摘要
目的:观察早期大剂量甲基强的松龙(MP)和胶质细胞源神经生长因子(GDNF)联合对大鼠急性脊髓损伤(SCI)后的治疗作用,通过对损伤区细胞凋亡及细胞内活性氧水平测定比较,探讨治疗脊髓损伤的机理及有效治疗方案。方法:将大鼠按照改良Allen's法建立脊髓急性损伤模型,分为单纯脊髓损伤组(A组),大剂量甲基强的松龙治疗组(B组),胶质细胞源神经生长因子组(C组),甲基强的松龙(MP)和胶质细胞源神经生长因子(GDNF)组(D组),损伤经治疗后1、4、7、14、21天处死每组中相应批次动物,心脏灌注后及固定后,取出损伤节段区脊髓组织,酒精梯度脱水后行石蜡包埋切片,切片采用TUNEL试剂盒进行细胞凋亡的检测(免疫组化法)以及用2',7'-二氯荧光素二脂(DCFH-DA)探针行活性氧表达的测定(流式细胞仪),采用统计学软件SPSS11.5对所得数据进行t检验分析。结果:①凋亡指数(凋亡指数(AI)=凋亡细胞核数/总细胞核数)在脊髓损伤后开始增高,至伤后第7天达最高,A组:43.89%,B组:32.65%,C组:34.16%,D组:30.00%(A＞C＞B＞D,P＜0.0001),至伤后14天仍维持在较高水平,14天之后便开始下降,至第21天仍维持在较高水平,A组:14.45%,B组:12.39%,C组:13.98%,D组:8.7%(A＞C＞B＞D,P＜0.0001),差异有统计学意义。②运动神经功能评分:采用斜板实验,对脊髓损伤后的运动神经功能评估,A组在受伤后运动神经功能恢复较差,第7天时评分为:2.25,21天时为:4.75,D组则恢复较好,第7天时为:5.50,21天时为:6.51,B、C组介于A、D组之间,结果:A＜C＜B＜D组,P＜0.017,差异有统计学意义。③取损伤区脊髓组织,制成细胞悬液,标本上流式细胞仪行活性氧表达的测定,检测结果:D组＜B组＜C组＜A组。D组细胞内活性氧水平最低,A组最高。结论早期大剂量MP联合GDNF治疗脊髓损伤,对损伤后脊髓结构和功能恢复有明显的促进作用,是治疗急性脊髓损伤的有效的治疗方案。
Objective:To investigate the effect of high dosemethylprednisolone(MP)combination with glial cell line-derivedneurotrophic factor(GDNF)on apoptosis after acute spinal cord injury(SCI) . Method: Experimental rats were divided into four groups:Allen's weight drop injury group (group A), Allen's weight drop spinalcord injury and high dose methylprednisolone group (group B), Allen'sweight drop injury group and glial cell line-derived neurotrophic factorgroup(C) , Allen's weight drop spinal cord injury and high dosemethylprednisolone combination with glial cell line-derived neurotrophicfactor group(D). After operation the rats were killed on 1,4,7,14and 21days. The apoptosis of spinal slice from the injured spinal cord wereexamined by the methods of the terminal deoxynucleotidaltransferase-mediated DUTP-biotin nick end labeling(TUNEL) reactionand the expression of active oxygen was measured by DCFH—DA kit.The positive cells were quantitatively analized by computer imageanalysis system.Result:The rate of apoptosis sequence and the strongexpression sequence of active oxygen expression were found in four categories; they were A>C>B>D(P<0.05); Conclusion:It is indicated that the high dose methylprednisolone combination with glial cell line-derived neurotrophic factor could inhibit apoptosis after SCI.
引文
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