摘要
背景:海马损伤被广泛认为与神经认知功能障碍有关,大鼠体外循环模型的建立使关于体外循环相关的海马损伤的研究得以进行。目的:旨在研究中度低温、血液稀释体外循环对大鼠海马bcl-2和bax基因表达及神经元凋亡的影响。设计:以实验动物为研究对象,完全随机分组设计,探索性研究。单位:一所大学医院麻醉科。材料:实验选用30只雄性SD大鼠,随机将大鼠分为体外循环组和假体外循环组,每组各15只。方法:体外循环组大鼠经历60min中度低温非搏动性体外循环通过使用蠕动泵和膜式氧合器,体外循环预充用20mL11晶胶液;另外15只假体外循环组大鼠除不进行体外循环外其他操作与体外循环组完全相同。每组中6只大鼠在手术后1h断头取海马,匀浆,采用逆转录聚链酶反应方法测定bcl-2和baxmRNA表达,表达强度以bcl-2或bax聚合酶链反应产物密度与看家基因β-actin比值来表示。每组中6只在手术后6h处死,采用免疫组化法检测Bcl-2和Bax蛋白表达,脱氧核糖核酸末端转移酶介导缺口末端标记法染色法测定神经元凋亡,蛋白表达强度以阳性面积占整个测量面积百分比表示。每组中其余3只在手术后6h采用电子显微镜观察海马神经元超微结构变化。主要观察指标:①两组大鼠海马bcl-2和bax基因表达、蛋白Bcl-2和Bax表达。②两组大鼠海马脱氧核糖核酸末?
BACKGROUND:Hippocampus injury is wildly believed to involve in neurocognitive dysfunction,the establishment of a rat model of cardiopulmonary bypass(CPB) allows us to investigate the mechanism of CPB related hippocampus injury.OBJECTIVE:To investigate the effects of moderate hypothermic CPB with a hemodilution on hippocampal bcl 2 and bax gene expression and neuronal apoptosis in rats. DESIGN:A randomized group division study based on the experimental animals.SETTING:Department of anesthesiology in a university hospital.MATERIALS:Thirty Sprague Dawley(SD) rats were randomly divided into two groups,CPB group and sham CPB group with 15 rats in each group.METHODS:Total 15 rats of CPB group were subjected to 60 minute moderate hypothermic nonpulsatile CPB using a peristaltic pump and a membrane oxygenator.The CPB circuit was primed with approximately 20 mL 1∶ 1 crystaloid colloid liquid,while another 15 rats of sham CPB group underwent identical anesthetic and surgical procedures(including cannulation) except CPB itself.At 1 hour post CPB,six rats in each group were decapitated,and hippocampi were removed,homogenized, and processed for apoptotic gene (bcl 2 and bax) mRNAs detection.Reverse transcriptase polymerase chain reaction(RT PCR) is used to detect expression of mRNA by comparing the PCR product of bcl 2 or bax to those of β actin housekeeping gene.Immunohistochemistry is used to detect bcl 2 and bax protein expressions and terminal deoxynucleiotidyl transferase mediated dUTP biotion nick end labeling(TUNEL) staining method was used to detect neuronal apoptosis at 6 hours post CPB (n= 6 in each group).The protein expression was quantitated as percentage of the positively stained area in the total stained.In addition,hippocampal neuronal ultrastructures were studied by electron microscopy at 6 hours post CPB(n=3 in each group).MAIN OUTCOME MEASURES:① Hippocampal bcl 2 and bax gene expressions,and Bcl 2 and Bax protein expressions.② Hippocampal neuronal apoptosis and ultrastructure changes between the two groups.RESULTS:At 1 hour post CPB,the expressions of bcl 2 and bax gene, and the ratio of bax to bcl 2 in CPB group were significantly increased compared with those of sham CPB group(P< 0.05).At 6 hours post CPB,the expressions of Bcl 2 and Bax protein in hippocampal CA1 region had significantly increased in CPB group(P< 0.05).TUNEL staining showed that hippocampal CA1 neuronal apoptosis was significantly increased in CPB group compared with sham CPB group at 6 hours post CPB(PElectron Microscopy demostrated that,at 6 hours post CPB,neuronal ultrastructures in CPB group had obvious abnormalities,many mitochondria being moderately to severely swollen with vacuolation as well as decreasing number of mitochondrial cristae,some neurons having characteristic morphological changes of earlier periods of apoptosis such as neuronal pycnosis,irregular nucleus, nuclear membrane with notchs, chromatin condensation,and nucleoli movement to the nuclear periphery,etc.CONCLUSION:Moderate hypothermic CPB with a hemodilution can induce hippocampal bax,bcl 2 gene expression and neuronal apoptosis in rats,which maybe partly explain the mechanism of post CPB neurocognitive dysfunction.
出处
《中国临床康复》
CSCD
北大核心
2005年第17期212-215,共4页
Chinese Journal of Clinical Rehabilitation
