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Molecular and pathological study on the expression of homeobox genes in mature and immature tissues.

成熟和未成熟组织中同源框基因表达的分子和病理学研究。

基本信息

批准号：
05454176
负责人：
MORIUCHI Tetsuya
金额：
$ 4.48万
依托单位：
Tokai University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

Polyclonal antibody against amino acid sequence encoded by first exon of HOXD4 gene was prerapred in rabbits. Distribution of HOXD4 protein was investigated in day-11, -13, -15, and -17 mouse embryos by using the HOXD4 antibody. (1) Skin ; Upper 1/3 of skin layrs was stained by anti-HOXD4. Lower 3/4 skin layrs was stained by anti-PCNA (marker of proliferating cells). Inter-digital skin was stained with anti-HOXD4 but not with anti-PCNA.(2) Cartilage ; Ossification center of the cartilage but not immature cartilage-forming cells were stained by anti-HOXD4. (3) Kidney ; Medullary region was stained by anti-HOXD4.HOXD3 gene was inserted at sense or antisense orientation in expression vector, and the construct was transfected in human erythroleukemia HEL cells. Two cell clones, E1 and E6, which were overexpressing HOXD3, were obtained. E1 and E6 expressed 7-10 fold amounts of HOXD3 mRNA compared to parental HEL cells, and C1 and C2 cell clones which had been, trasfected with antisense HOXD3 gene. Cell-cell and cell-extracellular matrix adhesions were markedly enhanced in E1 and E2 cells. Growth rate did not differ among sense-transfec-tants, parental HEL cells and antisense-transfectants. Overexpression of HOXD3 increased the mRNA level of integrin beta3. FACS analysis revealed that expression of alpha IIbbeta3 (GPIIb-IIIa) complex (=fibronectin receptor) on the cell surface was strongly increased in HEL sense-transfectants, E1 and E6.

制备了兔HOXD4基因第一外显子编码氨基酸序列的多克隆抗体。利用HOXD4抗体研究HOXD4蛋白在第11、-13、-15和-17天小鼠胚胎中的分布。(1)皮肤；抗hoxd4染色上1/3皮肤层。皮下3/4层采用抗pcna（增殖细胞标志物）染色。指间皮肤有抗hoxd4染色，但没有抗pcna染色。(2)软骨；抗hoxd4染色软骨成骨中心，未染色未成熟软骨形成细胞。(3)肾；髓质区抗hoxd4染色。将HOXD3基因以正义或反义方向插入表达载体，转染人红白血病HEL细胞。获得了两个过表达HOXD3的细胞克隆E1和E6。与亲本HEL细胞相比，E1和E6表达了7-10倍的HOXD3 mRNA，并且转染了反义HOXD3基因的C1和C2细胞克隆。E1和E2细胞的细胞间和细胞外基质黏附明显增强。正义转染、亲本HEL细胞和反义转染之间的生长速率没有差异。过表达HOXD3使整合素β 3 mRNA水平升高。FACS分析显示，在HEL感转染体E1和E6中，细胞表面α - IIbbeta3 （GPIIb-IIIa）复合物（=纤连蛋白受体）的表达明显增加。