摘要
背景:脑缺血再灌注早期,由于脾脏中有大量炎症因子浸润引发氧化应激损伤,导致脑缺血再灌注后脾细胞出现大量凋亡。目的:观察外源性过氧化氢对金属硫蛋白Ⅰ/Ⅰ敲除小鼠脾细胞活力的影响及N-乙酰-L-半胱氨酸对过氧化氢诱导的脾细胞氧化应激损伤的保护作用。方法:制备金属硫蛋白Ⅰ/Ⅰ敲除小鼠脾细胞悬液,分别用不同浓度(0.1,0.2,0.5,1,2mmol/L)过氧化氢处理2h后,MTT比色法检测细胞活力。根据MTT结果选择不同浓度过氧化氢(0.5,1mmol/L)诱导脾细胞凋亡,实验分为6组:对照组、N-乙酰-L-半胱氨酸组、0.5mmol/L过氧化氢组、1mmol/L过氧化氢组、N-乙酰-L-半胱氨酸+0.5mmol/L过氧化氢组、N-乙酰-L-半胱氨酸+1mmol/L过氧化氢组,2h后MTT比色法检测细胞活力,酶标仪法检测乳酸脱氢酶活力及紫外分光光度仪检测线粒体通透性转换孔的开放情况。结果与结论:随过氧化氢浓度增加脾细胞活力呈明显下降趋势(P<0.01),且0.2,0.5,1,2mmol/L过氧化氢组脾细胞活力下降幅度最大。与对照组相比,N-乙酰-L-半胱氨酸组脾细胞活力明显提高(P<0.01),且乳酸脱氢酶活力降低(P<0.01),线粒体通透性转换孔开放减少(P<0.01);分别于0.5,1mmol/L过氧化氢组相比,N-乙酰-L-半胱氨酸+0.5mmol/L过氧化氢组、N-乙酰-L-半胱氨酸+1mmol/L过氧化氢组脾细胞活力也明显提高(P<0.01),乳酸脱氢酶活力降低(P<0.01),线粒体通透性转换孔开放减少(P<0.01)。结果表明,金属硫蛋白Ⅰ/Ⅰ敲除小鼠随着过氧化氢浓度的升高,脾细胞活力逐渐下降,呈浓度依赖性,尤其对0.2,0.5,1,2mmol/L过氧化氢刺激最为敏感。N-乙酰-L-半胱氨酸使乳酸脱氢酶释放和线粒体通透性转换孔的开放减少,脾细胞活力增强,以此减轻过氧化氢诱导的金属硫蛋白Ⅰ/Ⅰ敲除鼠脾细胞的氧化应激损伤。
BACKGROUND： In the early stage of cerebral ischemia-reperfusion, there is a tremendous amount of inflammatory factor expression in the spleen. These inflammatory factor cause oxidative stress damage which leads to cell apoptosis in the spleen after cerebral ischemia-reperfusion. OBJECTIVE： To investigate the effect of exogenous hydrogen peroxide on spleen cell viability and the effect of N-acetyI-L-cytokine on protection of induced oxidative stress in the spleen of metallothionein Ⅰ/Ⅱ knockout mice. METHODS： Metallothionein Ⅰ/Ⅱ knockout mice spleen cell suspension was prepared and treated with various concentrations of hydrogen peroxide （0.1, 0.2, 0.5, 1, 2 mmol/L） for 2 hours, and the cell viability was detected by MTT colorimetric method. Based on the cells and mitochondria level, hydrogen peroxide induced spleen cells were divided into six group： control group, N-acetyI-L-cytokine group, 015 mmol/L hydrogen peroxide group, 1 mmol/L hydrogen peroxide group, N-acetyI-L-cytokine＋0.5 mmol/L hydrogen peroxide group, N-acetyI-L-cytokine＋l mmol/L hydrogen peroxide group. Then, the cell viability was detected by MTT colorimetric method, lactate dehydrogenate activity ware assayed by microplate reader; mitochondrial permeability transition pore was evaluated by ultraviolet spectrophotometer after 2 hours. RESULTS AND CONCLUSION： With the increase of the concentration of hydrogen peroxide, the spleen cell viability was significantly decreased （P 〈 0.01）, and 0.2, 0.5, 1,2 mmol/L hydrogen peroxide groups had the greatest reduction Compared with the control group, N-acetyI-L-cytokine could significantly increase spleen cell viability （P 〈 0.01）, decrease lactate dehydrogenate activity （P 〈 0.01） and decrease the opening state of mitochondrial permeability transition pore （P 〈 0.01）. Compared with 0.5 mmol/L hydrogen peroxide group and 1 mmol/L hydrogen peroxide group, N-acetyI-L-cytokine＋0.5 mrsol/L hydrogen peroxide group and N-acetyI-L-cytokine＋l mmol/L hydrogen peroxide group could also increase spleen cell viability （P 〈 0.01）, decrease lactate dehydrogenate activity （P 〈 0.01 ） and decrease the opening state of mitochonddal permeability transition pore （P 〈 0.01）, respectively. These findings suggest that with the increase of the concentration of hydrogen peroxide, the spleen cell viability is significantly decreased in a dose-dependent manner, especially for 0.2, 0.5, 1, 2 mmol/L hydrogen peroxide; N-acetyI-L-cysteine can relieve the oxidative stress damage induced by hydrogen peroxide in metallothioneinⅠ/Ⅱ knockout mice spleen cells, through reducing the lactate dehydrogenase release and the opening state of mitochondrial permeability transition pore, and increasing spleen cell viability.
出处
《中国组织工程研究》
CAS
CSCD
2013年第7期1243-1250,共8页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金(81273009)
天津市高等学校科技发展基金项目(20050107)
天津市应用基础及前沿技术研究计划(09JCYBJC11700)~~
关键词
组织构建
组织构建细胞学实验
金属硫蛋白
金属硫蛋白Ⅰ/Ⅰ基因敲除小鼠
脾
凋亡
N-乙酰-L-半胱氨酸
过氧化氢
氧化应激
线粒体通透性转换孔
脑缺血再灌注
乳酸脱氢酶
国家自然科学基金
tissue construction
cytology experiments in tissue construction
metallothionein
metallothionein Ⅰ/Ⅱ knockout mice
spleen
apoptosis
N-acetyI-L-cysteine
hydrogen peroxide
oxidative stress
mitochondrialpermeability transition pore
cerebral ischemia/reperfusion
lactate dehydrogenase
the National Natural ScienceFoundation of China
