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目的:观察神经调节素-1β(neuregulin-1β,NRG-1β)对大鼠脊髓缺血再灌注损伤后脊髓组织基质金属蛋白酶-9(MMP-9)、金属蛋白酶组织抑制剂-1(TIMP-1)表达的影响,并初步探讨其作用与机制。方法:48只SD大鼠随机分为对照组(n=16)、缺血再灌注组(模型组,n=16)、NRG-1β治疗组(n=16),对照组仅分离暴露腹主动脉,其余两组采用腹主动脉阻断法制备动物模型。治疗组在打开动脉夹后立即经尾静脉注射NRG-1β(10μg/kg),缺血再灌注模型组在打开动脉夹后立即经尾静脉注射等量的0.1mol/L的PBS缓冲液。于取材前3min根据改良Tarlov评分标准进行神经功能评分。分别于术后3h、6h、12h、24h取材(每个时间点4只),HE染色观察脊髓病理变化,免疫组化和Real-time PCR分别检测MMP-9、TIMP-1蛋白和m RNA水平的表达。结果:模型组在术后各时间点的Tarlov评分较对照组显著下降(P<0.05),治疗组在术后6h、12h、24h的Tarlov评分较模型组显著增高(P<0.05)。HE染色显示模型组和治疗组均存在脊髓组织损伤,但治疗组较模型组减轻。免疫组化结果显示,对照组未见MMP-9、TIMP-1阳性表达细胞;模型组MMP-9免疫阳性细胞数(个/高倍视野)在术后3h、6h、12h、24h分别为9.00±1.63、23.80±1.71、28.30±1.50、34.80±2.63,治疗组分别为8.50±0.58、17.80±0.96、20.80±3.50、30.00±2.16,其中6h、12h、24h治疗组与模型组相比阳性细胞数明显减少(P<0.05)。模型组TIMP-1免疫阳性细胞数(个/高倍视野)在术后3h、6h、12h、24h分别为11.80±0.96、12.30±1.50、7.80±0.96、7.80±1.50,治疗组分别为12.30±0.96、13.80±0.96、10.50±1.73、10.30±0.96,其中12h、24h治疗组与模型组相比阳性细胞数明显增多(P<0.05)。对照组MMP-9在术后3h、6h、12h、24h的m RNA表达量分别为4.93±0.21、4.95±0.24、4.96±0.25、4.98±0.23,模型组分别为5.38±0.25、6.53±0.31、6.87±0.29、7.53±0.33,治疗组分别为5.35±0.26、5.56±0.22、5.74±0.27、5.90±0.31,其中6h、12h、24h模型组与对照组比较MMP-9的m RNA表达量明显增加(P<0.05),6h、12h、24h治疗组MMP-9的m RNA表达量较模型组明显减少(P<0.05)。对照组TIMP-1在术后3h、6h、12h、24h的m RNA表达量分别为4.74±0.23、4.76±0.21、4.73±0.25、4.76±0.24,模型组分别为4.53±0.32、4.62±0.21、3.83±0.20、3.65±0.32,治疗组分别为4.55±0.26、4.65±0.27、4.28±0.22、4.25±0.24,其中12h、24h模型组与对照组比较TIMP-1的m RNA表达量明显减少(P<0.05),12h、24h治疗组TIMP-1的m RNA表达量较模型组明显增加(P<0.05)。结论 :神经调节素可在大鼠脊髓缺血再灌注损伤过程中发挥神经保护作用,该作用的实现可能与MMP-9的表达下调和TIMP-1的表达上调有关。
Objective: To observe the effect of neuregulin-1β (NRG-1β) on the expression of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP- 1) expression, and to explore its role and mechanism. Methods: Forty eight SD rats were randomly divided into control group (n = 16), ischemia reperfusion group (n = 16) and NRG-1β group (n = 16) Artery, the other two groups were prepared by abdominal aorta occlusion method animal model. In the treatment group, NRG-1β (10μg / kg) was injected through tail vein immediately after opening the artery clip. The ischemia-reperfusion model group was injected with 0.1mol / L PBS solution in the tail vein immediately after opening the artery clip. Neurological scores were assessed according to modified Tarlov score 3 minutes before drawing. The rats were sacrificed at 3h, 6h, 12h and 24h after operation respectively. The pathological changes of the spinal cord were observed by HE staining. The levels of MMP-9, TIMP-1 protein and m RNA were detected by immunohistochemistry and real-time PCR expression. Results: The Tarlov score of the model group was significantly lower than that of the control group at each time point (P <0.05). The Tarlov score of the treatment group at 6h, 12h and 24h after operation was significantly higher than that of the model group (P <0.05). HE staining showed that there was spinal cord injury in the model group and the treatment group, but the treatment group was less than the model group. Immunohistochemistry showed that MMP-9 and TIMP-1 positive cells were not found in the control group. The number of MMP-9 immunopositive cells (a / high power field) in the model group at 9h, 24h, 12h and 24h after operation were 9.00 ± 1.63,23.80 ± 1.71,28.30 ± 1.50,34.80 ± 2.63, the treatment group were 8.50 ± 0.58,17.80 ± 0.96,20.80 ± 3.50,30.00 ± 2.16, 6h, 12h, 24h treatment group compared with the model group, the number of positive cells Significantly reduced (P <0.05). The numbers of TIMP-1 immunoreactive cells in a model group were 11.80 ± 0.96, 12.30 ± 1.50, 7.80 ± 0.96 and 7.80 ± 1.50 at 3h, 6h, 12h and 24h after operation, respectively, and were 12.30 ± 0.96 , 13.80 ± 0.96,10.50 ± 1.73,10.30 ± 0.96 respectively, in which the number of positive cells in the treatment group at 12h and 24h increased significantly (P <0.05). The mRNA expression of MMP-9 in the control group at 3h, 6h, 12h and 24h after operation was 4.93 ± 0.21, 4.95 ± 0.24, 4.96 ± 0.25 and 4.98 ± 0.23, respectively, and the model group was 5.38 ± 0.25, 6.53 ± 0.31, 6.87 ± 0.29,7.53 ± 0.33, the treatment group were 5.35 ± 0.26,5.56 ± 0.22,5.74 ± 0.27,5.90 ± 0.31, 6h, 12h, 24h model group compared with the control group MMP-9 m RNA expression was significantly increased (P <0.05). Compared with the model group, the mRNA expression of MMP-9 in the 6h, 12h, 24h groups decreased significantly (P <0.05). The mRNA expressions of TIMP-1 in the control group at 3h, 6h, 12h and 24h after operation were 4.74 ± 0.23, 4.76 ± 0.21, 4.73 ± 0.25 and 4.76 ± 0.24, respectively, and those in the model group were 4.53 ± 0.32 and 4.62 ± 0.21, 3.83 ± 0.20 and 3.65 ± 0.32, respectively, and the treatment groups were 4.55 ± 0.26,4.65 ± 0.27,4.28 ± 0.22 and 4.25 ± 0.24 respectively. The mRNA expression of TIMP-1 in model group and control group at 12h and 24h were significantly decreased (P <0.05). Compared with the model group, the mRNA expression of TIMP-1 in the treated group was significantly increased at 12h and 24h (P <0.05). CONCLUSION: Neuromodulin plays a neuroprotective role in the process of spinal cord ischemia-reperfusion injury in rats. This effect may be related to the down-regulation of MMP-9 and the up-regulation of TIMP-1.