大鼠脊髓损伤后细胞凋亡及Caspase-3、Fas的表达和意义

Neural cell apoptosis and significant expression of Caspase-3、Fas expression after spinal cord injury in rats

目的:研究脊髓损伤后神经细胞凋亡表达及Caspase-3、Fas的表达变化规律。方法:使用改良Allen法制作大鼠急性SCI模型,实验分对照组(脊髓未受打击)和损伤5个组,致大鼠脊髓(T9、T10)中度撞击损伤,损伤后6h、1d、3d、7d、15d取材,共分6组,采用HE染色、荧光Hoechst33258染色、原位末端脱氧核糖核酸转移酶介导DUTP标记法(TUNEL)对损伤脊髓组织进行标记;免疫组化方法测Caspase-3及凋亡因子Fas,半定量逆转录-聚合酶链式反应(RT-PCR)测定各时间段Caspase-3的表达变化。结果:大鼠急性损伤后TUNEL标记阳性凋亡细胞6h开始出现,多位于脊髓白质;3d达到高峰,在灰质和白质均有表达,7d后开始降低,持续15d;免疫组化检测Fas表达的阳性细胞6h开始增高,2d达到高峰,15d下降;Caspase-3mRNA6h开始增高,3d达到高峰,7d后恢复正常。免疫组化检测Caspase-3表达的阳性细胞与TUNEL标记阳性凋亡细胞出现的时限相似。结论:脊髓损伤后存在神经细胞凋亡现象发生,Caspase-3参与了脊髓损伤细胞凋亡的调节。Caspase-3的表达和Fas的变化有一定的相关性。

Objective： To study the expressions of neural cell apoptosis and the changes of Caspase-3、Fas after spinal cord injury in rats. Method： With improved Allen＇s method, rats acute SCI at the level of T9, T10 in moderate degree were established. The study animals were divided into six groups including control group and injured 5 groups. The segments of injuried spinal cord were collected for morphological studies at 6, 24, 48 hours and 7, 15 days after injury, including HE staining, Hoechst 33258 staining, and TUNEL methods. The expression of caspase-3 was detected by immunohistochemical staining and RT-PCR. Result： After injury, TUNEL-positive cells were found in the compression region after 6h and mostly located in the gray matter. TUNEL-positive cells were present both in the gray matter and white matter with a maximum presence at 3d after injury. Its decreased from 3d to 7d in the white and gray matter. Fas increased at 6h and peaked at 3d. Caspase-3 mRNA increased at 6h and peaked at 48h after trauma, it was decreased at 7d. The protein expression of caspase-3 by immunohistochemical staining was similar to the TUNEL time-course date. Conclusion： There are neuronal apoptosis after spinal cord injury, Caspase-3 mRNA and protein expressions are enhanced in combination with neuronal apoptosis. The expression of Caspase-3 have some certainty correlation with Fas＇expression.