不同年龄大鼠坐骨神经损伤后睫状神经营养因子对脊髓组织中天冬氨酸特异酶切的半胱氨酸蛋白酶3表达的影响(英文)

Influence of ciliary neurotrophic factor on spinal cord caspase-3 expression in rats of different age following sciatic nerve damage

背景:天冬氨酸特异酶切的半胱氨酸蛋白酶3是一种促进细胞凋亡的半胱氨酸蛋白酶,睫状神经营养因子具有多种生物活性,能保护损伤后多种神经元特别是运动神经元,减少神经细胞损伤的发生。目的:观察睫状神经营养因子对天冬氨酸特异酶切的半胱氨酸蛋白酶3在坐骨神经损伤后不同年龄大鼠脊髓组织中的表达以及在不同年龄阶段的变化规律和调控特点。设计:随机对照动物实验。单位:解放军第三军医大学野战外科研究所大坪医院超声科、第五研究室。材料:实验于2000-04/2001-12在解放军第三军医大学野战外科研究所完成。Wistar大鼠,体质量30～100g的幼年大鼠(鼠龄20d)、200～350g的成年大鼠(鼠龄4个月)、400～800g的老年大鼠(鼠龄18～24个月)各270只,雌雄不限,共810只。方法:①实验动物分组:各年龄组大鼠随机分为正常组(n=18)、睫状神经营养因子组(n=126)和生理盐水组(n=126),睫状神经营养因子组和生理盐水组分为术后1d、3d、1周、2周、4周、8周、12周组。②动物模型制作:睫状神经营养因子组和生理盐水组切除2mm右侧坐骨神经,用硅胶管连接近、远侧神经残端制作神经再生小室,睫状神经营养因子组再生小室内注入25mg/L重组睫状神经营养因子15μL,生理盐水组再生小室内注入生理盐水15μL。③待测标本的制作及检测:分别取各组动物6只,取脊髓L3～5节段,进行天冬氨酸特异酶切的半胱氨酸蛋白酶3免疫组织化学检测、天冬氨酸特异酶切的半胱氨酸蛋白酶3活性检测和Westernblotting检测。主要观察指标:①免疫组织化学检测的各组大鼠脊髓天冬氨酸特异酶切的半胱氨酸蛋白酶3的表达。②Westernblotting检测脊髓天冬氨酸特异酶切的半胱氨酸蛋白酶3的分布与表达强度变化。③天冬氨酸特异酶切的半胱氨酸蛋白酶3活性变化。结果:810只大鼠进入结果分析。①免疫组织化学检测各组大鼠脊髓天冬氨酸特异酶切的半胱氨酸蛋白酶3的表达:损伤后各年龄组生理盐水组伤侧神经元天冬氨酸特异酶切的半胱氨酸蛋白酶3染色表达增强,伤后2周、4周明显增多、增强,8周、12周较少神经元表达阳性;睫状神经营养因子组损伤后各年龄组天冬氨酸特异酶切的半胱氨酸蛋白酶3表达增强,伤后2周、4周最为明显。②Westernblotting检测脊髓天冬氨酸特异酶切的半胱氨酸蛋白酶3的分布与表达强度变化:各年龄正常组天冬氨酸特异酶切的半胱氨酸蛋白酶3表达差异无显著性意义(P>0.05)。与同龄正常组及对侧未伤侧比较,各年龄组生理盐水组及睫状神经营养因子组损伤后1周、2周、4周天冬氨酸特异酶切的半胱氨酸蛋白酶3表达增强,差异有显著性意义(P<0.05～0.01);睫状神经营养因子组天冬氨酸特异酶切的半胱氨酸蛋白酶3幼年于损伤后1周、2周、4周,成年于2周、4周,老年于4周表达较生理盐水组减弱,差异有显著性意义(P<0.05～0.01)。各组对侧未伤侧与正常组比较,差异无显著性意义(P>0.05)。③天冬氨酸特异酶切的半胱氨酸蛋白酶3活性变化:幼年、成年、老年生理盐水组伤侧脊髓天冬氨酸特异酶切的半胱氨酸蛋白酶3活性升高,各时相点幼年大鼠天冬氨酸特异酶切的半胱氨酸蛋白酶3活性高于老年组及成年组(P<0.05～0.01),幼年、成年、老年组睫状神经营养因子组天冬氨酸特异酶切的半胱氨酸蛋白酶3活性低于生理盐水组(P<0.05～0.01)。损伤后生理盐水组及睫状神经营养因子组对侧未伤侧天冬氨酸特异酶切的半胱氨酸蛋白酶3活性,除幼年伤后12周较正常组低外(P<0.05),其余各组与正常组比较,差异无显著性意义(P>0.05)。结论:坐骨神经损伤后不同鼠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蛋白表达及活性增高,外源性睫状神经营养因子可减少脊髓神经元中天冬氨酸特异酶切的半胱氨酸蛋白酶3蛋白表达及活性,起保护脊髓神经细胞的作用,其作用在幼年组、成年组及老年组依次减弱。

