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[摘要] 目的 探讨糖尿病大鼠局灶性脑缺血再灌注模型中脑源性神经营养因子(BDNF)的表达及阻断泛素蛋白酶体途径(UPP)对它的影响。方法 将SD大鼠随机分成正常对照组(A组)、假手术组(B组)、非糖尿病组(C组)、糖尿病组(D组)和糖尿病治疗组(E组),免疫组织化学方法观察再灌注3h、6h、24h、48h及阻断UPP后脑组织中BDNF的动态变化。结果 脑缺血再灌注后,各脑梗死组BDNF表达均增加,24h达高峰,48h后下降; UPP阻断后E组脑组织中BDNF阳性细胞计数较C组、D组明显增加(P 1.3.4 MCAO模型神经功能评分 根据Longa等[8]报道的神经功能缺陷5分制标准,待大鼠麻醉苏醒后进行神经功能评分。1~3分为模型制作成功。制作成功的大鼠进行下一步实验。不具备上述体征及术后未生存至预定时间点的大鼠被淘汰。
1.4 取材及免疫组化染色
各组根据不同亚组的再灌注时间点,将大鼠深麻醉,经4%多聚甲醛PBS缓冲液灌注固定后 ,断头取脑。取视交叉后 2mm 脑组织层面进行脱水、石蜡包埋、切片4μm。免疫组化采用标准SABC法,DAB显色,按试剂盒说明进行操作,免疫染色阴性对照采用PBS液代替I抗。在额顶叶皮质,用Image-pro Plus 5.0图文分析系统测定BDNF免疫阳性细胞计数。每张切片用高倍镜(×200)随机观测皮质到白质5个不重复视野,计算BDNF免疫阳性细胞计数。
1.5 统计学方法
所有数据以χ±s表示,成组设计多样本均数比较用one-way ANOVA方差分析,多样本均数间两两比较用LSD法,检验水准为(P = 0.05),应用SPSS11.5统计软件进行分析。
2 结果
2.1 脑梗死各组不同时间点神经功能评分比较
见表1。A组、B组无神经功能受损,C、D、E三组在MACO术前Longa评分差异无统计学意义。各缺血时间点上D组神经功能评分较其他各脑梗死组高,差异有显著性(P [7] Nagasawa H,Kogure K. Correlation between cerebral blood flow and histologic changes in a new rat model of nfiddle cerebral artery oeclusion[J]. Stroke,1989,20(8):1037-1043.
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(收稿日期:2009-07-30)