大鼠肢体缺血再灌注后脊髓神经元Bcl-2和Caspase-3的表达及前列地尔的干预效应

发布时间：2018-02-26 20:20

本文关键词： 缺血再灌注肢体脊髓细胞凋亡前列地尔　出处：《山西医科大学》2009年硕士论文　论文类型：学位论文

【摘要】： 研究背景:肢体缺血再灌注损伤在临床外科中较多见,研究肢体缺血再灌注损伤具有重要的临床意义。肢体缺血再灌注除造成肢体自身的损伤外,还通过神经-内分泌-免疫机制导致全身炎症反应综合征,出现远隔脏器的功能障碍和损伤,甚至危及生命。肢体缺血再灌注后周围神经发生一系列结构和功能的异常变化,对中枢神经系统产生重要影响,导致脊髓神经元的损伤。目的:观察大鼠肢体缺血再灌注脊髓神经元损伤过程中细胞凋亡、Bcl-2和Caspase-3的变化及前列地尔对此过程的干预效应。方法:成年健康SD大鼠72只,随机分为3组:①假手术组(sham组,n=24),只进行麻醉并解剖股血管和股神经,不做其他处理。②肢体缺血3h再灌注组(IR组,n=24)。③肢体缺血3h再灌注+前列地尔组( IR+PGE1组,n=24),缺血前10min于大鼠尾静脉注射前列地尔5μg·ml-1·kg-1。每组按照再灌注时间(4h、12h、24h、48h)又分为4个时相。实验结束,采用心脏灌注处死大鼠,取脊髓腰段(L1~5),采用HE染色观察脊髓病理学改变;免疫组织化学法(SP法)观察Bcl-2和Caspase-3蛋白质表达情况; TUNEL法观察细胞凋亡的发生情况。结果:①脊髓组织中Bcl-2和Caspase-3的表达:免疫组织化学切片以阳性细胞率为指标进行定量分析。sham组Bcl-2和Caspase-3表达均较低。Bcl-2表达阳性细胞率定量分析:其它组与sham组比较,Bcl-2阳性细胞率明显增加,差异有统计学意义(P0.01);与IR组比较,IR+PGE1组阳性细胞率增加,差异有统计学意义(P0.05)。IR、IR+PGE1组Bcl-2表达均于12h达高峰,之后下降。Caspase-3表达阳性细胞率定量分析:其它组与sham组比较Caspase-3阳性细胞率均明显增加,差异有统计学意义(P0.01);与IR组比较,IR+PGE1组阳性细胞率降低,差异有统计学意义(P0.05)。IR、IR+PGE1组Caspase-3表达于24h达高峰,之后下降。②TUNEL法检测凋亡细胞:sham组较少见凋亡细胞。其余各组凋亡指数(AI)与sham组比较差异均具有统计学意义(P0.01);与IR组比较,IR+PGE1组各时相AI值明显减少,差别有显著性,具有统计学意义(P0.05)。相关性分析Caspase-3阳性细胞率与AI值成正相关。结论:①大鼠肢体缺血再灌注后可导致脊髓神经元细胞凋亡的发生,引起凋亡相关蛋白Bcl-2和Caspase-3的表达增加。②缺血前预防性给予前列地尔对大鼠肢体缺血再灌注引起的脊髓神经元细胞凋亡有一定的抑制作用,可能通过上调Bcl-2的表达,下调Caspase-3的表达,从而减轻脊髓神经元的损伤,起到保护作用。
[Abstract]:Background: limb ischemia-reperfusion injury is more common in clinical surgery. It has important clinical significance to study limb ischemia-reperfusion injury. It also leads to systemic inflammatory response syndrome through neuroendocrine and immunological mechanism, resulting in dysfunction and injury of distant organs, and even endangering life. A series of abnormal changes in structure and function of peripheral nerves occur after limb ischemia and reperfusion. Has an important effect on the central nervous system, resulting in spinal cord neuron injury. Aim: to observe the changes of apoptosis Bcl-2 and Caspase-3 during spinal cord neuron injury after limb ischemia reperfusion in rats and the effect of alprostadil on this process. Methods: Seventy-two adult healthy SD rats were randomly divided into 3 groups: sham group (n = 24), sham group (n = 3), anesthetized and dissected femoral vessels and femoral nerves. 2. No other treatment. 2 limb ischemia / reperfusion group / IR group / IR / PGE1 group after 3 h / 3 limb ischemia / reperfusion. Ten minutes before ischemia, alprostadil 5 渭 g 路ml-1 路kg ~ (-1) was injected into the tail vein of rats. Each group was divided into 4 groups according to the reperfusion time of 4 h, 12 h, 24 h and 48 h). At the end of the experiment, The rats were killed by cardiac perfusion. The pathological changes of spinal cord were observed by HE staining. The expression of Bcl-2 and Caspase-3 protein was observed by immunohistochemical method (SP method) and apoptosis was observed by TUNEL method. Results the expression of Bcl-2 and Caspase-3 in the spinal cord tissue of 1: 1: immunohistochemical section was used as an index to quantitatively analyze the positive cell rate. The expression of Bcl-2 and Caspase-3 in Sham group was lower than that in the sham group. The positive cell rate of Bcl-2 expression in the other groups was compared with that in the sham group. The rate of Bcl-2 positive cells increased significantly. Compared with IR group, the positive cell rate of IR PGE1 group increased, and the Bcl-2 expression of IR PGE1 group reached its peak at 12 h. After that, the positive cell rate of Caspase-3 decreased: compared with sham group, the positive cell rate of Caspase-3 increased significantly in other groups, the difference was statistically significant (P 0.01), and the positive cell rate in IR PGE1 group was lower than that in IR group. The expression of Caspase-3 in IR PGE1 group reached its peak at 24 h. The apoptosis index of other groups was significantly higher than that of sham group (P 0.01), and the AI value of IR PGE1 group was significantly lower than that of IR PGE1 group (P < 0.05). The correlation analysis showed that the positive cell rate of Caspase-3 was positively correlated with AI value. Conclusion the apoptosis of spinal cord neurons can be induced by ischemia and reperfusion of rat limbs. The expression of apoptosis-related proteins (Bcl-2 and Caspase-3) was increased by prophylaxis of prostadil before ischemia. 2 the apoptosis of spinal cord neurons induced by ischemia and reperfusion of rat limbs was inhibited to some extent, possibly by upregulating the expression of Bcl-2. The expression of Caspase-3 was down-regulated, which alleviated the injury of spinal cord neurons and played a protective role.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363

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