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EXTRACELLULAR MATRIX GENE EXPRESSION IN CORNEA

角膜细胞外基质基因表达

基本信息

批准号：
2161443
负责人：
BJORN R OLSEN
金额：
$ 20.52万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
1987
资助国家：
美国
起止时间：
1987-09-30 至 1995-09-29
项目状态：
已结题

项目摘要

The primary goals of the study proposed here are: 1) to examine the molecular organization of rabbit type VIII collagen, the major constituent of the Descemet's membrane, and to establish the structural and evolutionary relationship between the gene(s) encoding the alpha2(VIII) chain and the genes that code for the alpha1(VIII) and alpha1(X) chains; 2) to examine the synthesis, assembly and location of the type VIII collagen molecule in the highly organized extracellular matrices of the cornea and in extraocular tissues; 3) to isolate the human type VIII collagen genes and examine hereditary disorders that may be linked to abnormalities in type VIII collagen gene structure and/or expression; 4) to isolate the mouse alpha1(VIII) gene and disrupt the endogenous gene in embryonic stem cells by insertional mutagenesis, as a first step in developing mouse strains with defects in the expression of type VIII collagen. To accomplish these goals, we will use a combination of DNA cloning/sequencing, protein chemistry and immunologic techniques. cDNA coding for the alpha2(VIII) chain will be synthesized from rabbit corneal endothelial cell mRNA. The gene coding for the alpha2(VIII) chain will be isolated from rabbit genomic libraries. Specific antibodies against synthetic peptides deduced from the nucleotide sequence of rabbit cDNAs will be generated and utilized to examine the synthesis, assembly and the location of the type VIII collagen in adult cornea as well as in corneal development. The primary structure of human type VIII collagen will be determined by isolation and sequencing of human genomic DNA encoding alpha1(VIII) collagen and specific antibodies will be generated against synthetic peptides. We also propose to examine whether the accumulation of collagen posterior to Descemet's membrane observed in autosomal dominantly inherited Fuchs' dystrophy and/or cornea guttata is due to altered structure and/or expression of type VIII collagen genes. Finally, to provide a basis for studies of the consequences of alterations in alpha1(VIII) collagen gene structure and expression in an animal model, we propose to disrupt the endogenous gene by homologous recombination in mouse embryonic stem cells. If successful, these experiments may allow generation of transgenic mice that could be models of hereditary disorders of type VIII collagen.

本研究的主要目的是：1）研究兔VIII型胶原蛋白（主要成分）的分子组织后弹力膜，并建立结构和编码α 2（VIII）的基因之间的进化关系链以及编码α 1（VIII）和α 1（X）链的基因; 2）检查VIII型胶原的合成、组装和定位，角膜高度组织化的细胞外基质中的分子，眼外组织中; 3）分离人VIII型胶原基因并检查可能与异常有关的遗传性疾病， VIII型胶原基因结构和/或表达; 4）分离小鼠α 1（VIII）基因，并破坏胚胎干中的内源基因细胞通过插入诱变，作为第一步，在发展小鼠 VIII型胶原表达缺陷的菌株。完成这些目标，我们将使用DNA克隆/测序，蛋白质化学和免疫技术。编码α 2（VIII）链的cDNA将从兔中合成角膜内皮细胞mRNA。编码α 2（VIII）链的基因将从兔子基因组文库中分离。特异性抗体从兔cDNA核苷酸序列推导的合成肽将产生和使用，以检查合成，组装和 VIII型胶原在成人角膜以及角膜中的位置发展人VIII型胶原蛋白的一级结构将是通过分离和测序人类基因组DNA编码 α 1（VIII）胶原蛋白和特异性抗体将产生，合成肽此外，我们亦建议研究，在常染色体显性遗传中观察到后弹力膜后胶原遗传性Fuchs营养不良和/或角膜点滴状是由于改变了 VIII型胶原基因的结构和/或表达。最后为为研究改变的后果提供了基础， α 1（VIII）胶原基因结构和表达的动物模型，我们建议在小鼠中通过同源重组破坏内源基因胚胎干细胞如果成功，这些实验可能会产生转基因小鼠可以作为遗传性疾病的模型， VIII胶原蛋白。

项目成果

期刊论文数量（14）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（2）

Basement membrane assembly and differentiation of cultured corneal cells: importance of culture environment and endothelial cell interaction.

DOI：
10.1006/excr.1994.1300
发表时间：
1994-10
期刊：
Experimental cell research
影响因子：
3.7
作者：
J. Zieske;Valerie S. Mason;M. Wasson;Susan F. Meunier;C. Nolte;N. Fukai;Björn Olsen;N. Parenteau
通讯作者：
J. Zieske;Valerie S. Mason;M. Wasson;Susan F. Meunier;C. Nolte;N. Fukai;Björn Olsen;N. Parenteau

Secretion of collagen types I and II by epithelial and endothelial cells in the developing chick cornea demonstrated by in situ hybridization and immunohistochemistry.

原位杂交和免疫组织化学证实发育中的鸡角膜中上皮细胞和内皮细胞分泌 I 型和 II 型胶原蛋白。