BACKGROUND： Caspase-3 is a cysteine proteinase that can promote cell apoptosis. Ciliary neurotrophie factor has many kinds of biological activities, such as protecting various neurons from injury, especially motorial neurons, thereby reducing the occurrence of nerve cell injury. OBJECTIVE： To observe the Influence of ciliary neurotrophie factor on spinal cord caspase-3 expression in different ages rats following sciatic nerve injury, aiming to make further investigation about the changing regularity and modulating character of peripheral nerve damage at various ages rats. DESIGN： Randomized controlled experiment. SETTING： Ultrasound Department and the 5^th Research Institute of Daping Hospital, Field Surgery Research Institute of the 3^rd Military Medical University of Chinese PLA. MATERIALS： This experiment was carried out at the Field Surgery Re- search Institute of the 3^rd Military Medical University of Chinese PLA from April 2000 to December 2001. Altogether 810 wistar rats including infant rats with body mass of 30-100 g （birth age of 20 days）, adult rats of 200- 350 g （birth age of 4 m）, elder rats of 400-800 g （birth age of 18-24 m）, were selected with 270 rats in each age stage. Female and male does not limit. METHODS： ① Experimental animal grouping： Various age rats were randomly dividod into normal group （n=18）, ciliary neurotrophic factor group （n=126） and physiological saline group （n=126）, rats in ciliary neurotrophic factor group and physiological saline group were subdivided into 1 day, 3 days, 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks subgroups. ② Animal model preparation： In Ciliary neurotrophic factor group and physiological saline group, rats were cut off a piece of sciatic nerve of 2 mm long, both ends connected with silica tube for constructing neuranagenesis cab, in which 15μL recombined ciliary neurotrophic factor （25 mg/L） was injected in ciliary neurotrophic factor group, and 15μL physiological saline in physiological saline group. ③ Preparation and examination of specimen： Six rats were randomly selected from each group, lumbar L3-5 spinal cord were obtained for carryingonnation, caspase-3 activity examination and Western blotting examination. MAIN OUTCOME MEASURES： ① Expression of spinal cord caspase-3 with IHC assay. ② Distribution and expression intensity of spinal cordcaspase-3 by Western blotting technique. ③ Changes in caspase-3 activity. RESULTS： Altogether 810 rats completed the experiment, all data was en- tered the final result analysis. ① Expression of spinal cord caspase-3 with IHC assay： After injury, neuronal caspase-3 expression became strengthened at injured side of various age physiological saline groups, which obviously increased at posttraumatie 2 weeks and 4 weeks, but less expressed at 8 weeks and 12 weeks; while in ciliary neurotrophic factor group, posttraumatic caspase-3 expression was mostly obvious at 2 weeks and 4 weeks. ② Distribution and expression intensity of spinal cordcaspase-3 by Western blotting technique： Caspaser3 expression was not significant difference between various age subgroups in normal group （P 〉 0.05）. Comparing to the same age normal group and non traumatic group, caspase-3 expression was strengthened at 1 week, 2 weeks, 4 weeks in various age physiological saline group and ciliary neurotrophic factor group damages （P 〈 0.05-0.01）; in ciliary neurotrophic factor group, caspase-3 showed weaker expression at 1 week, 2 weeks, 4 weeks in infants; 2 weeks and 4 weeks in adults, 4 weeks in elders comparing to corresponding physiological saline group, difference was significant （P 〈 0.05-0.01）. The comparison between untraumatic side of each group and normal group showed no statistical difference （P 〉 0.05）. ③ Changes in caspase-3 activity： In child, adult and elder physiological saline groups, caspase-3 activity was increased in traumatic spinal cord, moreover caspase-3 activity was higher than elder and adult group at various age point （P 〈 0.05-0.01）; in child, adult and elder ciliary neurotrophic factor groups, caspase-3 activity is lower than physiological saline group （P 〈 0.05-0.01）. After damage, caspase-3 activity at traumatic side in physiological saline group and ciliary neurotrophic factor group was difference from normal group but without significant meaning （P 〉 0.05）, except the expression in child posttraumatic 12 weeks group was lower than normal group （P 〈 0.05）. CONCLUSION： After sciatic nerve damage, caspase-3 protein expression and activity were found to be increased in spinal cord of different age groups rats which can be reduced by extragenous ciliary neurotrophic factor, which playing protective role on spinal cord nerve cells, such protection would gradually attenuated in child group, adult group to elder group in turns.