摘要
目的探讨急性脊髓损伤后热休克蛋白70(heat shock prtein 70,HSP70)表达的变化规律;通过诱导急性脊髓损伤后早期HSP70的大量表达,探讨其诱导剂替普瑞酮(Geranylgeranylacetone,GGA)修复脊髓损伤的疗效,综合、客观评价脊髓功能恢复程度,从而寻求一种简单有效、可应用于临床的新方法,为临床治疗脊髓损伤提供实验学依据。
方法1、急性脊髓损伤后HSP70表达的变化。健康成年雌性Wistar大鼠50只,随机分为Ⅰ假手术组:单纯显露T10节段脊髓,不予打击损伤;Ⅱ损伤组:显露T10节段脊髓,使用MASCIS Impactor ModelⅡ脊髓损伤打击器打击脊髓(10g×25mm),建立急性脊髓损伤模型。分别于术后6、12、24、48、72小时,每组随机选取5只动物,取材、固定、切片,行HSP70免疫组织化学染色,观察HSP70表达变化情况。
2、GGA对急性脊髓损伤后HSP70表达、神经细胞凋亡及神经功能恢复的影响。健康成年雌性Wistar大鼠75只,随机分为3组,各25只。Ⅰ对照组:损伤前24小时、损伤后半小时以及损伤后的每24小时至伤后1周分别经胃管给予等量生理盐水;Ⅱ损伤后给药组:损伤前24小时经胃管给予等量生理盐水,损伤后半小时以及损伤后的每24小时至伤后1周均经胃管给予GGA(溶于生理盐水)600mg/kg;Ⅲ损伤前、后联合给药组:各时间点分别经胃管给予GGA(溶于生理盐水)600mg/kg。所有动物均手术显露T10节段脊髓,使用MASCISImpactor ModelⅡ脊髓损伤打击器打击脊髓(10g×25mm),建立急性脊髓损伤模型。免疫组织化学染色测定HSP70的表达情况,TUNEL法检测神经细胞凋亡情况,HE染色光学显微镜观察脊髓组织形态学改变,BBB评分法进行行为学功能评价。
结果1、急性脊髓损伤后HSP70表达水平迅速上升,至伤后24h达到高峰,之后转为下降,至伤后72h脊髓组织内已少见HSP70阳性细胞;而假手术组仅有低水平、短时间的HSP70表达。
2、各组术后12、24、48、72小时HSP70表达水平差异有统计学意义(ANOVA,P＜0.05),Ⅲ组、Ⅱ组均大于Ⅰ组。各组术后12、24、48、72小时损伤部位细胞凋亡水平差异有统计学意义(ANOVA,P＜0.05),Ⅲ组、Ⅱ组均小于Ⅰ组。所有大鼠在脊髓损伤后出现双下肢截瘫,BBB评分组间比较,损伤后7天至伤后28天,各组间差异有统计学意义(ANOVA,P＜0.05),Ⅲ组、Ⅱ组评分大于Ⅰ组。
结论急性脊髓损伤后HSP70表达自发性上调,但其上调的水平有限,持续时间较短。GGA可以增加急性脊髓损伤后HSP70的表达水平,减少急性脊髓损伤后神经细胞的凋亡,促进急性脊髓损伤后神经功能的恢复。
Objective To observe the expression of heat shock prtein 70(HSP70) after acute spinal cord injury(ASCI);and also observe the effect of a HSP70 inducer -Geranylgeranylacetone(GGA) on repairing of ASCI through up-regulation HSP70 expression in the earlier period after ASCI.Evaluation the functional recovery objectively in many ways to find an new effective way,which can be used in clinical practice easily,for providing an experimental evidence to clinical treatment of ASCI.
Methods 1.The expression of HSP70 after acute spinal cord injury.50 healthy female Wistar rats were separated into two groups randomly(25 rats in each group ):Ⅰcontrol group,rats were exposed T10 spinal cord,but not injured by the impactor;Ⅱstudy group,rats were exposed T10 spinal cord,which were then injured by the impactor(10g×25mm).Rats were sacrificed 6,12,24,48,72 hours after the operation separativly,and the injured spinal cord were taken out for fix,slice,and Immunohistochemistry staining was used to determin HSP70 expression.
2.The effect of GGA on expression of HSP70,apoptosis of neural cells and functional recovery after ASCI.75 healthy female Wistar rats were separated into three groups randomly(25 rats in each group):Ⅰcontrol group,Ⅱpost injury GGA administration group andⅢahead and post injury combined GGA administration group.Rats in groupⅠwere administrated equal saline by oral tube 24h ahead of injury,0.5h after injury and every 24h after injury until 1 week later;rats in groupⅡwere administrated equal saline 24h ahead of injury and GGA(600 mg/kg) 0.5h after injury and every 24h after injury until 1 week later;rats in groupⅢwere administrated GGA(600 mg/kg) on all the time points.Immunohistochemistry staining was used to determin HSP70 expression,TUNEL staining was used to detect the apoptosis level,HE staining and optics microscope was used to observe the histomorphology change of the spinal cord and the BBB(Basso,Beattie and Bresnahan) scales were used to evaluate hindlimb neurological function.
Results 1.The expression level of HSP70 were upgraded rapidly after ASCI,the peak value were presented at 24h after injury,then the value decreased,and to be rare 72h after injury.In the control group,the expression of HSP70 was in a lower lever, and for a shorter period.
2.The expression level of HSP70 among groups were statistically significance 12、24、48、72h after injury(ANOVA,P＜0.05),groupⅢandⅡexceed groupⅠ; the apoptosis level among groups were statistically significance 12、24、48、72h after injury(ANOVA,P＜0.05) too,groupⅢandⅡwere lower than groupⅠ.All the rats were inferior paraplegia after injury.The BBB score among groups were statistically significance since 7 days after injury(ANOVA,P＜0.05),groupⅢandⅡexceed groupⅠ.
Conclusions The expression level of HSP70 was upgraded spontaneously.But the degree of it was limited,and the period of upregulation is deficient.GGA can increase the level of HSP70 expression,reduce the apoptosis of neural cells and promote the functional recovery after acute spinal cord injury.
引文
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