神经营养因子和生长因子等对培养的成人视网膜神经细胞的影响

目的　探讨成人视网膜神经细胞体外培养条件 ,脑源性神经营养因子 (BDNF)、神经营养素 (NT 4 )、表皮生长因子 (EGF)、成纤维细胞生长因子 (FGF)、诱导分化因子全反式视黄酸 (RA)等对成人视网膜神经细胞生长、增殖、凋亡的影响及调控机制。方法　胰蛋白酶消化结合机械吹打分离成人视网膜神经细胞 ,在培养基中加入或不加入BDNF、NT 4、EGF、FGF、RA。根据细胞形态、生长方式及免疫细胞化学特征确定细胞类型。比较各组中神经元的数目、转录调控因子c fos、c jun及细胞凋亡调控因子Bcl 2、Bax表达水平。结果　与对照组相比 ,BDNF、FGF处理组存活的神经元特异性烯醇酶、Thy1.1抗体和Bcl 2、c fos及c jun表达阳性细胞数也增多 (均P <0 0 1) ,培养的视网膜神经细胞在体外存活时间可长达 8个月 ;RA处理组c fos、c jun阳性细胞数较对照组增多 (均P <0 0 1) ;而NT 4、EGF处理组各项指标与对照组比较 ,差异无显著意义 (均P >0 0 5 )。结论　BDNF、FGF、RA能显著提高体外培养的成人视网膜神经细胞的存活 ,其机制可能涉及上调转录调控因子c fos、c jun及凋亡抑制因子Bcl 2的表达 ,或下调凋亡促进因子Bax的表达。但EGF、NT 4对体外培养的视网膜神经细胞存活状态无明显改善作用。

Effect of neurotrophic factors and growth factors on adult human retinal cells in vitro

Objective To examine whether adult human retinal cells can be maintained in vitro over long time periods and to study the effects of brain-derived neurotrophic factor (BDNF),neurotrophin- 4 (NT- 4), epidermal growth factor (EGF), fibroblast growth factor (FGF), and retinoic acid (RA) on growth, proliferation, and apoptosis of the cultured adult human retinal cells. Methods Adult retinal cells were isolated from donor eyes and cultured. BDNF, NT- 4, EGF, FGF, and RA were added individually to the cultures. The cells were identified by morphologic criteria, growth patterns, and immunocytochemical staining. The number of neurons in each group was compared. Expression levels of c-fos, c-jun, Bcl-2, and Bax were examined. Results Adult human retinal cells survived for 240 days in vitro. The addition of BDNF and FGF promoted survival of adult retinal neurons, increased the number of cells positive for neuron specific enzyme (NSE), Thy1.1, c-fos, c-jun, and Bcl-2. More c-fos and c-jun positive cells were observed in cell cultures containing medium with RA. However, NT- 4 and EGF treatment groups showed no significant difference from control groups in that neuron cell survival was low and fewer cells were positive for NSE, Thy1.1, c-fos, c-jun, and Bcl-2. Conclusions BDNF, FGF and RA improve survival of adult human retinal neurons in vitro via a mechanism that may involve c-fos, c-jun, and Bcl-2 expression, However, EGF and NT- 4 do not improve surival of the adult human retinal neurons.