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目的探讨人穿孔素(PFP)、颗粒酶B(GrB)共表达是否可以诱导人的喉癌细胞系Hep-2的凋亡及其作用的机理。方法利用脂质体2000将PFP、GrB共表达载体pVAX1-PIG(即pVAX1-PFP-IRES-GrB)转染人喉癌Hep-2细胞,采用荧光染料Hoechst33342法、流式细胞仪(FCM)检测、透射电镜观察Hep-2细胞的凋亡情况。以激光共聚焦显微镜检测重组载体转染后的Hep-2细胞内[Ca2+]i浓度的变化,并探讨其作用机理。结果pVAX1-PIG转染组的Hep-2细胞大量凋亡且其凋亡率显著高于对照组(P<0.05),透射电镜研究显示单个Hep-2细胞内亦出现凋亡的特征。Hep-2细胞内[Ca2+]i的浓度发生了变化,且由FI值增大可知细胞胞浆内[Ca2+]i的浓度升高。结论PFP、GrB共表达能够诱导人Hep-2细胞的凋亡,且凋亡的发生与细胞胞浆内[Ca2+]i的浓度升高有关。
Objective To investigate whether perforin (PFP) and granzyme B (GrB) co-expression can induce the apoptosis of human laryngeal carcinoma cell line Hep-2 and its mechanism. Methods Human laryngeal carcinoma Hep-2 cells were transfected with pVAX1-PIG (PVAX1-PFP-IRES-GrB) using Lipofectamine 2000. Fluorescent dye Hoechst33342 assay and flow cytometry (FCM) The apoptosis of Hep-2 cells was observed by transmission electron microscope. The expression of [Ca2 +] i in Hep-2 cells transfected with the recombinant vector was detected by laser scanning confocal microscopy and its mechanism of action was also explored. Results The apoptosis of Hep-2 cells in the pVAX1-PIG transfected group was higher than that in the control group (P <0.05). Transmission electron microscopy revealed the apoptosis of Hep-2 cells in a single Hep-2 cell. The concentration of [Ca2 +] i in Hep-2 cells changed, and the concentration of [Ca2 +] i in cytoplasm of cells increased by the increase of FI value. Conclusions Co-expression of PFP and GrB can induce apoptosis of human Hep-2 cells, and the occurrence of apoptosis is related to the increase of intracellular [Ca2 +] i concentration.