に最も重要な分子の一つである。近年、VII型コラーゲンの遺伝子に変異で生ずる栄養障害型表皮水庖症やVII型コラーゲンに対する自己抗体が産生される後天性表皮水庖症では、創傷治癒が著名に遷延することが知られている。本研究の目的は、合成VII型コラーゲン外用によって創傷治癒が促進されることを証明し、合成VII型コラーゲンを用いる新しい創傷治療戦略を開発することであった。平成15年度は、1)大量VII型コラーゲン蛋白の合成と精製をはじめに行った。我々は、すでにVII型コラーゲンのcDNAの全長をクローニングし、ネオマイシン耐性の発現プラスミドを作成している。そこで、そのプラスミドを表皮細胞培養株であるHaCaT細胞に導入して抗生剤G418にて選択し、stableの系を作成した。現在、その培養上清には常にVII型コラーゲンが分泌されているので、培養液を大量に回収した。さらに、英国のLeigh教授からVII型コラーゲンのモノクローナル抗体LH7.2の産生ハイブリドーマーの供与を受けているので、その抗体でアフィニティーカラムを作成した。VII型コラーゲンを含む培地をカラムにかけ、吸着されるVII型コラーゲンを精製した。2)その合成VII型コラーゲンを用いて、単層培養細胞での創傷治癒促進作用を検討した。VII型コラーゲン発現の全くないノックアウトマウス、VII型コラーゲン遺伝子の発現のない重症劣性栄養障害型Hallopeau-Simens型患者の表皮細胞と線維芽細胞を単層培養した。それらの細胞遊走能を観察した所、正常コントロールに比較して低下していることが確認され、合成VII型コラーゲンを加えると、回復する傾向があることを確認した。

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 : anaylsis 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 ante-p200 pemphigoid is not a 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 updated 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 squamaous 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 IFNAR1 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 Sp1-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

合成VII型コラーゲンを用いる新しい創傷治療戦略の開発

Principal Investigator

清水 宏 北海道大学, 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 : anaylsis 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 ante-p200 pemphigoid is not a 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 updated 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 squamaous 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 IFNAR1 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 Sp1-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, 医学研究科, 教授 (00146672)