^(60)Coγ射线照射后豚鼠耳蜗细胞凋亡相关基因及蛋白表达分析

Experimental Study of Apoptotic Gene and Protein Expression in Guinea Pig Cochlea after Irradiation

目的探讨60 Coγ射线照射后豚鼠耳蜗细胞凋亡相关基因及蛋白的表达变化。方法健康白化豚鼠60只随机分为对照组12只和照射组48只。对照组不进行照射,照射组以右耳为照射耳,予60 Coγ射线一次性照射70Gy,于照射后第1、4、7和14天各处死12只豚鼠,行耳蜗中轴切片HE染色、逆转录聚合酶链式反应(reverse transcription polymerase chain reaction,RT-PCR)和耳蜗蛋白印迹法(western blot,WB)检测耳蜗细胞凋亡相关基因及蛋白的表达。结果对照组耳蜗组织结构正常,照射组照射后耳蜗组织结构均出现了不同程度的损伤,螺旋神经节细胞浆萎缩,细胞核出现凝集,Corti器塌陷萎缩,血管纹轻微水肿或空泡形成。对照组豚鼠耳蜗可见少量Fas、Bax、Bcl-2mRNA表达,照射组Fas mRNA表达上调,其中照射后第1天(2.13±1.23)和第4天(2.92±2.06)高于对照组(0.80±0.37),差异有统计学意义(P<0.05,P<0.01)。照射后第1、4天Bax mRNA表达(1.90±1.20和1.81±1.54)高于对照组(0.65±0.42),差异有统计学意义(P<0.01)。照射后各时间点Bcl-2mRNA表达与对照组比较差异无统计学意义。照射组照射后第1、4、7天Caspase 8表达(0.68±0.09、0.75±0.19和0.95±0.25)高于对照组(0.17±0.02),差异有统计学意义(P<0.01);照射组Caspase 3、Caspase 9的表达上调,其中照射后第1、4、14天高于对照组,差异有统计学意义(P<0.05或P<0.01)。结论 60 Coγ射线照射可引起豚鼠耳蜗细胞损伤,耳蜗细胞凋亡相关基因Fas、Bax mRNA及凋亡蛋白Caspase 3、Caspase 8、Caspase 9表达上调。

Objective To explore the apoptotic gene and protein expression in guinea pig cochlea after irradia- tion of Cobalt--60. Methods Sixty guinea pigs were randomly divided into control group and irradiation group. The control group （n=12） received no irradiation and was sacrificed. The right ear was the radiation ear in irradiation group （n=48）. The guinea pigs were sacrificed at 1, 4, 7 and 14 days after irradiation of 70 Gy. Cochlea sections were stained with haematoxylin and eosin. The protein and gene expressions were detected by reverse transcription polymerase chain reaction （RT--PCR） and western blot （WB）. Results The cochlear structure was normal in the cortrol group,while cytoplasmmic and nuclear condensation, nucleus and neuron loss,hydropic and loss of hair cell, edema, vacuolization and loss of stria vascularis were observed in irradiation group cochlear. The Fas, Bax, Bcl--2 genes showed a little expression in the control group. While there were a large amont Fas mRNA expression in cochlea at 1（2.13±1.23）, and 4 （2.92±2.06）days after irradiation, and Fas mRNA expression significantly increased at 4 days （P〈0. 01）. In irradiation group, Bax mRNA expression significantly increased at 1, 4 days after the irradia- tion （P〈0.01）, but there was no significant Change in Bcl--2 mRNA expression. There was a significant difference on radiation--induced group compared with control group （0.17±0.02） with WB examination （P%0.01）. The protein value of Caspase 8 of cochlea was 0. 68±0. 09 at 1 day, the value was 0.75±0.19 at 4 days and 0.95±0.25 at 7 days. Conclusion 60Co irradiotion caused damage of guinea pigs cochlea, Fas,Bax mRNA expression, Caspase 3 ,Caspase 8 and Caspase 9 protein expression increased after the irradiation.