脑源性神经营养因子(BDNF)对高糖环境下视网膜Müller细胞的保护作用

Effect of brain-derived neurotrophic factor on high glucose induced apoptosis of retinal müller cell

目的探讨脑源性神经营养因子(brain derived neurotrophic factor,BDNF)对高糖环境下视网膜Müller细胞的保护作用。方法该研究实验分为3组,对照组、高糖组(对照组中添加25 mmol·L-1葡萄糖)、BDNF组(高糖组添加100 mg·L-1BDNF),实验干预24 h后,在倒置显微镜下观察各组细胞形态和生长状态,并进行各项指标检测。用倒置荧光显微镜观察各组视网膜Müller细胞的生长状态并检测细胞内活性氧(reactive oxygen species,ROS)荧光强度;流式细胞仪检测视网膜Müller细胞凋亡率,Western blot检测Bax、Bcl-2蛋白表达情况。结果与对照组相比,高糖组Müller细胞生长状态差,细胞突起减少,细胞失去原有形态,由原来的不规则形变为类圆形,折光性增强;而BDNF组与对照组相比无明显差异;与高糖组相比,BDNF组细胞生长状态明显改善,细胞突起增多,细胞折光性减弱。与对照组相比,高糖组Müller细胞内ROS含量显著升高(P<0.01),BDNF组细胞内ROS含量亦升高(P<0.01);与高糖组相比,BDNF组ROS含量显著减少(P<0.01)。与对照组相比,高糖组细胞凋亡率显著升高(P<0.01),BDNF组凋亡率仍高于对照组(P<0.01);与高糖组相比,BDNF组凋亡率明显减少(P<0.01)。与对照组相比,高糖组Bcl-2蛋白相对表达量显著减少,Bax蛋白相对表达量明显升高,Bcl-2/Bax二者比值降低(P<0.01),BDNF组与对照组相比相对表达量差异无统计学意义(P>0.05);与高糖组相比,BDNF组Bcl-2蛋白相对表达量明显升高,Bax蛋白表达减少,Bcl-2/Bax比值升高(P<0.01)。结论高糖环境能诱导视网膜Müller细胞凋亡,而给予外源性BDNF能够显著抑制高糖环境诱导的视网膜Müller细胞凋亡,对视网膜Müller细胞起到保护作用。

Objective To explore the protective effect of brain-derived neurotrophic factor （BDNF） on retinal Müller cells under high glucose environment.Methods The experiment was divided into three groups：control group,high glucose group （the addition of 25 mmol·L-1 glucose） and BDNF group （the addition 100mg·mL-1 BDNF to the high glucose group）.After 24 hours of intervention,the growth state of each group retinal Müller cell was observed by inverted microscope,meanwhile the fluorescence intensity of reactive oxygen species （ROS） was detected.Flow cytometry was used to detect the apoptosis rate of retinal Müller cells,the protein expression levels of Bax and Bcl-2 was detected by Western blot.Results Compared with the control group,the growth state of Müller cell in high glucose group was poor,and the cell processes was decreased,their original form were lost,then their state changed from original irregular shape to a quasi-circular,as well as the refractivity was increased.There was no obvious difference between BDNF group and control group.Compared with the high glucose group,the growth state of Müller cell in BDNF group improved significantly,the cell process was increased,and the refractivity was weakened.Compared with the control group,the content of ROS in retinal Müller cells was increased significantly in high glucose group（P〈0.01）,the content of ROS was still elevated in BDNF group（P〈0.01）;Compared with the high glucose group,the content of ROS in BDNF group was decreased（P〈0.01）.Compared with the control group,rate of apoptosis was increased significantly（P〈0.01）,rate of apoptosis was still elevated in BDNF group（P〈0.01）;Compared with the high glucose group,the rate of apoptosis was decreased significantly.Compared with the control group,Bcl-2 protein relative expression was decreased significantly and Bax protein relative expression was increased significantly,the ratio of Bcl-2/Bax protein content was decreased （P〈0.01）,the relative expression has no statistics significance（P〉0.05）.Compared with the high glucose group,Bcl-2 protein relative expression was increased significantly,Bax protein relative expression was decreased,the ratio of Bcl-2/Bax protein content was increased（P〈0.01）.Conclusion High glucose can induce the apoptosis of retinal Müller cells,but the exogenous BDNF can significantly inhibit retinal Müller cell apoptosis induced by high glucose,and can protect the retinal Müller cells.