缺血后处理对高血脂大鼠缺血再灌注心肌Bcl2及Bax蛋白表达的影响

1、缺血后处理对高血脂大鼠缺血再灌注心肌Bcl2及Bax蛋白表达的影响         10-09-08 11:51:00     编辑：studa20                      作者：霍玉娥 尹博英 刘胜辉【摘要】  目的 观察缺血后处

2、理对高血脂大鼠缺血再灌注心肌Bcl2及Bax蛋白表达的影响。方法 选择高血脂SD大鼠36只，随机分为3组：假手术组、缺血再灌注组、缺血后处理组，每组12只。制备大鼠心肌缺血再灌注模型。缺血再灌注组:收紧结扎线缺血40 min，放松结扎线再灌注240 min;缺血后处理组:缺血40 min后，再灌注10 s,缺血10 s,连续3个循环，然后再灌注240 min;假手术组:开胸后穿线做套环，但不收紧结扎线。再灌注结束后自右颈动脉采血测定血清肌酸激酶(CK)活性,用TUNEL法检测再灌注心肌凋亡程度，采用免疫组织化学方法检测Bcl2及Bax蛋白的表达情况。结果 血清中CK活性的测定：再灌注结束后缺血

3、后处理组和缺血再灌注组CK活性明显高于假手术组分别为(789.68±67.34)，(932.86±84.17)，(252.48±19.78)U/L，P<0.05，缺血后处理组明显低于缺血再灌注组(P<0.05)。心肌凋亡细胞计数：再灌注结束后假手术组未见明显细胞凋亡(<5%)，缺血后处理组心肌细胞凋亡率明显低于缺血再灌注组分别为(11.9±2.7)%，(21.2±3.5)%，P<0.05。与缺血再灌注组相比，缺血后处理组Bcl2蛋白表达增加(P<0.05)，Bax蛋白表达减低(P<0.05)。结论 缺血后处理

4、可以增加高血脂大鼠缺血再灌注心肌Bcl2蛋白表达、降低Bax蛋白表达，进而抑制凋亡。 【关键词】  再灌注损伤;缺血后处理;凋亡【Abstract】 Objective To study the effect of ischemic postconditioning on expression of Bcl2 and Bax protein in hyperlipemia rats with myocardial ischemia reperfusion.Methods 36 hyperlipemia SD rats were selected and randomly divide

5、d into 3 groups：sham operation，ischemic reperfusion and ischemic postconditioning groups(n=12)，and rats were made into MIR models.Rats in the ischemic reperfusion group were ligated for 40 minutes ischemia，and then reperfused for 240 min.Rats in ischemic postconditioning group were treated for 40 mi

6、nutes ischemia，reperfused for 10 s and 10 s ischemia for 3 cycles，and then reperfused reperfussed for 240 min.Rats in sham operation group were opened the chest to braid for ringer without deligation.The presence of apoptotic myocytes was detected by(TUNEL). Immunohistochemistry was used to detect t

7、he Bcl2 and Bax protein.Meanwhile，the serum creatine kinase(CK) activity was measured.Results Determination of serum CK activity：it was obviously higher in the ischemic postconditioning group and ischemic reperfusion group after reperfusion than that of the sham operation group(789.68±67.34)，(9

8、32.86±84.17)，(252.48±19.78)U/L respectively, P<0.05， and it was significantly lower in the ischemic postconditioning group than that in the ischemic reperfusion group(P<0.05). Number of apoptotic myocardial cells：there was no significant apoptosis found after the reperfusion in the s

9、hamoperation group(<5%)，and the apoptotic rate in the ischemic postconditioning group was remarkably lower than that in the ischemic reperfusion group (11.9±2.7)%，(21.2±3.5)% respectively， P<0.05. The expression of Bcl2 protein was higher in the ischemic postconditioning group than t

10、hat in the ischemic reperfusion group (P<0.05).The expression of Bax protein was lower in the ischemic postconditioning group than that in the ischemic reperfusion group (P<0.05).Conclusions Ischemic postconditioning may increase the expression of Bcl2 protein and decrease the expression of Ba

11、x protein which may be related with the decreased myocardial apoptosis.【Key words】 Reperfusion injury;Postcondioning;Apoptosis 研究表明缺血后处理可以缩小心肌梗死面积，减少再灌注心律失常，具有心肌保护作用13。急性心肌梗死患者常合并高血脂，缺血后处理对高血脂大鼠的作用尚未见研究报道,本文旨在探讨在体情况下缺血后处理对高血脂大鼠缺血/再灌注心肌Bcl2及Bax蛋白表达的影响。1 材料与方法1.1 实验动物 选择健康清洁级SD大鼠36只，雄性，喂养高血脂造模饲料(2%胆固醇

12、、10%猪油、0.2%丙基硫氧嘧啶、87.8%基础饲料) 4 w,体重(250±30)g，甘油三酯(TG)为(2.84±0.68)mmol/L，总胆固醇(TC)为(3.71±0.23)mmol/L;较正常大鼠(n=16)的TG(1.23±0.32)mmol/L和TC(1.96±0.39)mmol/L明显升高(P<0.05)。1.2 动物模型制备及分组1.2.1 动物模型制备 实验大鼠以30 g/L戊巴比妥钠(45 mg/kg)腹腔注射麻醉，仰卧固定于鼠台，行气管切开,用呼吸机进行机械通气,通气频率60次/min，潮气量2030 ml/kg

13、，取左胸第3肋间进胸，暴露心脏,于左心耳根部下方1 mm处进针，50 丝线(结扎线) 穿过心肌表层在肺动脉圆锥处稍下方穿线，线两端各穿一小缝合垫片后再并线一起经内径约2 mm的聚乙烯管中穿出，用血管钳推压小管压迫左冠状动脉前降支造成缺血，放松即可恢复血流形成再灌注，收紧结扎线心电图出现ST段抬高，放松结扎线ST段下降1/2以上为模型成功。1.2.2 动物分组 随机分为3组，每组12只：假手术组：开胸后穿线做套环，但不收紧结扎线;缺血再灌注组：收紧结扎线缺血40 min，放松结扎线再灌注240 min;缺血后处理组：缺血40 min后，再灌注10 s,缺血10 s,连续3个循环，然后再灌注240

14、 min。1.3 检测指标1.3.1 肌酸激酶(CK)的检测 再灌注结束后自右颈动脉分别采血2 ml,3 000 r/ min离心10 min，-70冰箱保存待测。采用7180型全自动生化分析仪(日本日立公司)检测CK的含量，参照试剂盒(北京中山生物技术有限公司)说明书操作。1.3.2 凋亡细胞原位标记与凋亡指数计算 再灌注结束后，将缺血区心肌组织剪下，40 g/L多聚甲醛固定24 h,常规石蜡包埋,用TUNEL技术标记凋亡的细胞核，在免疫荧光显微镜下观察心肌细胞凋亡,紫外激发光下，所有细胞核发出蓝色荧光，蓝色激发光下，凋亡心肌细胞核发出绿色荧光，每个标本选取5张切片，每张切片选取5个视野(×400)，计算细胞总数和凋亡细胞数，以平均阳性细胞数所