α晶体蛋白对视网膜神经节细胞作用的初步研究

Effect of α-crystallin on retinal ganglial cells

提要：目的探讨α晶体蛋白对体外培养视网膜神经节细胞（retinal ganglion cells，RGCs）的作用。方法原代培养RGCs，Thy1．1免疫荧光法鉴定RGCs并进行纯度检测，10^-7、10^-6、10^-5、10^-4g／Lα晶体蛋白加入后于1、3、12h、1、2d和3d在相差倒置显微镜下观察RGCs形态学变化；计数突起数目和轴突长度；应用免疫荧光及激光共聚焦扫描技术观察实验组α晶体蛋白在体外培养RGCs上的定位并进行荧光强度半定量分析。结果Thy1．1鉴定RGCs纯度为90％～95％，RGCs10^-4、10^-5g／L组突起数目及轴突长度显著大于对照组，以10^-4g／L组变化最显著。免疫荧光结果显示10^-4g／Lα晶体蛋白组RGCs与α晶体蛋白抗体呈阳性结合，并呈先升高后降低趋势。结论α晶体蛋白能够促进RGCs生长，最佳浓度为10^-4g／L，细胞膜上抗体结合阳性，表明α晶体蛋白可与RGCs膜结合，这在α晶体蛋白促进RGCs存活和轴突再生的反应中可能起重要作用。

To study the effeα of α-crystallin on retinal ganglial cells （RGCs） and the possible mechanisms. Methods The changes of RGCs in vitro stimulated by10^ -7, 10^ -6 ,10 ^-5 , or 10 ^-4g/L α-crystallin for 1, 3, 12 h, 1, 2, 3 d were observed and identified by Thyl. 1 antibody. The number and the length of outgrowing RGCs axons were calculated under microscope and compared with the control group. The effeα of α- crystallin on RGCs was deteαed by immunofluorescence with confocal laser scanning microscope. Results The length and number of RGCs axons in 10 -4, 10-Sg/L α-crystallin solution were more than in the control on day 1, 2, 3 after α-crystallin stimulation, most significant difference in 10^-4g/L group. The interaαion be-tween α-crystallin and RGCs was proven positive by immunofluorescence with confocal laser scanning microscope. It increased firstly, then reduced. It reached the maximum at 3 h after α-crystallin stimulation. Conclusion α-crystallin enhanced the survival of RGCs and the regeneration of axons, suggesting α-crystallin might be related to the changes of RGCs membrane signal transduαion pathway. The mechanism might be involved in the interaαion between α-crystallin and RGCs.