ObjectiveTo detect the induction of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production in immunostimulated retinal pigment epithelial (RPE) cells to seek for the supplying of the arginine, a substrate for NOS; as well as the effects of produced NO on the tight junction of RPE-J cells. MethodsRat′s RPE-J cells were treated with interferon-γ(INF-γ), tumor necrosis factor-α(TNF-α) and lipopolysaccharide (LPS), and Northern and Western blotting were applied to analyze the expression of the citrulline-NO cycle enzymes and related enzymes and the effect of dexamethasone and cyclic adenosine monophosphate (camp) on the expression of iNOS. Immunocytochemistry reaction and Western blotting were used to evaluate the effect of produced NO on the tight junctions of RPE-J cells.ResultsiNOS and argininosuccinate synthetase (AS) were highly induced at both mRNA and protection levels in immunostimulated RPE cells while arginiosuccinate lyase (AL) was not induced. NO was produced by cells after stimulation with TNFα, IFNγ and LPS. The induction of iNOS mRNA and the production of NO by these immunostimulated cells was further enhanced by cAMP. NO was produced from citrulline as well as from arginine. And the produced NO impaired the tight junction of RPE-J cells, decreased the production of tight junction related protein ZO-1.ConclusionIn activated RPE-J cells, citrullinearginine recycling is important for NO production, and the produced NO weakened the function of tight junction of RPE-J cells.(Chin J Ocul Fundus Dis, 2005,21:32-36)
Objective To investigate the effect of the 8-bromum-cyclic adenosine monophosphate (8-Br-cAMP) on the telomerase activity and changes of cell cycle in retinoblastoma （RB） cells. Methods The cultured RB cells were divided into the experimental group (8-Br-cAMP) and control group. After cultured for 24, 48 and 72 hours in vitro, the telomerase activity of RB cells was detected by polymerase chain reaction enzyme-linked immunosorbent assay (PCR-ELISA) and the changes of cell cycle were detected by flow-cytometry. Results The difference of telomerase activity was significant between the experimental groups and control group (Plt;0.01). There was a negative correlation between the A value of absorbance and the time in the experimental groups (r=-0.778 9, F=33.936, Plt;0.01). The changes of the cell cycle were that the percentages increased in G1 phase and decreased in S phases. Conclusion 8-Br-cAMP may weaken telomerase activity, affect the cell cycle, and inhibit the proliferation of RB cells. (Chin J Ocul Fundus Dis，2004，20：358-360)