Ngn2基因转染对小鼠体外培养三维视杯结构视网膜神经元分化的调控作用

Regulation effects of Ngn2 gene transfection on retinal neuron differentiation in three-dimensional optic vesicle of mice

目的探讨Ngn2基因对小鼠体外培养三维视杯结构视网膜神经元分化的调控作用。方法采用小鼠诱导多能干细胞(iPSC)在特定条件下体外培养获取视泡结构(OV),OV培养过程中通过慢病毒介导Ngn2基因多次转染OV,并在OV发育成熟后进行诱导,促进OV内细胞向视网膜神经细胞的特异性分化。以绿色荧光蛋白(EGFP)基因作为对照,免疫荧光法检测OV内视网膜神经细胞的分化情况,采用逆转录PCR和Western blot法定量检测OV内视网膜神经细胞特异性蛋白Pax6、Islet1和Brn3b基因和蛋白的表达情况。结果成功培养出小鼠iPSC来源的OV。慢病毒介导下经Ngn2基因诱导的OV组织内部神经细胞的分化数量增加。逆转录PCR和Westernblot法定量检测结果显示,Ngn2转染组OV内视网膜神经细胞特异性蛋白Pax6、Islet1和Brn3b在基因及蛋白水平的相对表达量均明显高于EGFP转染组,差异均有统计学意义(均P<0.05)。结论Ngn2基因转染可有效提高OV内视网膜神经细胞的分化数量,使体外培养的OV发育更为成熟,形成更为完善的视网膜细胞神经环路。

Objective To investigate the regulation effects of Ngn 2 gene transfection on retinal neuron differnetion in three-dimentional optic vesicle(OV)of mice.Methods OV was cultured in vitro using mouse induced pluripotent stem cells(iPSC)under specific conditions.During OV culture,it was transfected multiple times by lentivirus-mediated Ngn2 gene and then it was induced after maturation.The cells were specificly differentiated toward retinal nerve cells in OV.Using the green fluorescent protein(EGFP)gene as control,the differentiation of retinal nerve cells in OV was detected by immunohistochemistry.Reverse transcription PCR and Western blot were used to quantitatively detect the expressions of retinal neuron-specific proteins Pax6,Islet1 and Brn3b.Results The mouse iPS-derived OV was successfully cultured.The number of neural cells in the OV transfected with the Ngn 2 gene was increased by the lentiviral-mediated lentivirus.The expressions of PAX6,Islet1 and Brn3b in the Ngn2 transfection group were significantly higher at the gene and protein levels than those in the control group,with significant differences between the two groups(P<0.05).Conclusions The Ngn2 gene can effectively increase the number of retinal neuron differentiation in OV and make in vitro cultured OV more mature and form a more perfect retinal cell neural circuit.