Abstract
Abstract: :
Purpose:The pP344 gene was identified as a marker of chick retinal pigment epithelium. Sequence analysis showed that the product of pP344 gene may be a secretory protein with serine protease inhibitory activity. Immunohistochemical study revealed the distribution of PP344 protein in neural retina. We investigated the effects of the pP344 gene product on the retinal cells as a neurotrophic activity. Methods: The 1648 bp pP344 cDNA including the initiation codon was subcloned into the plasmid pcDNA4/TO/myc–His C which must be induced in the transfected cells by tetracycline to produce recombinant PP344 protein. The pcDNA4 –pP344 was transfected into T–RExTM–HeLa cells using lipofectoamine 2000 Reagent, and the expression of recombinant PP344 protein was induced by tetracycline. The purified recombinant protein was investigated by Western blot analysis. Embryonic chick retinal cells were cultured in the presence of varying amounts of recombinant protein to determine whether the protein promotes morphological differentiation by examining the ability of the protein to stimulate neurite outgrowth. Results: A band of 70kDa was detected in pcDNA4–pP344 transfected Hela cells. The cultured non–photoreceptor cells treated with 0, 2, 10, and 50 ng/ml showed the length of neuron processes of 33.0±16.8, 52.2±16.3 µm, 54.2±13.7 µm, and 70.4±28.2 µm, respectively. The PP344 fusion protein significantly stimulated neurite outgrowth compared with control (P=0.01, P=0.003, P<0.0001). Conclusions: These results indicated that PP344 fusion protein exerts trophic effects on retinal cells. The PP344 protein may facilitate diffrentiation of retinal cells.
Keywords: retinal pigment epithelium • retinal culture • retinal development