大鼠脑缺血再灌注损伤后重组人粒细胞集落刺激因子对AKT/caspase9信号通路的影响  

RhG-CSF ameliorates neuronal apoptosis through anti-apoptotic AKT/caspase9 signal pathway following cerebral ischemia/reperfusion injury in rats

作　　者：黄姗姗[1] 马静萍[2] 

机构地区：[1]山西医科大学第一临床医学院,太原030001 [2]山西医科大学第一医院神经内科,太原030001

出　　处：《中华临床医师杂志（电子版）》2016年第6期819-825,共7页Chinese Journal of Clinicians(Electronic Edition)

摘　　要：目的探讨大鼠脑缺血再灌注损伤后重组人粒细胞集落刺激因子(rh G-CSF)的神经保护作用机制及动态观察p-AKT、caspase9、caspase3蛋白表达变化。方法 74只成年雄性SD大鼠,随机分为四组:假手术组、模型组、治疗组、观察组。采用longa线栓法制作大脑中动脉闭塞再灌注模型(MCAO/R),治疗组于再灌注时及之后每6 h腹腔注射50μg/kg rh G-CSF,模型组和假手术组同时间给予等量的生理盐水,采用longa 5分制法行神经功能缺损评分,光镜下观察缺血区病理学变化,运用免疫组化及Western blot蛋白印迹测定p-AKT、caspase9、caspase3蛋白表达,用原位末端转移酶标记(TUNEL)法检测细胞凋亡情况。结果 (1)再灌注24 h时,假手术组未见神经功能缺损,模型组神经功能缺损评分(2.25±0.62)高于治疗组(1.58±0.67,P=0.022);免疫组化及Western blot测定p-AKT、caspase9蛋白表达结果一致,与假手术组相比,模型组和治疗组p-AKT、caspase9蛋白表达均增加(P<0.01),治疗组p-AKT蛋白表达较模型组升高(P<0.01),模型组caspase9蛋白表达比治疗组升高(P<0.01);假手术组偶见凋亡细胞,模型组凋亡细胞比例[(31.80±2.91)%]较治疗组[(22.13±2.74)%]显著增多。(2)观察组:再灌注后不同时间点(2 h、6 h、24 h、48 h、72 h)p-AKT、caspase9及caspase3蛋白表达不同。结论 rh G-CSF可能对大鼠脑缺血再灌注损伤有保护作用,其机制可能通过AKT/caspase9信号通路减少神经细胞凋亡。ObjectiveTo discuss the nerve protective mechanism of recombinant human granulocyte colony-stimulating factor （rhG-CSF） and dynamic observation of p-AKT, caspase9, caspase3 protein expression changes after cerebral ischemia-reperfusion injury in rats.Methods 74 adult male SD rats were randomly divided into four groups： the sham-operated （SO） group, the middle cerebral artery occlusion/reperfusion （MCAO/R） group, rhG-CSF group and observation （OV） group. The temporary middle cerebral artery occlusion reperfusion model was made by the suture method. In rhG-CSF group, rats were injected a single dose of 50 μg/kg rhG-CSF subcutaneously at the time of reperfusion and per 6 hours after reperfusion. The SO group and MCAO/R group received the same volume of saline. At the 24-hour time point, some rats were executed after scored with longa 5-point scale. Then the morphological changes of cortical neurons were observed by HE, the expressions of p-AKT, caspase9 were detected by immune-histochemistry and Western blot analysis, the apoptosis cells were shown by TUNEL method. The others were executed at the different time of reperfusion （2 h, 6 h, 24 h, 48 h, 72 h）, and the p-AKT,nbsp;caspase9 and caspase3 proteins were detected by immune-histochemistry.Results（1）At the 24-hour time point, the rats were normal in the SO group, and neurological outcome scores of the rhG-CSF group （1.58±0.67） were lower than the MCAO/R group （2.25±0.62,P=0.022）. The results of p-AKT, caspase9 proteins’ expression were consistent between immune-histochemistry with Western blot. Compared with the SO group, the mean gray values of the expressions of p-AKT, caspase9 increased significantly in the rhG-CSF group and in the MCAO/R group （P〈0.01）. The mean gray values of the expressions of p-AKT in the rhG-CSF group were significantly increased compared with MCAO/R group （P〈0.01）, caspase9 instead. Compared with the MCAO/R group [（31.80±2.91）%], the percentage of apoptotic cells decreased significa

关 键 词：再灌注损伤 粒细胞集落刺激因子 重组 细胞凋亡 丝/苏氨酸蛋白激酶 半胱氨酸天冬氨酸蛋白酶 

分 类 号：R743[医药卫生—神经病学与精神病学]