人肝细胞缺血再灌注损伤模型的建立与验证  

Establishment and validation of human hepatocyte ischemia-reperfusion injury model

作　　者：吴昊 齐炳才 张锦锐 张赛 白易 张雅敏[4] 沈中阳[3] Wu Hao;Qi Bingcai;Zhang Jinrui;Zhang Sai;Bai Yi;Zhang Yamin;Shen Zhongyang(Tianjin First Central Hospital Clinic Institute,Tianjin Medical University,Tianjin 300070,China;Tianjin Third Central Hospital Clinic Institute,Tianjin Medical University,Tianjin 300070,China;Department of Organ transplant center,Tianjin First Central Hospital,Tianjin 300192,China;Department of Hepatobiliary Surgery,Tianjin First Central Hospital,Tianjin 300192,China)

机构地区：[1]天津医科大学一中心临床学院,300070 [2]天津医科大学三中心临床学院,300070 [3]天津市第一中心医院器官移植中心,300192 [4]天津市第一中心医院肝胆外科,300192

出　　处：《中华实验外科杂志》2022年第12期2363-2366,共4页Chinese Journal of Experimental Surgery

基　　金：天津市科技计划项目(19ZXDBSY00010);天津市自然科学基金(20JCYBJC01310)。

摘　　要：目的:建立以及验证人肝细胞L-02缺血再灌注损伤(IRI)模型。方法:分别利用液体石蜡、氯化钴(CoCl 2)以及缺氧培养箱构建人肝细胞株L-02 IRI模型,采用细胞计数试剂盒(CCK-8)法检测细胞活力,利用相关试剂盒检测细胞超氧化物歧化酶(SOD)、丙二醛(MDA)水平,利用异硫氰酸荧光素标记的膜联素V/碘化丙锭(Annexin V-FITC/PI)细胞凋亡检测试剂盒检测细胞凋亡水平,蛋白质印迹(Western blot)法测定凋亡相关蛋白天冬氨酸特异性半胱氨酸蛋白酶-3(Caspase-3)以及B细胞淋巴瘤-2相关X蛋白(bax)表达水平,通过以上方法对3种方式构建肝细胞IRI效果进行验证以及统计分析。两组间的比较采用t检验,多组间比较采用单因素方差分析。结果:利用液体石蜡、CoCl 2以及缺氧培养箱构建的肝细胞IRI模型的细胞活力均低于对照组,各组细胞450 nm吸光度分别为(0.698±0.021、0.622±0.029、0.725±0.025比1.086±0.123),差异有统计学意义(F=30.02,P<0.01),其中液体石蜡组、缺氧培养箱组的细胞活性要高于CoCl 2组(t=3.660、4.605,P均<0.05);各组SOD水平均低于对照组[(117.678±9.797)、(100.095±5.588)、(105.614±4.880)U/mg蛋白比(172.378±6.208)U/mg蛋白,F=92.56,P<0.01],其中液体石蜡组SOD水平高于CoCl 2组(t=3.118,P<0.05);各组MDA水平高于对照组[(62.684±4.584)、(70.931±1.849)、(63.472±2.316)nmol/mg蛋白比(16.163±2.106)nmol/mg蛋白,F=219.90,P<0.01],CoCl 2组MDA高于液体石蜡组和缺氧培养箱组(t=2.889、4.347,P均<0.05);IRI各组细胞凋亡率高于对照组[(26.087±4.107)%、(36.797±3.453)%、(31.945±2.859)%比(1.840±0.489)%,F=103.50,P<0.01],其中CoCl 2组凋亡率高于液体石蜡组(t=3.992,P<0.01)。各组P-Caspase-3相对表达水平较对照组升高(2.460±0.102、2.629±0.059、2.574±0.107比1.000,F=291.23,P<0.01],bax相对表达水平同样高于对照组(1.506±0.012、1.618±0.055、1.631±0.080比1.000,F=90.80,P<0.01),液体石蜡、CoCl 2和缺氧培养箱三组之�Objective To establish and validate human liver cell L-02 Ischemia reperfusion injury(IRI)model.Methods Human liver cell line L-02 IRI model was constructed using liquid paraffin,cobalt chloride(CoCl2)and hypoxia incubator,respectively.Cell viability was detected by CCK-8 method.The levels of Superoxide Dismutase(SOD)and Malondialdehyde(MDA)were detected by oxidative stress kit,and apoptosis was detected by Annexin-V fluorescein isothiocyanate(FITC)/propidiumiodide(PI)apoptosis Detection Kit.Apoptosis-related aspartic acid specific cysteine proteinase-3(Caspase-3)and B-cell lymphoma-2-associated X protein(bax)were determined by Western blotting.The above methods were used to verify and statistically analyze the effects of constructing hepatocyte IRI in three ways.T test was used for comparison between the two groups,one-way ANOVA was used for comparison between multiple groups,and P<0.05 was considered statistically significant.Results Compared with the control group,the activity of hepatocyte IRI model constructed by liquid paraffin,CoCl2 and hypoxia incubator was significantly decreased,and the absorbance at 450 nm was 0.698±0.021,0.622±0.025 and 0.725±0.025 compared with 1.086±0.123,respectively.The difference was statistically significant(F=30.02,P<0.01).The cell activity of liquid paraffin hypoxia and incubator group were higher than that of CoCl2 group(t=3.660,4.605,P<0.05).SOD levels in all groups were lower than control group[(117.678±9.797),(100.095±5.588),(105.614±4.880)U/mg protein vs.(172.378±6.208)U/mg protein,F=92.56,P<0.01].SOD level in liquid paraffin group was higher than that in CoCl2 group(t=3.118,P<0.05).MDA levels in each group were significantly higher than control group[(62.684±4.584),(70.931±1.849),(63.472±2.316)vs.(16.163±2.106)nmol/mg protein,F=219.90,P<0.01).MDA in CoCl2 group was higher than that in liquid paraffin group and hypoxia incubator group(t=2.889,4.347,P<0.05).Compared with the control group,the apoptosis rate was increased[(26.087±4.107)%,(36.797±3.453)%,(31.94

关 键 词：缺血再灌注损伤 L-02肝细胞 细胞模型 缺氧 

分 类 号：R575[医药卫生—消化系统]