磷酸肌酸对脑缺血再灌注损伤大鼠海马c-fos基因表达的影响

发布时间：2018-04-16 21:14

  本文选题：缺血再灌注 + 脑损伤　； 参考：《蚌埠医学院》2014年硕士论文

【摘要】：目的：建立脑缺血再灌注损伤模型，观察磷酸肌酸对脑细胞损伤的保护效果，通过对大鼠海马组织即刻基因c-fosmRNA基因表达的检测，探讨损伤时磷酸肌酸对脑的保护作用和机制。 方法：本实验选用体重在300-350g之间的27只健康成年雄性Sprague-Dawley（SD）大鼠作为研究对象，随机分组:1、假手术组(Sham组，n=9)：麻醉（10%水合氯醛，，350mg·kg-1腹腔注射)后颈部正中切口，分离出两侧颈总动脉但不夹闭，暴露30min；2、缺血再灌注组(I/R组，n=9)：麻醉后分离并夹闭双侧颈总动脉30min，松开动脉夹后再灌注24h；3、药物处理组(Drug组，n=9)：麻醉后分离并夹闭双侧颈总动脉30min时松开动脉夹，同时从尾静脉缓慢注射磷酸肌酸（PCr，80mg·100g-1)，再灌注24h。术后三组大鼠均放笼饲养24h后直接断头，在冰屑上迅速开颅取海马组织，左侧海马进行液氮速冻后放入-80℃冰箱保存以备采用逆转录聚合酶链式反应（RT-PCR)对c-fos mRNA的检测，右侧海马放入4%的甲醛保存以备免疫组织化学法进行检测。 结果：两只大鼠因呼吸衰竭死亡，其余均度过观察期。Durg组的大鼠比Sham组大鼠精神状态佳，活力强。1、HE染色法结果比较：Sham组的神经细胞排列紧密、规则，形态结构基本正常；与Sham组相比，I/R组的神经元细胞排列散乱，细胞结构破坏，核碎裂、核固缩；与I/R组相比，Drug组大部分神经元细胞结构完整，排列有序；2、免疫组化染色观察c-fos基因表达结果比较：c-fos mRNA呈棕黄色的免疫阳性细胞在Sham组海马组织仅有少量表达，染色浅；I/R组大鼠海马组织中染色较深，且表达水平高；Drug组大鼠海马组织c-fos mRNA在细胞核内有所表达，与I/R组相比，染色减轻。3、RT-PCR技术检测结果比较：电泳图中三组β-actin电泳条带宽度和亮度无明显差别，I/R组的c-fos电泳条带宽厚、明亮；Sham组的c-fos mRNA条带模糊、暗淡；Drug组条带亮度介于两者之间。凝胶成像分析系统结果显示：I/R组的c-fos/β-actin比值大于Drug组(p0.05)；Drug组的c-fos/β-actin比值大于Sham组(p0.05)。 结论：1、脑缺血再灌导致的脑损伤注可以增加神经细胞c-fosmRNA的表达；2、磷酸肌酸可以降低c-fosmRNA的表达，保护脑细胞；3、C-fosmRNA可以作为脑损伤早期诊断的一个指标。
[Abstract]:Aim: to establish a model of cerebral ischemia-reperfusion injury and to observe the protective effect of creatine phosphate on brain cell injury, and to investigate the protective effect and mechanism of creatine phosphate on brain by detecting the expression of immediate gene c-fosmRNA gene in rat hippocampal tissue.Methods: 27 healthy adult male Sprague-Dawley SD rats weighing between 300g and 350g were randomly divided into two groups: 1, sham group, 10% chloral hydrate and 350 mg kg-1 intraperitoneal injection).Separating bilateral common carotid arteries without clipping,After 30 minutes of exposure, I / R group: after anesthesia, the bilateral common carotid arteries were separated and clamped for 30 minutes, and then re-perfused for 24 hours. In the drug treatment group, when the bilateral common carotid artery 30min was separated and clipped after anesthesia, the clamp was released.At the same time, 80 mg / 100g ~ (-1) of creatine phosphate was injected slowly into the tail vein and perfused for 24 h.After 24 hours of cage feeding, the rats in the three groups were cut off directly, and the hippocampus tissue was quickly removed from the ice chip. The left hippocampus was frozen with liquid nitrogen and stored in a refrigerator at -80 鈩

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