体外L02肝细胞氧化损伤模型的构建

赵辉; 孟炜; 易述红; 陈规划

doi:10.3969/j.issn.1674-7445.2014.04.010

体外L02肝细胞氧化损伤模型的构建

doi: 10.3969/j.issn.1674-7445.2014.04.010

510630 广州, 中山大学附属第三医院肝胆外科

基金项目:

广东省自然科学基金面上项目 S2012010009333

广州市科技计划项目重大民生公关专项 2001Y1-00033-2

广州市科技计划项目 2010J-E121、2010U1-E00551-1

详细信息

通讯作者:
易述红,Email:yishuhong@163.com

中图分类号: R617
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- 被引次数: 0
出版历程
- 收稿日期: 2014-04-01
- 网络出版日期: 2021-01-19
- 刊出日期: 2014-07-15

Establishing a model of oxidative damage of L02 hepatocytes in vitro

Department of Hepatobiliary Surgery, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China

摘要

摘要: 目的探讨L02肝细胞氧化损伤模型的构建方法。方法将L02肝细胞培养后分为损伤3 h组、6 h组和12 h组,每组再根据加入不同浓度的过氧化氢(H₂O₂)分为100、200、300、500、750、1 000 μmol/L 6个亚组,对照组不加入H₂O₂,分别在培养箱中继续孵育3 h、6 h或12 h后进行细胞计数试剂盒(CCK)-8检测。另1组L02肝细胞培养后分为损伤3 h组、6 h组和12 h组,损伤3 h组再根据加入H₂O₂浓度不同分为100、200、300、500、750 μmol/L 5个亚组,损伤6 h组分为100、200、300、500 μmol/L 4个亚组,损伤12 h组分为100、200、300 μmol/L 3个亚组,对照组不加入H₂O₂,分别在培养箱中继续孵育3 h、6 h或12 h后进行Annexin V-异硫氰酸荧光素(FITC)/碘化丙啶(PI)双染流式细胞检测。损伤模型的建立及鉴定:L02肝细胞培养后损伤组加入200 μmol/L H₂O₂,对照组不加入H₂O₂。在培养箱中继续孵育6 h后检测细胞线粒体膜电位、丙二醛(MDA)、活性氧自由基(ROS)、超氧化物歧化酶(SOD)、丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)等指标。结果损伤3 h组中,200、300、500、750、1 000 μmol/L等各亚组的细胞存活率与对照组比较,差异有统计学意义(均为P<0.01);损伤6 h组中,各亚组细胞存活率与对照组比较差异均有统计学意义(均为P<0.01);损伤12 h组中,各亚组存活率与对照组比较差异均有统计学意义(均为P<0.01)。3 h H₂O₂浓度与细胞存活率的相关系数为-0.993,6 h组为-0.955,12 h组为-0.819。Annexin V-FITC/PI双染检测不同浓度、不同作用时间的H₂O₂损伤情况下细胞凋亡/坏死率:损伤3、6、12 h组中,损伤各亚组与对照组比较,差异均有统计学意义(均为P<0.01)。3 h组H₂O₂浓度与细胞凋亡或坏死率的相关系数为0.971,6 h组为0.992,12 h组为0.986。与对照组比较,200 μmol/L H₂O₂作用6 h处理L02肝细胞损伤组的线粒体膜电位、MDA、ROS、SOD、ALT、AST差异均有统计学意义(均为P<0.01)。结论 200 μmol/L H₂O₂作用6 h处理L02肝细胞是体外模拟缺血-再灌注或氧化损伤的良好模型。
- 肝细胞 /
- 氧化损伤 /
- 缺血-再灌注损伤 /
- 凋亡
Abstract: Objective To investigate the method of establishing a model of oxidative damage of L02 hepatocytes. Methods L02 hepatocytes were cultured divided into 3 h damage group, 6 h group and 12 h group. Each group was divided into 6 subgroups according to different concentrations of hydrogen peroxide (H₂O₂) added: 100, 200, 300, 500, 750, 1 000 μmol/L. The control group was treated without H₂O₂. All groups were tested with cell counting kit (CCK)-8 after incubating for 3 h, 6 h or 12 h respectively. The other L02 hepatocytes were cultured and divided into 3 h damage group, 6 h group and 12 h group. The 3 h group was divided into 5 subgroups according to different concentrations of H₂O₂ added: 100, 200, 300, 500 and 750 μmol/L, the 6 h group was divided into 4 subgroups according to different concentrations of H₂O₂ added: 100, 200, 300 and 500 μmol/L, and the 12 h group was divided into 3 subgroups according to different concentrations of H₂O₂ added: 100, 200 and 300 μmol/L. The control group was treated without H₂O₂. All groups were tested by flow cytometry with Annexin V-fluorescein isothiocyanate(FITC)/propidium iodide(PI)double staining after incubating for 3 h, 6 h or 12 h respectively. The model of oxidative damage of L02 hepatocytes was established and identified. The L02 hepatocytes in damage group were treated with 200 μmol/L H₂O₂ after culture and L02 hepatocytes in control group were treated without H₂O₂ after culture. Indicators such as mitochondrial membrane potential, malondialdehyde (MDA), oxygen free radical (ROS), superoxide dismutase (SOD), alanine aminotransferase (ALT), aspartate aminotransferase (AST) were tested after L02 hepatocytes were incubated for 6 h. Results Compared with control group, significant differences were observed in cell survival rates of 200, 300, 500, 750 and 1 000 μmol/L subgroup in 3 h damage group (all in P<0.01). So were in 6 h damage group and 12 h damage group (all in P<0.01). The correlation coefficient between H₂O₂ concentration and cell viability was -0.993 in 3 h group, -0.955 in 6 h group, and -0.819 in 12 h group. Flow cytometry with Annexin V-FITC/PI double staining was used to detect the apoptosis/necrosis of L02 hepatocytes when treated with different concentrations and actuation duration of H₂O₂. In 3 h, 6 h and 12 h damage group, significant differences were observed between damage subgroups and control groups(all in P<0.01). The correlation coefficients between H₂O₂ concentration and cell apoptosis/necrosis in 3 h, 6 h and 12 h group were 0.971, 0.992 and 0.986 respectively. Compared with control group, significant differences were observed in mitochondrial membrane potential, MDA, ROS, SOD, ALT, and AST in damage group after treated with 200 μmol/L H₂O₂ for 6 h(all in P<0.01). Conclusions L02 hepatocytes treating with 200 μmol/L H₂O₂ for 6 h is an appropriate model simulating the ischemia-reperfusion or oxidative damage in vitro.
- Hepatocytes /
- Oxidative damage /
- Ischemia-reperfusion injury /
- Apoptosis

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图 1 不同浓度、不同作用时间的H₂O₂损伤情况下细胞存活率的比较

Figure 1. Comparison of cell survival rates among different H₂O₂ concentrations and actuation duration groups

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图 2 不同浓度、不同作用时间的H₂O₂损伤情况下细胞凋亡或坏死率比较

Figure 2. Comparison of cell apoptosis/necrosis rates among different H₂O₂ concentrations and actuation duration groups

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