Immunoelectron microscopic analysis of basement membrane components in type VII collagen knockout skin

VII型胶原基因敲除皮肤基底膜成分的免疫电镜分析

• 批准号: 12670836
• 负责人: ISHIKO Akira
• 金额: $ 1.98万
• 依托单位: Keio University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670836/
• 关键词: type VII collagen; epidermolysis bullosa; immunoelectron microscopy; BPAG2; laminin 5; basement membrane; mutation analysis

Receive dystrophic epidermolysis bullosa (RDEB) is a congenital bullous disease caused by lack of type VII collagen that anchors epidermal basement membrane (lamina densa) to the dermis. Molecular relationship between the type VII collagen and the other epidermal basement membrane molecules is not fully elucidated. The purpose of this study is to elucidate the in vivo molecular ultrastructural relationship between the type VII collagen and the other basement membrane molecules using the skin of RDEB patients, which were the type VII knockout human skin. For this, the precise ultrastructural localization of basement membrane molecules including type VII collagen, BPAG2, laminin 5 were compared between the type VII collagen knockout human skin and the normal human skin. The diagnosis of RDEB confirmed by electron microscopy and the lack of expression of LH7.2 antigen, N-terminus domain of type VII collagen. Blood samples from the five patients with RDEB were processed for genomic DNA elution. COL7A1 gene that encodes type VII collagen was amplified and DNA sequences were analyzed. Among ten alleles, three mutations on four alleles were identified: 6573+1G>C, 5504delA and 5818delC. Skin samples were obtained from three RDEB patients by consent. Localizations of N and C terminus of BPAG2, and laminin 5 were observed with post-embedding immunogold electron microscopy and compared statistically with those of normal human skin. As results, the N terminus of BPAG2 was localized at the hemidesmosome and laminin 5 was localized at lamina densa. There was no significant difference in localization of these molecules between normal and RDEB skin. However, C-terminus of BPAG2 were significantly shifted from lamina densa to the epidermal side. These results indicated that type VII collagen may have some molecular interaction between BPAG2 via C-terminal domain of BPAG2, both of which were localized at lamina densa in the normal skin.

接受营养不良的表皮溶液Bullosa（RDEB）是一种先天性大胆疾病，原因是缺乏VII型胶原蛋白，将表皮基底膜（Lamina Densa）锚定在真皮上。 VII型胶原蛋白与其他表皮基底膜分子之间的分子关系尚未完全阐明。这项研究的目的是使用RDEB患者的皮肤，即VII型敲除人类皮肤，阐明VII型胶原蛋白与另一个地下膜分子之间的体内分子超微结构关系。为此，比较了VII胶原蛋白敲除人类皮肤和正常的人类皮肤，比较了基底膜分子的精确超微结构定位。通过电子显微镜证实的RDEB的诊断和VII型胶原蛋白的N末端结构域的LH7.2抗原的表达不足。从五个患有RDEB患者的血液样本进行了基因组DNA洗脱的处理。扩增了编码VII型胶原蛋白的COL7A1基因并分析了DNA序列。在十个等位基因中，发现了四个等位基因的三个突变：6573+1g> c，5504dela和5818delc。通过同意，从三名RDEB患者那里获得皮肤样品。用后膜后的免疫元电子显微镜观察到BPAG2和层粘连蛋白5的N和C末端的局部定位，并与正常的人皮肤相比。结果，BPAG2的N末端位于Hemidesmosmosmosmosmosmoss，层粘连蛋白5位于Lamina Densa。正常皮肤和RDEB皮肤之间这些分子的定位没有显着差异。然而，BPAG2的C末端从Lamina Densa显着转移到表皮侧。这些结果表明，VII型胶原蛋白通过BPAG2的C末端结构域之间的BPAG2之间的分子相互作用可能存在一定的分子相互作用，而BPAG2的C末端结构域则位于正常皮肤中的Lamina Densa。

项目成果

Akira Ishiko, Hiroshi Shimizu: "Electron microscopy in diagnosis of autoimmune bullous disorders"Clinics in Dermatology. 19. 631-637 (2001)

Akira Ishiko、Hiroshi Shimizu：“电子显微镜诊断自身免疫性大疱性疾病”皮肤科诊所。

Akira Ishiko and Hiroshi Shimizu: "Electron microscopy in diagnosis of autoimmune bullous disorders"Clinics in Dermatology. 19. 631-637 (2001)

Akira Ishiko 和 Hiroshi Shimizu：“电子显微镜诊断自身免疫性大疱性疾病”皮肤科诊所。

Yasuko Takizawa., et al.: "Compound heterozygosity for a point mutation and a deletion located at slice acceptor sited I the LAMB3 gene leads to generalized atrophic benign epidermolysis bullosa"J Invest Dermatol:, 2000. 115. 312-316 (2000)

Yasuko Takizawa., et al.：“位于 LAMB3 基因的切片受体位点的点突变和缺失的复合杂合性导致全身性萎缩性良性大疱性表皮松解症”J Invest Dermatol：，2000. 115. 312-316 (2000)

Yasuko Takizawa, Yoshiki Hiraoka, Hayato Takahashi, Akira Ishiko, Isamu Yasuraoka, Isao Hashimoto, Sadakazu Also Takeji Nishikawa, Hiroshi Shimizu: "Compound heterozygosity for a point mutation and a deletion located at slice acceptor sited I the LAMB3 ge

Yasuko Takizawa、Yoshiki Hiraoka、Hayato Takahashi、Akira Ishiko、Isamu Yasuraoka、Isao Hashimoto、Sadakazu Also Takeji Nishikawa、Hiroshi Shimizu：“位于 LAMB3 ge 切片受体位点的点突变和缺失的复合杂合性

Detlef Zillikens, Akira Ishiko, Marcel F. Jonkman, lakov Chimanovich, Hiroshi Shimizu, Takashi Hashimoto, and Eva-B. Brocker: "Autoantibodies in anti-p200 pemphigoid stain skin lacking laminin 5 and type VII collagen"Br J Dermatol. 143. 1043-1049 (2000)

Detlef Zillikens、Akira Ishiko、Marcel F. Jonkman、lakov Chimanovich、Hiroshi Shimizu、Takashi Hashimoto 和 Eva-B。