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目的观察逆转录病毒及睫状神经营养因子(CNTF)诱导的成人视网膜色素上皮(RPE)细胞转分化过程中的形态特征与标志物变化。方法成人RPE细胞经携带绿色荧光蛋白(GFP)基因的逆转录病毒感染后,进一步用脂质体介导CNTF表达质粒进行转染。倒置荧光显微镜观察细胞形态变化,酶联免疫吸附实验、免疫组织化学染色及Westernblotting方法检测细胞产生和分泌CNTF的能力、CNTF受体(CNTFR)、多种神经细胞标志物及信号传导分子一类蛋白酪氨酸激酶(JAK)的表达水平。结果体外培养的成人RPE细胞经GFP逆转录病毒感染后,RPE细胞的形态无明显变化,但开始表达神经元和胶质细胞的某些标志物,如神经丝蛋白(NF)和胶质纤维酸性蛋白(GFAP);经CNTF表达质粒转染后,持续高水平表达CNTF的RPE细胞出现明显的神经细胞分化,CNTFR的表达量虽无明显变化,但其分布发生改变,主要分布在细胞膜上;此外,信号传导分子JAK表达明显上调。结论体外培养的成人RPE细胞在逆转录病毒及CNTF的影响下可出现明显的神经细胞形态分化,转分化与CNTF-CNTFR-JAK信号传导通路有关。
Objective To observe the morphological changes and markers of retrovirus and ciliary neurotrophic factor (CNTF) -induced retinal pigment epithelium (RPE) cell transdifferentiation. Methods Adult RPE cells were infected with retroviral vector carrying green fluorescent protein (GFP) gene and further transfected with liposome-mediated CNTF expression plasmid. Inverted fluorescence microscopy was used to observe the changes of cell morphology, enzyme-linked immunosorbent assay (ELISA), immunohistochemical staining and Western blotting to detect the ability of cells to produce and secrete CNTF, CNTF receptor (CNTFR), various neural cell markers and signal transduction molecules Tyrosine kinase (JAK) expression levels. Results In vitro cultured RPE cells showed no morphological changes after transfection with GFP retrovirus, but began to express certain markers of neurons and glial cells, such as neurofilament (NF) and glial fibrillary acidic (GFAP). After transfection with CNTF expression plasmid, RPE cells with high level of CNTF continued to show obvious neuronal differentiation. Although the expression of CNTFR did not change significantly, the distribution of CNTFR changed mainly on the cell membrane. In addition, , JAK expression of signal transduction molecules was significantly up-regulated. CONCLUSION: Adult RPE cells cultured in vitro have obvious morphological differentiation of neural cells under the influence of retrovirus and CNTF. Transdifferentiation is related to the CNTF-CNTFR-JAK signal transduction pathway.