大鼠脑挫伤后脑组织NF-κB表达与损伤时间关系

发布时间：2018-06-24 23:18

  本文选题：临床法医学 + 损伤时间推断　； 参考：《山西医科大学》2010年硕士论文

【摘要】： 目的：应用免疫组化(SABC法)、免疫印迹(Western blot)法及实时荧光定量RT-PCR技术检测大鼠脑挫伤后脑组织中NF-κB的表达规律,探讨NF-κB表达与脑损伤经过时间的关系,以期为法医学实践中早期推断脑损伤时间提供科学的实验依据。 方法：选取64只健康成年SD大鼠,雌雄不限,体重250±10g(由山西医科大学实验动物中心提供),随机分为实验组(n=56)及对照组(n=8),实验组依据损伤时间不同再分为7个亚组。对大鼠实施麻醉后,实验组采用自由落体打击法于颅骨中线旁3mm处造成脑挫伤模型,于损伤后1h、4h、12h、48h、72h、7d、14d七个时间点(每个时间点8只)取挫伤处脑组织用常规苏木素-伊红染色技术(hematoxylin and eosin,HE);对大鼠脑挫伤后14d内继发性脑损伤的病理学改变、NF-κB的分布特征进行光镜观察,同时另取100mg,置于-80℃液氮中保存备用；对照组大鼠仅钻孔处理,但不受打击,其余处理与实验组相同。应用免疫组化(SABC)法、免疫印迹(Western blot)法及实时荧光定量RT-PCR技术结合图像分析技术半定量检测大鼠脑组织NF-κB含量的变化,并与对照组作比较。将所得数据进行统计学分析。 结果：(1)紫外-可见光分光光度计检测RNA纯度、浓度及完整性较好,可以满足下一步实验的要求;(2) NF-κB分别与内参基因rpL32扩增效率一致,说明内参基因选择正确,引物设计特异性强、反应性能良好；(3)免疫组化(SABC)法、大鼠脑挫伤后在脑组织中于伤后1h即有少量NF-κB阳性细胞,48h阳性细胞明显增加并达到峰值,72h随后逐渐减少,7d后偶见阳性细胞,14d后未见阳性细胞。(4)免疫印迹(Western blot)法,大鼠脑挫伤后在脑组织中于伤后1h NF-κB蛋白增加,12-48达高峰,72h后下降,14d基本恢复到正常水平(5)实时荧光定量RT-PCR法,大鼠脑挫伤后在脑组织中于伤后1h NF-κBmRNA表达即快速增加,4h后逐渐下降,在到48h达低谷,而在72h增加并达高峰,7d和14d时表达弱。 结论：大鼠脑挫伤后脑组织中NF-κB表达随着损伤时间呈规律性变化,可望用于法医学鉴定及早期法医学损伤时间推断。免疫组化技术操作简单且易行,免疫印迹(Western blot)法、实时荧光定量RT-PCR技术检测分子水平的变化敏感而且精确,均适合法医学研究及检案的需要。
[Abstract]:Objective: to detect the expression of NF- 魏 B in brain tissue of rats after brain contusion by immunohistochemistry (SABC method), Western blotting (blot) and real-time fluorescence quantitative RT-PCR (RT-PCR), and to explore the relationship between NF- 魏 B expression and brain injury duration. In order to provide scientific experimental basis for early estimation of brain injury time in forensic practice. Methods: 64 healthy adult SD rats, weighing 250 卤10g (provided by Experimental Animal Center, Shanxi Medical University), were randomly divided into two groups: experimental group (n = 56) and control group (n = 8). The experimental group was subdivided into 7 subgroups according to the time of injury. After anaesthesia, the brain contusion model was established in the experimental group at 3mm near the midline of skull by free falling body attack method. The pathological changes of NF- 魏 B in rat brain contusion were observed under light microscope by routine hematoxylin-eosin staining technique (hematoxylin and eosinhe), and the pathological changes of NF- 魏 B were observed under light microscope at 14 days after injury, and the brain tissue of contusion was taken from 7 time points (8 rats at each time point), and the pathological changes of NF- 魏 B in rats were observed by light microscope, and the pathological changes of NF- 魏 B in brain contusion were observed by light microscope. At the same time, 100 mg was taken and stored in liquid nitrogen at -80 鈩，

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