甲泼尼松对兔急性脊髓损伤后MMP-212表达及神经功能影响.docVIP

甲泼尼松对兔急性脊髓损伤后MMP-212表达及神经功能影响 　　【摘要】 目的 探讨兔急性脊髓损伤后应用大剂量甲泼尼松(methylprednisolone)干预基质金属蛋白酶-2、12(MMP-2、12)表达及神经功能的影响。方法 家兔随机分为急性脊髓损伤后应用生理盐水对照组(A组)和应用甲泼尼松治疗组(B组),损伤后不同时间点取材,用HE染色观察损伤脊髓组织病理变化;用免疫组化染色检测MMP-2及Real-Time PCR检测MMP-12的表达变化;采用Tarlov评分评价神经功能。结果 HE染色镜检发现脊髓组织病理学改变B组明显轻于A组。A、B两组均发现MMP-2、12的表达,MMP-2、12表达A组B组(P 　　【关键词】 脊髓损伤;甲泼尼松;MMP-2、12 　　 　　Effect of methylprednisolone on expression of MMP-2、12 and on nerve function after acute spinal cord injury in rarebits 　　 　　ZHOU Liang,WANG Xing-sheng,LIANG Ke-jiang,et al. 　　Department of Orthopaedics and Traumatology,the 175th Hospital of PLA,Zhangzhou 363000,China 　　 　　【Abstract】 Objective To investigate the effect of methylprednisolone on expression of MMP-2、12 and nerve function after acute spinal cord injury (ASCI) in rarebits.Results Rarebits were divided at random into two groups: with pressure to cause acute spinal cord injury and use methylprednisolone a group (B group),and use normal saline group (group A). After operation the rarebits were sacrificed at 8 h,24 h,48 h, 72 h,7 d after surgery,and then the tissue sections were stained with hematoxylin and eosin(HE),immunohistochemistry and Real-Time PCR method,and the injured spinal cord tissue was evaluated. Results The histopathology changes in spinal cord tissue observed with HE staining microscopic examination in group A was worse than that in group B. Expression of MMP-2、12 were observed in two groups,and expression of MMP-2、12 in group A was higher than that in group B(P 　　1.5.2 实施荧光定量PCR 按照试剂盒说明操作,①通过逆转录将样品RNA转变为cDNA;②制备标准曲线样品:进行相对定量,需要将样品的目的基因及管家基因进行PCR 扩增,其产物进行梯度稀释用于做标准曲线;③将标准曲线样品与待测样品分别假入到Real-Time PCR反应液中,进行Real-Time PCR 扩增与检测;④检测结果进行标准曲线分析。 　　1.6 HE染色病理学观察 对假手术组及损伤组在损伤后按时间点进行脊髓取材,取材部位应在打击中央,以4%多聚甲醛固定,梯度乙醇脱水、二甲苯透明、石蜡包埋,4 μm连续切片,将做好的切片按以下顺序脱蜡:(1)二甲苯(2～3 min)→二甲苯(2～3 min)→二甲苯(2～3 min);脱过蜡的切片再按以下顺序用乙醇洗去二甲苯:无水乙醇→无水乙醇→95%乙醇→80%乙醇→蒸馏水;(2)苏木素中浸泡5 min,用蒸馏水冲洗,放入盐酸乙醇中浸泡20 s,用蒸馏水冲洗,再放入蒸馏水中浸泡15 min;(3)伊红染色1 min,依次在75%、80%、95%及无水乙醇中浸泡1 min。用中性树脂封片