不同年龄大鼠脊髓组织中天冬氨酸特异酶切半胱氨酸蛋白酶3的活性及坐骨神经损伤后的表达变化

Activity of caspase 3 in spinal cord of rats of different age and changes of expression after injury of sciatic nerve

目的:观察不同年龄大鼠脊髓组织中天冬氨酸特异酶切的半胱氨酸蛋白酶3表达情况及其在坐骨神经损伤后的表达变化,并分析其与脊髓细胞凋亡的关系。方法:实验于2004-03/09在解放军第三军医大学大坪医院野战外科研究所完成。选择Wistar大鼠324只,成年、幼年、老年大鼠各108只。不同年龄组动物均随机分为对照组和手术组,对照组12只为非手术组;手术组共96只,分为术后1d,3d,1周、2周、3周、4周、5周、6周8个时间组,每组12只。各手术组大鼠均在戊巴比妥钠麻醉后,切除股方肌下缘约6mm的坐骨神经。大鼠脊髓细胞凋亡情况采用原位缺口末端标记法及流式细胞仪AnnexinⅤ/PI双标检测法,原位缺口末端标记阳性细胞及AnnexinⅤ阳性细胞即凋亡细胞;大鼠脊髓组织中天冬氨酸特异酶切的半胱氨酸蛋白酶3表达情况应用免疫组化法检测;大鼠脊髓组织中天冬氨酸特异酶切的半胱氨酸蛋白酶3活性应用ActivityAssay试剂盒检测。结果:所有大鼠全部进入结果分析,无脱失。①脊髓细胞凋亡情况的原位缺口末端标记及流式细胞仪AnnexinⅤ/PI双标检测结果:正常成年及老年大鼠未见明显标记的阳性细胞,正常幼年大鼠可见个别阳性标记细胞,坐骨神经损伤诱导脊髓神经细胞凋亡的高发时间为伤后2～4周,老年大鼠细胞凋亡持续时间较长。各手术组正常侧与对照组相比差异无显著性意义(P>0.05)。不同年龄大鼠AnnexinⅤ阳性细胞出现时间均早于原位缺口末端标记阳性细胞。②天冬氨酸特异酶切的半胱氨酸蛋白酶3活性及表达情况比较:幼年正常组天冬氨酸特异酶切的半胱氨酸蛋白酶3表达及天冬氨酸特异酶切的半胱氨酸蛋白酶3活性均高于成年及老年大鼠(P<0.01),损伤后各年龄组均有明显变化,幼年正常组损伤后1d天冬氨酸特异酶切的半胱氨酸蛋白酶3开始升高,升高时间早于成年及老年组。各手术组正常侧与对照组相比差异无显著性意义(P>0.05)。结论:脊髓细胞凋亡情况与天冬氨酸特异酶切的半胱氨酸蛋白酶3活性测定结果有较好的相同改变趋势,提示天冬氨酸特异酶切的半胱氨酸蛋白酶3的活化是细胞凋亡的早期生物化学指标。不同年龄大鼠正常时脊髓组织中天冬氨酸特异酶切的半胱氨酸蛋白酶3表达以其在坐骨神经损伤后的表达变化存在差异,说明针对不同年龄段发生的周围神经损伤,其促进神经再生调控时期不同。

AIM：To observe the expression of caspase 3 in spinal cord of rats of different age as well as its changes after injury of sciatic nerve and analyze its relationship with apoptosis in spinal cord.METHODS： The experiment was conducted in the Institute of Battle Surgery, Daping Hospital of Third Military Medieal University from Mareh to September 2004. 342 Wistar rats were selected including 108 adult rats, 108 young rats and 108 senile rats. Rats of different age were randomly divided into control group （n=12） and operation group （n=96）： Operation group was subdivided into 8 time groups aecnrding to： 1 day, 3 days, 1 week, 2 weeks, 3 weeks. 4 weeks, 5 weeks, 6 weeks and 8 weeks after operation, with 12 rats in each subgroup. Rats in all operation groups received anesthesia with soluble pentobarbitone, then 6ram sciatic nerve of inferior boarder on quadratus femoris was cut off. Condition of cell apoptosis in spinal cord was detected with terminal deoxynucleotidyl trausferease-mediated nick end labelling （TUNEL） and flow cytomehry （Annexin V/PI）, expression of caspase 3 was assayed with immunohistochemical method; Activity of caspase 3 in spinal cord of rats was assayed with activity assay kit. RESULTS： All rats were involved iu the analysis of results, no subject withdrew from the experiment. ①Detection of apnptosis in spinal cord with TUNEL and flow eytomehry Annexin V/PI： No otwious tagged positive cell was found in adult rats and senile rats. but iudividual ones were found in normal young rats, high incidence time of spinal nerves apoptosis induced by injury, of sciatic nerve was 2-4 weeks after injury, which was much longer in apoptosis of senile rats. There were no significant differences in normal side between all operation groups and cnntrnl group （P 〈 0.05）. The emergence time of Annexin V positive cells in rats of different age were earlier than positive cells of TUNEL.②Comparison of expression of caspase 3 and its activity： They were higher ira the normal young group than those in the adult group and senile group （P 〈 0.01）, no significant change was found in rats of different age from the injury group, level of caspa.se 3 in the young group one day after injury began to increase, which was earlier than that in the senile group. There were no significant differences between each operation group and control group （P 〈 0.05）. CONCLUSION： Status of apoptosis in spinal cord is positively related with the aetivity of caspase 3. which indicates that the activation of caspase 3 is a biochemical index in early period. There are differences in changes of expression between caspase 3 and that ＇after injury of sciatic nerve, which means that aiming at peripheral nerve injury in patients of different age, there are different modulation periods for improving regeneration of nerve.