Department of Hematology, School of Allied Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Bosn J Basic Med Sci. 2017 Nov 20;17(4):315-322. doi: 10.17305/bjbms.2017.2103.
The positive role of platelet gel (PG) in tissue regeneration is well known, however, other characteristics of PG still remain to be determined. We investigated cellular and molecular changes in cultured human retinal pigment epithelial (hRPE) cells when treated with different concentrations of PG named PG1, PG2, and PG3. hRPE cells were isolated from donor eyes of two newborn children, within 24 hours after their death. The cells were treated with three concentrations of PG for 7 days: 3 × 104/ml (PG1), 6 × 104/ml (PG2), and 9 × 104/ml (PG3). Fetal bovine serum was used as a control. Immunocytochemistry was performed with anti-RPE65 (H-85), anti-Cytokeratin 8/18 (NCL-5D3), and anti-PAX6 antibody. We used MTT assay to determine cell viability. Gene expressions of PAX6, MMP2, RPE65, ACTA2, MKI67, MMP9, and KDR were analyzed using real-time PCR. A significant increase in viability was observed for PG3-treated cells compared to control (p = 0.044) and compared to PG1 group (p = 0.027), on day 7. Cellular elongation together with dendritiform extensions were observed in PG-treated cells on days 1 and 3, while epithelioid morphology was observed on day 7. All cells were immunoreactive for RPE65, cytokeratin 8/18, and PAX6. No significant change was observed in the expression of MKI67 and PAX6, but the expressions of MMP2, MMP9, ACTA2, and KDR were significantly higher in PG2-treated cells compared to controls (p < 0.05). Our results indicate that increased concentration of PG and extended exposure time have positive effects on viability of hRPE cells. PG may be useful for hRPE cell encapsulation in retinal cell replacement therapy.
血小板凝胶（PG）在组织再生中的积极作用是众所周知的，但 PG 的其他特性仍有待确定。我们研究了不同浓度的 PG（命名为 PG1、PG2 和 PG3）处理培养的人视网膜色素上皮（hRPE）细胞时的细胞和分子变化。hRPE 细胞从两个新生儿死亡后 24 小时内捐献的眼球中分离出来。将细胞用三种浓度的 PG 处理 7 天：3×104/ml（PG1）、6×104/ml（PG2）和 9×104/ml（PG3）。胎牛血清用作对照。用抗 RPE65（H-85）、抗细胞角蛋白 8/18（NCL-5D3）和抗 PAX6 抗体进行免疫细胞化学染色。我们使用 MTT 测定法来确定细胞活力。使用实时 PCR 分析 PAX6、MMP2、RPE65、ACTA2、MKI67、MMP9 和 KDR 的基因表达。与对照组（p=0.044）和 PG1 组（p=0.027）相比，PG3 处理的细胞在第 7 天的活力显著增加。在 PG 处理的细胞中，在第 1 天和第 3 天观察到细胞伸长和树突状延伸，而在第 7 天观察到上皮样形态。所有细胞对 RPE65、细胞角蛋白 8/18 和 PAX6 均呈免疫反应性。MKI67 和 PAX6 的表达没有明显变化，但与对照组相比，PG2 处理的细胞中 MMP2、MMP9、ACTA2 和 KDR 的表达明显更高（p<0.05）。我们的结果表明，增加 PG 浓度和延长暴露时间对 hRPE 细胞的活力有积极影响。PG 可能对视网膜细胞替代疗法中 hRPE 细胞的包封有用。
