【作者】 朱琳；

【导师】 胡治平；

【作者基本信息】 中南大学 ， 神经病学， 2010， 硕士

【摘要】 目的：观察大鼠脑缺血再灌注损伤以及丁苯酞干预后Smad4的表达变化,以及Smad4亚细胞定位,探讨丁苯酞的神经保护机制。方法：(1)分组：健康SD大鼠50只,随机分为正常对照组、假手术组、缺血再灌注组(I／R)、丁苯酞干预组(NI/R)；观察在脑缺血再灌注后6h、1d、3d、7d的各相应时间点Smad4的表达：(2)模型制作：缺血再灌注组大鼠予以夹闭双侧颈总动脉15min以及硝普钠控制低血压后再灌注；假手术组大鼠仅分离双侧颈总动脉不予以夹闭;丁苯酞干预组在实验前两天开始丁苯酞灌胃(80mg/kg,BID)直到处死,余步骤同缺血再灌注组；(3)HE染色观察病理变化；(4)免疫组织化学染色观察Smad4表达变化。(5)采用免疫荧光共聚焦的方法观察Smad4在额叶皮质细胞高尔基体的定位情况。全部数据采用SPSS13.0进行统计分析。结果：(1)HE染色：1.大鼠海马CA1区神经元细胞减少,神经元变性、坏死,这种改变在缺血再灌注后3天及7天最为明显。丁苯酞干预后,干预组中存活的海马CA1区神经细胞较非干预组明显增加。(2)免疫组化：Smad4在神经细胞胞核及胞质中均有阳性表达。正常对照组与假手术组大鼠海马CA3区神经元中Smad4阳性细胞表达较少,Smad4在缺血再灌注损伤后在各时间点表达显著上调,1d时明显升高,在3d和7d时达到高峰,与对照组比较,有显著性差异(P<0.05)。丁苯酞干预组Smad4表达在各相应时间点较对照组表达上调,趋势与缺血再灌注组一致,阳性率明显低于缺血再灌注组(P<0.05)。(3)高尔基体特异性标志物TGN46的免疫荧光和Smad4的免疫荧光在大鼠皮质细胞内明显发生重叠。结论：1. Smad4在缺血再灌注的脑组织中表达升高并可能发挥神经保护作用。2.丁苯酞可能通过与Smad4的协同作用来减轻脑组织损伤程度,发挥神经保护作用。3.在大鼠皮质细胞内,Smad4存在于高尔基体,表明Smad4的分泌依赖高尔基体,同时Smad4可能对高尔基体具有保护作用。更多还原

【Abstract】 Objective:To investigate the protective mechanisms of NBP after transient brain ischemia, and further study of expression changes and subcellular localization of smad4 in ratsMethods:(1) Animal Groups:Fifty healthy male SD rats, devided into four groups:Normal Group (n=5); Sham Operation Group (n=5); Ischemia Reperfusion Group (I/R) (n=20); NBP Intervention Group (NI/R) (n=20); I/R Group and NI/R Group are devided into four subgroups (each group n=5):6 hours,1 day,3 days,7 days after I/R ingury; (2) Models:15 minutes’common carotid artery occlusion(CAO) of both sides and reperfusion for 6 hours,1 day,3 days,7 days,Sodium Nitroprusside (SNP)control low blood pressure in Rats; The Sham Operation Group only give segregation but no occlusion; gavage of NBP(80mg/kg,BID) until death,;(3)HE Staining; (4)Immunohistochemistry.(5) Confocal Immuno-fluorographs of Smad4 and TGN46 were taken in rats models postocclusionResults:There were significant histological changes and elevated expression of Smad4 protein in I/R group compared with sham operation group in the hippocampus of the Rats,6h expression increased, 1d moderately increased,3d and 7d highest.P<0.05compared with the sham operation group; The histological changes in hippocampus of CA1 were significantly increased in NBP group compared with I/R group(P<0.05). Smad4 was colocalized with TGN46,a marker molecule for the Golgi apparatus.Conclusions:1. Expressions of Smad4 in hippocampus of CA3 of rats were significantly increased following cerebral ischemia-reperfusion, this suggested that the increased expressions of Smad4 could have protected the brain tissue from damage.2. NBP can relieve the brain damage induced by focal I/R in rats, which may be correlated with the inhibition of expression of Smad4.3. Smad4 was presented in the Golgi apparatus, suggesting that both continuous protein synthesis and transportation via the Golgi complex were required for Smad4 secretion and Smad4 may be essential for physiological functions involving GA更多还原