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目的探讨神经营养素-4(NT4)-神经短肽(NAP)对兔虹膜色素上皮细胞(IPECs)的保护作用。方法分离培养兔IPECs,采用非对称互补引物/模板方法构建神经短肽NAP和神经营养因子NT4的融合基因,构建重组腺相关病毒(rAAV)-绿色荧光蛋白(GFP)和rAAV-NAP,并以此转染体外培养的兔IPECs,以荧光表达来检测病毒的转染情况。对体外培养的兔IPECs进行紫外线光损伤,并用MTT法和流式细胞仪来检测NAP对兔IPECs的保护作用。结果转染了rAAV-GFP的兔IPECs表达了明显的GFP荧光。与对照细胞相比,转染了rAAV-NAP的兔IPECs经紫外线光损伤后,细胞的增殖正常,细胞凋亡率降低。结论本研究所构建的rAAV-NAP可有效地转染体外培养的兔IPECs,NAP对兔IPECs有光保护作用。(中华眼科杂志,2006,421008-1012)
Objective To investigate the protective effect of neurotrophin-4 (NT4) -neural short peptide (NAP) on rabbit iris pigment epithelial cells (IPECs). Methods Rabbit IPECs were isolated and cultured, and the fusion gene of neuropeptide NAP and NT4 was constructed by asymmetric primer / template method. Recombinant adeno-associated virus (rAAV) - green fluorescent protein (GFP) and rAAV- This transfection in vitro cultured rabbit IPECs, fluorescent expression to detect the virus transfection. Rabbit IPECs cultured in vitro were exposed to ultraviolet light. MTT assay and flow cytometry were used to detect the protective effects of NAP on rabbit IPECs. Results The rabbit IPECs transfected with rAAV-GFP expressed significant GFP fluorescence. Compared with the control cells, rabbit IPECs transfected with rAAV-NAP showed normal cell proliferation and decreased apoptosis rate after UV light damage. Conclusion The rAAV-NAP constructed in this study can effectively transfect rabbit IPECs cultured in vitro. NAP can protect the IPECs from light. (Chinese Journal of Ophthalmology, 2006,421008-1012)