人喉鳞癌Hep-2细胞血红素加氧酶1表达及其对卡铂作用的影响

HO-1 expression in human laryngeal squamous-cell carcinoma Hep-2 cells and its influence on Carboplatin's effects

目的探讨人喉鳞癌Hep-2细胞血红素加氧酶1(HO-1)表达对卡铂作用的影响及其可能的作用机制。方法将对数生长期Hep-2细胞分为六组。空白对照组不干预,Zn PP(HO-1抑制剂锌原卟啉)组加入Zn PP 10μmol/L;Hemin(HO-1诱导剂氯化血红素)组加入Hemin 10μmol/L;卡铂组加入卡铂10μg/m L;Hemin联合卡铂组先加入Hemin 10μmol/L,作用1 h后再加入卡铂10μg/m L;Zn PP联合卡铂组先加入Zn PP 10μmol/L,作用1 h后再加入卡铂10μg/m L。每组设5个复孔,加药后培养24 h。采用MTT法观察各组细胞存活率;RT-PCR、Western blotting法检测各组细胞内HO-1 mRNA和蛋白表达情况;DCFH-DA探针检测各组细胞内活性氧(ROS);流式细胞术检测各组细胞凋亡率。结果 1细胞存活率:与空白对照组比较,卡铂组和Zn PP组明显降低,Hemin组明显升高(P均<0.05);与卡铂组比较,Zn PP联合卡铂组明显降低,Hemin联合卡铂组明显升高(P均<0.05)。2细胞HO-1 mRNA和蛋白相对表达量:与空白对照组比较,卡铂组和Hemin组明显升高,Zn PP组明显下降(P均<0.05);与卡铂组比较,Zn PP联合卡铂组明显降低,Hemin联合卡铂组明显升高(P均<0.05)。3细胞凋亡率:与空白对照组比较,卡铂组、Zn PP组明显升高(P均<0.05);与卡铂组比较,Zn PP联合卡铂组明显升高,Hemin联合卡铂组明显降低(P均<0.05);4细胞内ROS活性:与空白对照组比较,卡铂组、Zn PP组显著增高(P均<0.05);与卡铂组比较,Zn PP联合卡铂组明显升高,Hemin联合卡铂组明显降低(P均<0.05)。结论 HO-1在Hep-2细胞内的高表达可影响卡铂对Hep-2细胞的促凋亡作用,应用Zn PP后HO-1表达下调,同时提高了人喉鳞癌Hep-2细胞在卡铂作用下的凋亡率,这一过程可能与增强细胞内ROS水平相关。

Objective To investigate the influence of heme oxygenase-1( HO-1) expression in human laryngeal squamous-cell carcinoma Hep-2 cells on the Carboplatin’s effects and its possible mechanism. Methods Hep-2 cells in the logarithmic phase were divided into six groups. The blank control group was not treated,Zn PP group was treated with 10μmol /L HO-1 inhibitor,Hemin group was treated with 10 μmol /L HO-1 inducer,and Carboplatin group was treated with10 μg/m L,Hemin combined with Carboplatin group was treated with 10 μmol/L Hemin alone for 1 h and then 10 μg/m L Carboplatin,Zn PP combined with Carboplatin group was treated with 10 μmol/L Zn PP alone for 1 h and then 10 μg/m L Carboplatin. These six groups all had five complex holes and were cultured for 24 h after dosing. The survival rates were evaluated by MTT assay. The expression of HO-1 mRNA and protein was detected by RT-PCR and Western blotting. Intracellular ROS activity was examined by DCFH-DA probe. The apoptosis rate was performed by flow cytometry.Results 1 the cell survival rate: compared with the control group,the survival rates of Hep-2 cells in the Zn PP and Carboplatin groups were significantly decreased,but the survival rate in the Hemin group was increased( all P < 0. 05); compared with the Carboplatin group,the survival rate of the Znpp combined with Carboplatin group was decreased and the Hemin combined with Carboplatin group was increased( all P < 0. 05). 2 HO-1 in mRNA and protein levels: compared with the control group,the expression of HO-1 mRNA and protein was increased in the Hemin and Carboplatin groups and was decreased in the Zn PP group( all P < 0. 05); compared with the Carboplatin group,the Znpp combined with Carboplatin decreased,but HO-1 expression was increased in the Hemin combined with Carboplatin group( all P < 0. 05). 3 the apoptosis rate: compared with the control group,the apoptosis rates in the Carboplatin and Zn PP groups were increased( all P< 0. 05); compared with the Carboplatin group,Znpp combined with Carboplatin increased the apoptosis rate,but Hemin combined with Carboplatin decreased the apoptosis rate( all P < 0. 05). 4 the ROS activity: compared with the control group,the the ROS activities in the Carboplatin and Zn PP groups were increased( all P < 0. 05); compared with the Carboplatin group,Znpp combined with Carboplatin increased the ROS activity,but Hemin combined with Carboplatin decreased the ROS activity( all P < 0. 05). Conclusions HO-1 overexpression may affect the pro-apoptotic effects of Carboplatin on Hep-2 cells,and the application of Zn PP down-regulates the HO-1 expression and increases the apoptosis rate of Hep-2 cells with treatment of Carboplatin. In this process,the elevation of intracellular ROS activity may be involved.