创伤性脑损伤后大鼠脑组织CtyC与AIF的变化及其对神经细胞的影响

目的 探讨创伤性脑损伤后大鼠脑组织中细胞色素C(ctyC)与凋亡诱导因子(AIF)的变化及其对神经细胞的影响.方法 70只SD大鼠按随机数字表法分为假损伤组(n=10)和脑损伤组,脑损伤组按伤后1、6、24、48、72和168 h观察时间点分为6个亚组(n=10).用Feeny氏自由落体撞击法制作重型颅脑损伤模型.利用免疫荧光和共聚焦显微镜技术测定损伤后不同时间点CtyC和AIF的荧光表达强度.用免疫荧光双标记技术观察损伤后24 h时CtyC及AIF对神经细胞的影响.结果 (1)脑损伤后脑组织中CtyC变化:伤后1、6、24、48 h,皮层CtyC分别增高为1.89±0.03、2.69±0.07、2.99±0.06、3.05±0.05,海马CtyC分别增高为1.34±0.04、1.87±0.03、2.60±0.03、2.80±0.06,明显高于假损伤组,差异有统计学意义(P<0.05).72h皮层与海马CtyC分别降为1.94±0.05及1.12±0.04,亦明显高于假损伤组,差异有统计学意义(P<0.05).168 h皮层与海马CtyC与假损伤组比较差异无统计学意义(P>0.05).(2)脑损伤后脑组织中AIF变化:伤后1、6、24、48 h,皮层AIF分别增高为1.82±0.16、2.16±0.34、2.75±0.22、2.87±0.12,海马AIF分别增高为1.27±0.06、2.01±0.05、2.49±0.02、2.62±0.05,明显高于假损伤组,差异有统计学意义(P<0.05).72 h皮层与海马AIF分别降为1.35±0.09及1.32±0.05,亦明显高于假损伤组,差异有统计学意义(P<0.05).168 h皮层与海马AIF恢复正常水平,与假损伤组比较差异无统计学意义(P>0.05).(3)CtyC与AIF都能引起神经细胞凋亡.结论 创伤性脑损伤后CtyC和AIF可从线粒体释放,并起到凋亡因子的作用,诱导神经细胞发生凋亡.

Changes of CtyC and AIF in brain and effects of them on neuronal cells in rats after traumatic brain injury

Objective To study the changes of cytochrome C (CtyC) and apoptosis-induced factor (AIF) in the brain and effects of them on neuronal cells in rats after traumatic brain injury (TBI). Methods Seventy Sprague-Dawley (SD) rats were randomly divided into sham-injury group (n=10) and brain injury group. The brain injury group was divided into 6 subgroups according to 1, 6, 24, 48, 72, 168 h after traumatic brain injury (n=10). Rat models with contusion and laceration of brain were established by Feeney's impact with flee falling. Fluorescence intensity of CtyC and AIF were observed by immnnofluorescence and confocal laser scanning microscopy at different time points. The effects of CtyC and AIF on neuronal cells were observed by double-labeled immunofluorescence. Results (1)The changes of CtyC in the brain after TBI: at 1, 6, 24 and 48 h following TBL CtyC was 1.89±0.03, 2.69±0.07, 2.99±0.06 and 3.05±0.05 in the cortex and 1.34±0.04, 1.87±0.03, 2.60±0.03 and 2.80±0.06 in the hippocampus, respectively, being significantly higher than that of sham-injury group (P<0.05); at 72 h, CtyC declined to 1.94±0.05 in the cortex and to 1.12±0.04 in the hippocampus, respectively, being significantly higher than that of sham-injury group (P<0.05); at 168 h, CtyC recovered to the normal level in the cortex and hippocampus, being not significantly different between injury group and sham-injury group. (2) The changes of AIF in the brain after TBI: at 1, 6, 24 and 48 h following TBI, AIF was 1.82± 0.16, 2.16±0.34, 2.75±0.22, 2.87±0.12 in the cortex and 1.27±0.06, 2.01±0.05, 2.49±0.02, 2.62±0.05 in the hippocampus, respectively, being significantly higher than that of sham-injury group (P<0.05); at 72h, AIF declined to 1.35±0.09 in the cortex and to 1.32±0.05 in the hippocampus, respectively, being significantly higher than that of sham-in jury group (P<0.05); at 168 h, AIF recovered to the normal level in the cortex and hippocampus, being not significantly different between injury group and sham-injury group (P<0.05). (3) CtyC and AIF triggered neuronal cell apoptosis. Conclusion CtyC and AIF might release from mitochondria after TBI, and can induce neuronal cell apoptosis as apoptosis-inducing factors.