不同剂量脑源性神经营养因子作用下视网膜色素变性组织中bcl-2蛋白的动态表达特征

Characteristic of bcl-2 dynamic expression in retinitis pigmentosa tissues affected by different dosages of brain derived neurotrophic factor

目的探讨不同剂量脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)作用下,出生后不同时间视网膜色素变性(retinitis pigmentosa,RP)小鼠视网膜组织内bcl-2蛋白表达的动态变化特征。方法分别取生后(postnatal,PN)2周龄、3周龄的rd小鼠各16只,其中各8只玻璃体内注射浓度分别为100μg·L-1和200μg·L-1BDNF,另各8只中4只玻璃体内注射相同体积磷酸盐缓冲液(PBS)作为治疗对照组,另4只玻璃体内不注射任何物质作为空白对照组。取PN-2周龄、3周龄C57BL/6小鼠(各4只)作为正常对照组。在注射后2周时即分别为PN-4周龄和PN-5周龄时将小鼠处死。在小鼠处死后30s内取出眼球,进行冰冻切片的制作和免疫荧光染色(按常规荧光染色程序进行)。Leica DM4000B荧光显微镜观察并照相,Im-age Pro Plus6.0图像分析软件对图像进行半定量分析,选用平均荧光强度为分析指标。结果 100μg·L-1和200μg·L-1BD-NF作用2周后即PN-4周龄rd小鼠视网膜组织内bcl-2的表达:正常对照组视网膜外核层(outer nuclear layer,ONL)外侧面、外丛状层(outer plexiform layer,OPL)、内丛状层(inner plexiform layer,IPL)及神经纤维层(nerve fiber layer,NFL)均有bcl-2蛋白(25.30±2.07)表达;而空白对照组(20.20±5.40)、治疗对照组(15.00±3.70)和BDNF治疗组(102.35±29.89、162.25±30.21)在OPL、IPL和NFL均有表达,其中治疗对照组低于空白对照组,BDNF治疗组高于空白对照组和治疗对照组。100μg·L-1和200μg·L-1BDNF作用2周后即PN-5周龄rd小鼠视网膜组织内bcl-2的表达:正常对照组ONL外侧面、OPL、IPL及NFL均有bcl-2蛋白(28.18±8.64)表达;而空白对照组(15.77±2.05)、治疗对照组(16.76±5.66)和100μg·L-1及200μg·L-1BDNF治疗组(76.00±30.14、136.62±23.52)在OPL、IPL和NFL均有表达,其中空白对照组与治疗对照组相比差异无统计学意义,BDNF治疗组高于空白对照组和治疗对照组。结论 BDNF对不同病变时期RP组织内抗凋亡蛋白的表达有不同效果,注射剂量越高其表达越高,干预时间越早其表达越高。

Objective To explore the characteristic of bcl-2 dynamic expression in retinitis pigmentosa tissues affected by different dosages of brain derived neurotrophic factor（BDNF）.Methods The rd mice at postnatal（PN）-2 weeks and 3 weeks were chosen,16 cases in each age.Eight cases in each aged mice received intravitreal injections of BDNF with a concentration of 100 μgL-1 and 200 μgL-1.Four cases in other 8 cases in each aged mice received intravitreal injections of PBS as treated control group and the rest injected nothing were set as blank group.The C57BL/6 mice at PN-2 weeks and 3 weeks were set as normal control group.Two weeks later eyes were enucleated after sacrifice within 30 seconds and frozen sections were made up for immunofluorescence staining.Images were captured by Leica DM4000B fluorescence microscope and analyzed semi-quantitively by Image Pro Plus 6.0 with the indicator of intensity.Results Two weeks after intravitreal injections of 100 μgL-1 and 200 μgL-1 BDNF,the expression of bcl-2 in the retina of PN-4 weeks rd mouse was significantly different from the other groups.At this stage,the normal group had expression（25.30±2.07） in the outer side of outer nuclear layer（ONL）,outer plexiform layer（OPL）,inner plexiform layer（IPL）,and nerve fiber layer（NFL） whereas there were expressions（20.20±5.40,15.00±3.70,102.35±29.89,162.25±30.21） in OPL,IPL and NFL in the blank group,treated control group and BDNF groups.The expression of treated group was higher than that of blank group,and BDNF groups were higher than the other two groups.Two weeks after intravitreal injections of 100 μgL-1 and 200 μgL-1 BDNF,the expression of bcl-2 in the retina of PN-5 weeks rd mouse was significantly different from the other groups.At this stage,the normal group had expression（28.18±8.64） in the outer side of ONL,OPL,IPL and NFL whereas there were expressions（15.77±2.05,16.76±5.66,76.00±30.14,136.62±23.52） in OPL,IPL and NFL in the blank group,treated control group and BDNF groups.The expression of treated group had no difference compared with blank group,and BDNF groups were higher than the other two groups.Conclusion BDNF has different kinds of influence on the expression of anti-apoptosis protein in the RP tissue at various time points.Higher dosages and earlier time points of intravitreal injections lead to more expression of the protein.