大鼠脊髓损伤后TIMP-2、Collagen Ⅰ和Collagen Ⅳ的表达情况及其肢体运动功能的研究

Expression changes of tissue inhibitor of metalloproteinase and Collagens after spinal cord injury

目的观察大鼠脊髓损伤后TIMP-2、Collagen Ⅰ和Collagen Ⅳ的表达情况,探讨其对大鼠肢体运动功能的影响。方法将60只雌性SD(2个月龄)大鼠按随机数表法随机分为假手术组、术后3天组、术后5天组、术后7天组和术后9天组,每组12只。假手术组仅行椎板切除术,其余组采用NYU脊髓打击器建立脊髓损伤模型。术后1、3、5、7、9天应用BBB肢体运动功能评分对大鼠肢体运动功能进行评分;采用尼式染色观察脊髓神经元形态;采用免疫组织化学染色法检测脊髓组织TIMP-2、Collagen Ⅰ和Collagen Ⅳ表达;Western blot检测TIMP-2、Collagen Ⅰ和Collagen Ⅳ的蛋白表达量。结果 (1)脊髓损伤后,大鼠BBB肢体运动功能评分显著下降,随着时间延长,大鼠BBB肢体运动功能评分逐渐增高,与术后1天相比,术后7天和术后9天大鼠肢体BBB运动功能评分增高,差异有统计学意义(P<0.05);(2)尼氏染色观察发现,假手术组脊髓和神经元结构完整,尼氏体饱满,脊髓损伤各组脊髓和神经元结构被破坏,但术后9天大鼠脊髓和神经元的形态有所改善;(3)免疫组化检测显示假手术组TIMP-2、Collagen Ⅰ和Collagen Ⅳ表达量不高,脊髓损伤各组TIMP-2、Collagen Ⅰ和Collagen Ⅳ表达均有不同程度升高,与假手术组相比差异有统计学意义(P<0.05),术后5天TIMP-2的表达量最高且差异有统计学意义(P<0.05),术后7天Collagen Ⅰ和Collagen Ⅳ的表达量最高且差异有统计学意义(P<0.05)。结论脊髓损伤后大鼠肢体运动功能的变化可能与TIMP-2,Ⅰ、Ⅳ型胶原蛋白表达的变化有关。大鼠脊髓损伤后,脊髓组织中TIMP-2、Collagen Ⅰ和Collagen Ⅳ表达增高,TIMP-2表达量在伤后5天达到高峰,Collagen Ⅰ和Collagen Ⅳ表达量在伤后7天达到高峰,有助于促进脊髓损伤模型大鼠肢体运动功能恢复。

Objective To observe the expressions of TIMP-2, Collagen Ⅰ and Collagen Ⅳ after spinal cord injury to study if expression changes influence the limb locomotion of rats with spinal cord injury. Methods Sixty SD rats were randomly divided into the sham group(n = 12）, 3 days post-injury group(n = 12）, 5 days post-injury group(n = 12）, 7 days post-injury group(n = 12） and 9 days post-injury group(n = 12）. The sham group underwent laminectomy. In other groups, NYU was applied to establish the spinal cord injury model. BBB score was applied to estimate the limb locomotion of rats 1 day, 3 days, 5 days, 7 days and 9 days post-injury. Immunohistochemistry was used to detect the expressions of TIMP-2, Collagen Ⅰ and Collagen Ⅳ. Western blot was applied to observe the TIMP-2, Collagen Ⅰand Collagen Ⅳ protein expressions. Results(1） BBB score declined significantly when spinal injury occurred. As the time went on, BBB score increased gradually. BBB scores 7 days post-injury, 9 days post-injury were better than that of the sham group with statistical significance(P 0.05）.(2） Nissl staining showed that the structure of neurons in the sham group was complete and the structure of neurons in other groups was destroyed. The structure of neurons 9 days post-injury was refined. 3. Immunohistochemistry showed that expressions of TIMP-2, Collagen Ⅰ and Collagen Ⅳ in sham group were on the low levels, while in other groups TIMP-2, Collagen Ⅰ and Collagen Ⅳ expressions were higher than that of the sham group with statistical significance(P 0.05）. TIMP-2 expression was the highest 5 days post-injury with statistical significance(P 0.05）. Collagen Ⅰ and Collagen Ⅳ expressions were the highest 7 days post-injury with statistical significance(P 0.05）. Conclusions Changes of limb motor function in rats of spinal cord injury may be related to the changes of TIMP-2, Collagen Ⅰ and Collagen Ⅳ expressions. Expression changes are good for the limb motor function recovery.