阻断血管内皮细胞生长因子表达并诱导视网膜色素上皮细胞转分化成为神经元样细胞

In vitro transdifferentiation of adult human retinal pigment epithelium cells to neuro-like cells induced by blocking VEGF expression

目的探讨阻断培养的成人视网膜色素上皮(retinalpigmentepithelium,RPE)细胞产生血管内皮细胞生长因子(vascularendotheliumgrowthfactor,VEGF)后对RPE细胞形态及生长的影响。方法利用脂质体将编码反义VEGF的表达质粒导入RPE细胞,再利用新霉素(neomycin,又称G418)筛选稳定转导反义VEGF的RPE细胞克隆,采用倒置显微镜观察、免疫荧光及免疫组化染色、酶联免疫吸附(ELISA法)技术,观察细胞形态、产生VEGF的能力及细胞标志物的变化。结果持续阻断成人RPE细胞产生VEGF后,RPE细胞的生长受到抑制,同时细胞外基质分泌增加,形态发生转变,酷似神经细胞。RPE细胞产生VEGF减少后,细胞标志物表达也发生变化,视网膜神经元样细胞特异性标志物Thy11及神经丝蛋白转变为阳性,VEGF的受体Flk1表达也明显上调。结论VEGF信号传导通路在调控RPE细胞转分化方面起重要作用。

Objective To investigate the effects of blocking VEGF expression on the retinal pigment epithelium (RPE) in vitro. Methods RPE was transfected with plasmid encoding anti-sense of VEGF by lipofectin. RPE clones with stable expression of anti-sense of VEGF were screened by neomycin (G418). The changes of RPE and the level of VEGF secreted by RPE were observed by immunofluorescence immunohistochemical staining, Elisa and inverted-microscope. Results Transdifferentiation of cultured adult human retinal pigment epithelium cells to neurons was induced by blocking VEGF expression. The transdifferentiation was a direct phenotypic change from RPE cell to neurons without cell differentiation. The transdifferentiated neurons showed up-regulated expression of VEGF receptor 2 (Flk-1) and expressed several markers of retinal ganglion cell, including Thy1.1 and neurofilament. Conclusion Blocking of VEGF expression and up-regulation of Flk-1 can induce transdifferetiation of adult human retinal pigment epithelium cells into ganglion cells.(Chin J Ophthalmol,2005,41:114-117)