摘要
目的了解转化生长因子β2(TGFβ2)和细胞外基质(ECM)调节人胚胎视网膜色素上皮(hfRPE)细胞向肌纤维母细胞分化的作用,确定这种调节的信号传导机制。方法hfRPE细胞培养于由ECM包被或未包被的培养皿上,培养液中加入或不加入TGFβ2,用免疫组织化学、流式细胞仪、蛋白印迹技术检测α-平滑肌机动蛋白(α-SMA)的表达。在培养液中分别加入calphostinC,染料木黄铜(genistein),PD98059和渥曼青霉素(Wortmannin),测定α-SMA的表达。结果hfRPE细胞培养于ECM包被的培养板上,培养液中加入TGFβ2后,出现明显的形态学改变,包括细胞伸展变长,可见肌动蛋白微丝的形态。流式细胞仪及免疫细胞化学检测结果显示,培养液中加入TGFβ2可明显增加α-SMA的表达,并与剂量成正相关。当TGFβ2的刺激浓度为50ng/ml时,与培养于未包被的培养板上的hfRPE细胞相比,用纤维连接蛋白(FN)包被培养板后,总的平均荧光强度(TMFI)增加(38.01±1.14)%(P<0.05)。蛋白印迹技术检测显示同样的结果。而上述效应可以大部分被蛋白激酶C(PKC)通路抑制剂calphostinC(10nmol/L)抑制(P<0.01)。结论TGFβ2可诱导hfRPE细胞向肌纤维母细胞转化,并与其剂量成正相关,这种作用可被FN加强。TGFβ2和ECM的这种调节作用可能是通过PKC细胞内信号传导通路而发挥作用。
Objective To investigate the modulating effect of transforming growth factor β2 （TGFβ2） and extracellular matrix （ECM） on the transdifferentiation of human fetal RPE （hfRPE） cells into myofibroblast like cells, and to determine the mechanism of signal transduction. Methods hfRPE cells were cultured on ECM coated or uncoated petri dish with or without TGFβ2 in the medium. The expression of α-smooth muscle actin （α-SMA） were detected by immunocytochemistry examination, flow cytometry and Western blotting via calphostin C, genistein, PD98059, and Wortmannin. Results After cultured on ECM coated petri dish with TGFβ2 in the medium ,there were obvious morphological changes of hfRPE cells including cellular elongating and appearing of actin microfilaments. The results of flow cytometry and immunocytochemistry examination showed that expression of α-SMA obviously increased after TGFβ2 was added in the medium in a dose-dependent manner. Compared with which of hfRPE cells cultured on the uncoated surface of culture plates, the total mean fluorescence intensity （TMFI） of hfRPE cells cultured on FN-coated surface increased （38. 01±1. 14）% when the stimulation concentration of TGFβ2 was 50 ng/ml （P〈0.05）. Western blotting further confirmed the effects. The changes mentioned above could be inhibited mostly by protein kinase C （PKC） and calphostin C （10 nmol/L）（P〈0.01）. Conclusion TGFβ2 may induce the transdifferentiation of hfRPE cells into myofibroblast like cells in a dose dependent manner, which could be intensified by FN. These mediated effects of TGFβ2 and ECM may act via the PKC signal transduction pathway.
出处
《中华眼底病杂志》
CAS
CSCD
北大核心
2006年第5期328-332,共5页
Chinese Journal of Ocular Fundus Diseases
关键词
细胞外基质
转化生长因子Β2
色素上皮
眼肌动蛋白
Extracellular matrix
Transforming growth factor beta
Pigment epithelium of eye, actin
