论文部分内容阅读
目的观察碘海醇诱导的人肾小管上皮细胞(HK-2)凋亡及氧化应激所起的作用。方法将体外培养的HK-2细胞分为4组:①对照组;②碘海醇组(100mgI/ml);③N-乙酰半胱氨酸(NAC 10mmol/L)组;④共作用组:NAC10mmol/L预处理1h+碘海醇100mgI/ml。各组以相应药物孵育6h。采用细胞增殖/毒性检测法(CCK-8)测定细胞存活率,核染色、流式Annexin Ⅴ-FITC/PI双染法检测细胞凋亡,DCFH-DA荧光结合流式细胞法检测细胞内活性氧(ROS)的产生,IF、Western blot检测相关信号转导途径。结果碘海醇降低HK-2细胞存活率至(63±5%)(P<0.01),细胞凋亡率升至24.41%(对照组0.9%,P<0.05),细胞内活性氧升高至对照组的1.3倍(P<0.05)。NAC预处理与碘海醇共作用后,HK-2细胞存活率比碘海醇组明显增高(118%vs.63%,P<0.05),凋亡率比碘海醇组明显降低(13.46%vs.24.41%,P<0.05),活性氧降为对照组的0.93倍,比碘海醇组(1.3倍)明显降低(P<0.05)。碘海醇作用于HK-2细胞,引起p53磷酸化,上调凋亡相关蛋白(Bax),下调Bcl-2的表达,促进线粒体内细胞色素C(CytC)释放到胞质,进而引起Caspase-3活化,导致细胞凋亡。NAC降低细胞内活性氧浓度,下调Bax、上调Bcl-2、减少Caspase-3活化,从而减轻细胞损伤。结论碘海醇引起的氧化应激在其诱导的HK-2细胞凋亡中起重要作用。
Objective To observe the effect of iohexol on apoptosis and oxidative stress in human renal tubular epithelial cells (HK-2). Methods HK-2 cells cultured in vitro were divided into 4 groups: ① control group; ② iohexol group (100mgI / ml); ③ N-acetylcysteine (NAC 10mmol / L) group; / L pretreatment 1h + iohexol 100mgI / ml. Each group with the corresponding drug incubation 6h. Cell viability and nuclear staining were determined by cell proliferation / toxicity assay (CCK-8). Apoptosis was detected by flow cytometry with Annexin Ⅴ-FITC / PI double staining. DCFH-DA fluorescence combined with flow cytometry was used to detect intracellular reactive oxygen species (ROS) production, IF, Western blot detection related signal transduction pathway. Results Iohexol reduced the viability of HK-2 cells to (63 ± 5%) (P <0.01), and the apoptosis rate increased to 24.41% (0.9%, P <0.05) The control group 1.3 times (P <0.05). After pretreatment with iohexol, the survival rate of HK-2 cells was significantly higher than that of iohexol (118% vs.63%, P <0.05), and the apoptosis rate of HK-2 cells was significantly lower than that of iohexol (13.46% vs.24.41%, P <0.05). The reactive oxygen species decreased to 0.93 times that of the control group and 1.3 times lower than that of the iohexol group (P <0.05). Iohexol can induce the phosphorylation of p53, up-regulate Bax, down-regulate the expression of Bcl-2, and release cytochrome C (CytC) into the cytoplasm of HK-2 cells, which in turn leads to caspase-3 Activated, leading to apoptosis. NAC reduces intracellular reactive oxygen species, down-regulates Bax, up-regulates Bcl-2, reduces Caspase-3 activation and thus reduces cell damage. Conclusion Ioxo-alcohol-induced oxidative stress plays an important role in the apoptosis induced by HK-2.