糖尿病早期大鼠视网膜神经细胞凋亡与caspase-3表达的关系

目的:观察糖尿病早期大鼠,视网膜神经细胞凋亡与caspase-3表达变化的关系。方法:健康雄性8周龄SD大鼠36只,建立糖尿病大鼠模型18只,正常对照18只。再各自分为4,8,12wk3组,每组6只大鼠。TdT介导DNA缺口末端的dUTP标记(TUNEL)法检测视网膜神经细胞的凋亡程度;免疫组织化学方法检测半胱氨酸天冬氨酸蛋白酶3(caspase-3)蛋白表达水平,实时荧光定量聚合酶链反应(PCR)法检测caspase-3mRNA表达情况。结果:正常对照组4,8,12wk大鼠均未见视网膜神经细胞凋亡。糖尿病组大鼠于建模后4wk即出现视网膜神经细胞凋亡,8wk和12wk大鼠视网膜神经凋亡程度较同期正常对照组大鼠均明显增强。神经细胞凋亡主要位于视网膜神经节细胞层及内核层。正常对照组4,8,12wk大鼠视网膜均未见caspase-3蛋白阳性表达,糖尿病组大鼠4wk即可见caspase-3蛋白阳性表达,8和12wk时表达增强。正常对照组4,8,12wk大鼠caspase-3mRNA表达RQ值分别为1.6±0.6,1.5±0.5,1.6±0.3;糖尿病组4,8,12wk大鼠caspase-3mRNA表达RQ值分别为5.7±1.2,12.6±2.3,14.3±2.1;较同期对照组均明显升高,差异有统计学意义(P<0.05)。结论:糖尿病早期视网膜神经细胞凋亡可能与capase-3表达增强有关。

Relationship between the apoptosis of retinal neurons and change of caspase-3 expression in early diabetic rats

·AIM:To probe the relationship between the apoptosis of diabetic rats’ retinal neurons and change of caspase-3 expression in early diabetic rats.·METHODS:Eighteen male 8-week-aged SD rats were injected with a single dose of streptozotocin(60mg/kg)to induce the diabetic model,and then they were divided into 3 groups at 4,8 and 12 weeks(each group,n=6).Another 18 male 8-week-aged SD rats were the normal control group,and were raised at the same time without any intervertion,also divided into 3 groups at 4,8 and 12 weeks(each group,n=6).Apoptosis of retinal neurons was detected by TdT-mediated dUTP nick end label(TUNEL)assay.The protein expression of caspase-3 was detected by immunohistochemistry.Caspase-3 mRNA levels were determined by SYBR Green Realtime PCR Master Mix.·RESULTS:There were no apoptosis neurons in control groups’ retina at 4,8,12 weeks.Apoptosis of the retinal neurons occurred 4 weeks after the onset of diabetes,and the apoptosis degrees were significantly higher than that of age-matched control groups at 8 and 12 weeks.The apoptosis of the retinal neurons located in the rentinal ganglion cells layer(GCL)and the inner nuclear layer(INL).Caspase-3 protein expression was not observed in the control rats’ retina at 4,8,12 weeks.Positive staining of caspase-3 occurred 4 weeks after the onset of diabetes,and enhanced at 8 and 12 weeks.In control rats at 4,8 and 12 weeks,caspase-3 mRNA levels were 1.6±0.6,1.5±0.5,1.6±0.3 respectively.Caspase-3 mRNA levels in diabetic rats that had diabetes for 4,8 and 12 weeks were 5.7±1.2,12.6±2.3,14.3±2.1 respectively,which were greater than that in the control groups(P<0.05).·CONCLUSION:The apoptosis of the retinal neurons in diabetic rats may be related with the enhance of the caspase-3 expression.·