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目的转染pcDNA3HO1入人血管内皮细胞(HUVEC)并表达,研究血红素加氧酶_1(HO_1)保护细胞凋亡的作用。方法将构建好的质粒用DOTAP包裹转入细胞中表达。用CCLR破碎细胞后,SDS_PAGE鉴定表达量,采用TNF_α诱导细胞凋亡,以流式细胞仪测定细胞的凋亡率;采用Hemin和SnPP分别刺激细胞,促进和抑制HO_1在细胞中的表达。结果经Hemin处理后细胞的凋亡率均在20%以下,而SnPP处理后细胞的凋亡率大幅上升,最高可达到95%以上。实验数据显示HO_1基因表达被抑制时细胞凋亡率是诱导时的5~20倍。结论细胞凋亡率与质粒转染效率和HO_1表达量均成反比,提示HO_1对细胞有保护作用,可以抑制细胞凋亡,在临床具有广泛的应用前景。
Objective To transfect pcDNA3HO1 into human umbilical vein endothelial cells (HUVECs) and express the same to study the effect of heme oxygenase-1 (HO_1) on apoptosis. Methods The constructed plasmid was transfected into cells with DOTAP. After the cells were broken by CCLR, the expression of SDS-PAGE was identified, and the apoptosis was induced by TNF-α. The apoptosis rate was determined by flow cytometry. Hemin and SnPP were used to stimulate the cells to promote and inhibit the expression of HO-1. Results After Hemin treatment, the apoptosis rate of cells was below 20%, while the apoptosis rate of SnPP treatment cells increased significantly up to 95%. The experimental data show that when the HO 1 gene expression is inhibited, the apoptosis rate is 5 to 20 times when induced. Conclusions The rate of apoptosis is inversely proportional to the efficiency of plasmid transfection and the expression of HO_1, suggesting that HO_1 has a protective effect on cells and can inhibit apoptosis. It has wide application prospect in clinic.