大剂量甲泼尼龙对脊髓损伤早期基因表达谱的影响  

IMPACTS OF HIGH-DOSE METHYLPREDNISOLONE ON GENE EXPRESSION PROFILING IN ACUTE SPINAL CORD INJURY

作　　者：黄颖[1] 王辉[2] 张春强[2] 王兵[2] 黄河[2] 赵学凌[2] 

机构地区：[1]昆明医学院第一附属医院血液科,昆明65003212 [2]昆明医学院第一附属医院骨科,昆明65003212

出　　处：《中国修复重建外科杂志》2011年第3期327-333,共7页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：昆明医学院第一附属医院博士启动基金资助项目(2009bs03;2009bs04)~~

摘　　要：目的甲泼尼龙(methylprednisolone,MP)是治疗急性脊髓损伤(spinal cord injury,SCI)的惟一有效药物,但其作用机制尚未明确。通过观察MP对SCI早期基因表达谱的影响,从基因表达层面探讨其机制。方法采用重复样本和重复基因芯片检测的方法进行实验。取9只健康成年日本大耳白兔,体重(3 100±140)g,随机分为对照组(A组)、模型组(B组)和药物组(C组),每组3只。A组仅进行椎板切除术,B、C组行椎板切除后采用Allen法建立急性SCI模型。C组在造模后2 h按人-兔等效剂量给予大剂量MP冲击治疗,B组注射等量生理盐水,A组不作处理。造模后8 h处死实验动物,分别获取以损伤区为中心长约8 mm的脊髓组织,采用Trizol法提取总RNA,用9张Agilent兔脊髓组织全基因4×44 K芯片进行检测。采用GeneSpring11.0统计软件,以P<0.05且倍数变化(fold change,FC)≥2筛选出差异表达基因,并对部分差异表达基因行RT-PCR验证。结果成功建立SCI动物模型并获得相应的组织标本。各组总RNA质量均能满足基因芯片检测要求。基因芯片结果显示:在SCI早期大剂量MP治疗后许多基因发生表达变化,主要涉及炎症、先天性免疫、离子通道、物质转运和转录因子等,IL-1α、IL-1β和防御素(defensin 4,NP-4)的RT-PCR检测结果与基因芯片结果相符。其中,先天性免疫相关基因如NP-3、NP-4、皮质素6、内源性抗菌肽CAP-18、抗菌肽等呈现显著的差异性表达。结论大剂量MP可能主要通过免疫调节机制发挥神经功能保护作用,其主效应细胞可能是中性粒细胞。Objective Methylprednisolone （MP） is the only active drug for acute spinal cord injury （SCI）, but the molecular mechanism is still further studied. To investigate the pathophysiology of SCI and the molecular mechanism of MP in treating SCI. Methods Nine rabbits were randomly divided into 3 groups, weighing （3 100 ± 140） g： sham operation group （group A, n=3）, model group （group B, n=3）, and drug treatment group （group C, n=3）. After laminectomy was performed in 3 groups, no treatment was given in group A, and the model of SCI was establ ished with modified Allen’s fall ing strike method in groups B and C at L4; then high-dose MP equivalent with human dose was adopted in group C at 2 hours after SCI and the normal sal ine in group B. All rabbits were sacrificed at 8 hours after SCI, and then the spinal cord tissues about 8 mm long which included the injuried site were obtained. Total RNA was isolated with Trizol one-step method to examine the gene expression profile by using Ogl io technologies with standard operating procedures and qual ity control as recently described respectively. GeneSpring11.0 analyzer software was used to filter potential candidate genes for statistical significance using Welch’s t test, and only genes with P 〈 0.05 and fold change （FC） ≥ 2 were retained for further analysis. Some differentially expressed genes were also verified by RT-PCR to ensure the rel iabil ity of microarray results. Results The SCI model was set up and the samples of spinal cord tissues were acquired successfully at 8 hours after SCI. The qual ify of total RNA from each group met the requirement for the microarray examination and data analysis. These differentially expressed genes involved inflammation, immunity, ion transportation, transcription factors, and so on. The results of genes IL-1α, IL-1β, and defensin 4 （NP-4） by RTPCR were consistent with that of gene-chips. The immuno-related genes included NP-3, NP-4, corticostatin 6, CAP-18, and antimicrobial peptide, whi

关 键 词：脊髓损伤 甲泼尼龙 先天性免疫 抗菌肽 基因芯片 

分 类 号：R651.2[医药卫生—外科学]