视网膜色素上皮细胞吞噬纤维连接蛋白时细胞内信号传导通路的研究

摘要

Objective　To deterimine the phagocytosis of fibronectin (FN) by retinal pigment epithelial (RPE) cells and its related intracellular signal transduction pathways. Methods　Fluorescent latex beads were coated by FN and then incubated with RPE cells at 37℃ for 3 hours. Phagocytosis was quantified by a flow cytometric assay. Experiments were also performed in the presence of inhibitors of various intracellular signaling pathways ［tyrosine kinase (TK), phosphatidylinositol 3-kinase (PI 3-K), protein kinase C (PKC)］. Results FN-coated beads produced significantly increase in the phagocytic index (P<0.05) when compared to the uncoated control. The PKC inhibitors, phorbol 12-myristate 13-acetate (PMA, 100 nmol/L) or calphostin C (400 nmol/L), non-specifically increased the phagocytosis of both FN-coated (P<0.01) and uncoated beads (P<0.01). Inhibitors of TK (genistein, 100 μg/ml) and PI 3-K (wortmannin, 5 μmol/L), significantly inhibited FN phagocytosis (P<0.001) but did not affect the uncoated control. While a combination of different inhibitors (genistein plus wortmannin, genistein plus PMA, wortmannin plus PMA) was used, the results showed that genistein or wortmannin can counteract the effect of PMA, and that genistein plus wortmannin have an additive inhibitory effect. Conclusions　Our results suggest that the FN phagocytosis by RPE cells appear to be regulated, at least in part, by some signal transduction pathways. The knowledge of the signaling pathways that mediate FN phagocytosis by RPE cells may provide novel therapeutic targets for molecular pharmacology of proliferative RPE disorders such as proliferative vitreoretinopathy.%目的　探讨视网膜色素上皮（retinal pigment epithelium, RPE）细胞对纤维连接蛋白(fibronectin, FN)的吞噬及相关细胞内信号传导通路的作用。方法　以FN包被的聚苯乙烯微球(微球)作为吞噬标记物, 建立人胎儿RPE细胞吞噬模型。所研究的细胞内信号传导通路包括蛋白激酶C(protein kinase C, PKC)通路、酪氨酸激酶(tyrosine kinase, TK)通路和磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase, PI 3-K)通路, 分别使用这3种信号通路的抑制剂预处理RPE细胞，37℃吞噬3 h，流式细胞仪定量检测吞噬指数。 结果　与对照组比较, RPE细胞对FN包被微球有明显吞噬作用（P<0.05）。PKC抑制剂PMA（phorbol 12-myristate 13-acetate, 100 nmol/L）或Calphostin C (400 nmol/L)非特异性地增加了RPE细胞对FN包被或未包被微球的吞噬指数（P<0.01）; TK和PI 3K的抑制剂Genistein （100 μg/ml）与Wortmannin（5 μmol/L）分别降低了FN包被微球的吞噬指数（P<0.001），但对对照组无影响。联合使用不同抑制剂Genistein或Wortmannin可对抗PMA对FN吞噬的增强作用，但二者联合使用则具有相累加的抑制吞噬作用。结论　RPE细胞对FN的吞噬受细胞内PKC、TK和PI 3-K信号传导通路的调节，此结论将为进一步研究治疗增殖性玻璃体视网膜病变的药物提供理论依据。