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

在兔体内制备了HOXD4基因第一外显子编码的氨基酸序列的多克隆抗体。用HOXD4抗体研究了HOXD4蛋白在小鼠胚胎中的分布。(1)皮肤：上1/3皮肤层用抗HOXD4染色。下3/4皮肤层用增殖细胞标记物--抗增殖细胞核抗原染色。(2)软骨：抗HOXD4抗体阳性，未成熟的软骨形成细胞阳性。(3)肾脏：用抗HOXD4抗体对延髓区进行染色，将HOXD3基因正、反义插入表达载体，并将其导入人红白血病HEL细胞。获得了两个高表达Hoxd3的细胞克隆：E1和E6。与亲本HEL细胞和转反义Hoxd3基因的细胞克隆相比，E1和E6细胞表达的Hoxd3mRNA是亲本细胞的7-10倍。细胞与细胞之间、细胞与细胞外基质的黏附在E1和E2细胞中明显增强。正义转染体、亲本HEL细胞和反义转染体的生长速度无明显差异。Hoxd3基因的过表达增加了整合素Beta3的mRNA水平。流式细胞仪分析显示，在HEL正义转染体E1和E6中，细胞表面αIIbbeta3(GPIIb-IIIa)复合体(=纤维连接蛋白受体)的表达显著增加。

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.