RESUMO
Background and purpose: Non-homologous end joining [NHEJ] is the major pathway for removing DNA double strand breaks lesions. NHEJ is considered to be a resistant factor against chemotherapy induced neuropathy. beta-Lapachone [beta-Lap] is one of the antineoplastic agents which is shown to have anti neuroinflammatory effects. Extremely low frequency [<300 Hz] electromagnetic field [EMF] is shown to decrease NHEJ genes expression. Morphine [Mor] is associated with reducing effect on DNA repair and induce DNA damages. The goal of this study was to evaluate the effect of combination treatment of beta-Lap, morphine [Mor] and EMF on expression of NHEJ related genes [XRCC4, Ku70, Ku80, DNA-PKcs and LIG4]
Materials and methods: SH-SY5Y cells [epithelial neuroblasts] were treated with four combinational treatments of beta-Lap [2.0 and 3.2 microM], Mor [5.0 microM] and EMF [50 Hz, 0.50 mT, "15 min field on/15 min field off"] and mRNA levels of XRCC4, Ku70, Ku80, DNA-PKcs and LIG4 were evaluated by quantitative real-time PCR and primers specific for the examined genes. The experiments were done in triplicates
Results: No significant alteration in the mRNA levels of NHEJ related genes was observed in "beta-Lap alone" and "beta-Lap + Mor" treated cells. The expression levels of NHEJ related genes were significantly increased in "beta-Lap + EMF" and "beta-Lap + Mor + EMF". Multiple linear regression analysis showed that the effect of EMF and Mor on NHEJ related genes expression is opposite to the effect of beta-Lap
Conclusion: In overall, combination of beta-Lap, Mor and EMF leads to increased expression of NHEJ related gene expression. This effect may lead to decreased sensitivity of SH-SY5Y cells against beta-Lap and can improve its neuroprotective property which might be hopeful for its clinical applications
RESUMO
To establish human retinal pigment epithelial [RPE] cell culture as a source for cell replacement therapy in ocular diseases Human cadaver globes were used to isolate RPE cells. Each globe was cut into several pieces of a few millimeters in size. After removing the sclera and choroid, remaining tissues were washed in phosphate buffer saline and RPE cells were isolated using dispase enzyme solution and cultured in Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 supplemented with 10% fetal calf serum. Primary cultures of RPE cells were established and spheroid colonies related to progenitor/stem cells developed in a number of cultures. The colonies included purely pigmented or mixed pigmented and non-pigmented cells. After multiple cellular passages, several types of photoreceptors and neural-like cells were detected morphologically. Cellular plasticity in RPE cell cultures revealed promising results in terms of generation of stem/progenitor cells from human RPE cells. Whether the spheroids and neural-like retinal cells were directly derived from retinal stem cells or offspring of trans-differentiating or de-differentiating RPE cells remains to be answered