重构型天冬氨酸特异性半胱氨酸蛋白酶3表达载体诱导人肺腺癌细胞凋亡的意义

目的构建天冬氨酸特异性半胱氨酸蛋白酶3(Caspase3)表达载体,观察Caspase3表达载体对人肺腺癌A549细胞凋亡的影响。方法应用基因重组方法,建立重构型Caspase3基因的真核表达系统pcDNA3.1revCaspase3质粒和野生型pcDNA3.1Caspase3质粒。将实验细胞分为3组转染pcDNA3.1revCaspase3质粒组,转染pcDNA3.1Caspase3质粒组,转染pcDNA3.1质粒空白对照组。前2组用Caspase3抑制剂DEVDfmk干预。应用Caspase3酶活性分析、流式细胞仪及甲基偶氮唑蓝(MTT)比色法检测A549细胞Caspase3酶活性变化及细胞凋亡和增殖情况。结果(1)pcDNA3.1revCaspase3质粒组、pcDNA3.1Caspase3质粒组的A549细胞Caspase3酶活性分别是(11.87±0.92)%、(5.34±0.38)%(t=16.02,P<0.01);用Caspase3抑制剂DEVDfmk干预后,pcDNA3.1revCaspase3质粒组酶活性为(7.04±0.48)%,仍明显高于野生型pcDNA3.1Caspase3质粒组的(4.51±0.20)%(t=11.86,P<0.01)。(2)流式细胞分析结果显示,pcDNA3.1revCaspase3质粒组、pcDNA3.1Caspase3质粒组和pcDNA3.1质粒组的A549细胞凋亡率分别是(20.1±3.5)%、(7.8±2.8)%、(1.4±0.3)%,3组差异有统计学意义(F=44.01,P<0.01)。(3)MTT比色检测显示pcDNA3.1revCaspase3质粒组、pcDNA3.1Caspase3质粒组和pcDNA3.1质粒组的A549细胞生存率分别是(35.7±1.1)%、(72.8±2.9)%、(85.4±4.8)%,转染pcDNA3.1revCaspase3质粒组生存率明显低于其他两组(F=375.07,P<0.01)。结论pcDNA3.1revCaspase3在A549细胞内有较强的自身活化能力,对Caspase3抑制剂抵抗作用较强,可明显诱导A549细胞凋亡并抑制A549细胞生长。

Construction of reversed caspase-3 and it'''' s effect on apoptosis induction in human lung adenocarcinoma cell line A549

OBJECTIVE: To investigate the effect of reversed caspase-3 expression vector on apoptosis of human lung adenocarcinoma cell line A549. METHODS: Reversed pcDNA 3.1-rev-caspase-3 plasmid and wild type pcDNA3.1-caspase-3 plasmid were constructed by genetic recombination. Three experiments (groups) with A549 cells were carried out: a group of transfection with pcDNA 3.1-rev-caspase plasmid, a group of transfection with pcDNA 3.1-caspase plasmid, and a control group of transfection with pcDNA 3.1 plasmid. Intervention with DEVD-fmk, an inhibitor of caspase-3, was carried out in the first two groups. The activity of caspase-3, the apoptosis and the growth of A549 cells were measured. RESULTS: (1) The activity of caspase-3 in the pcDNA 3.1-rev-caspase-3 plasmid group and pcDNA 3.1-caspase-3 plasmid group was (11.87 +/- 0.92)% and (5.34 +/- 0.38)%, respectively, significantly higher in the former group (t = 16.02, P < 0.01). After intervention with DEVD-fmk, the activity of caspase-3 in pcDNA 3.1-rev-caspase-3 plasmid group and in pcDNA 3.1-caspase-3 plasmid group was (7.04 +/- 0.48)% and (4.51 +/- 0.20)%, respectively, also significantly higher in the former group (t = 11.86, P < 0.01). (2) Flow cytometry showed that the apoptosis rate of A549 cells in pcDNA 3.1-rev-caspase-3 plasmid group, pcDNA 3.1-caspase-3 plasmid group and pcDNA 3.1 plasmid group was (20.1 +/- 3.5)%, (7.8 +/- 2.8)% and (1.4 +/- 0.3)%, respectively. The difference of the apoptosis rate was significant (F = 44.01, P < 0.01). (3) The survival rates of A549 cells in the pcDNA 3.1-rev-caspase-3 plasmid group, pcDNA 3.1-caspase-3 plasmid group and pcDNA 3.1 plasmid group were (35.7 +/- 1.1)%, (72.8 +/- 2.9)% and (85.4 +/- 4.8)%, respectively. The survival rate of A549 cells in pcDNA 3.1-rev-caspase-3 plasmid group was lower than those in the other two groups (F = 375.07, P < 0.01). CONCLUSIONS: pcDNA 3.1-rev-caspase-3 in A549 cells has a strong activity and can resist caspase-3 inhibitor in A549 cells. It can also induce A549 cell apoptosis and inhibit the growth of A549 cells.