GLS／IL-12对黑色素瘤细胞B16的增殖及凋亡的影响

Effect of granulysin (GLS) and IL-12 genes' expression products on proliferation and apoptosis of melanoma B16 cell

目的:体外检测人GLS活性肽和小鼠IL-12基因的共表达产物对黑色素瘤细胞B16的增殖及凋亡的影响。方法:用脂质体将含GLS活性肽和小鼠IL-12基因的共表达质粒转染黑色素瘤细胞B16并用RT-PCR检测其表达,再用MTT试验、Hoechst33258染色、AO/EB染色和AnnexinⅤ—FITC流式细胞分析检测基因表达产物对B16细胞的增殖抑制与促凋亡作用。结果:GLS活性肽和小鼠IL-12基因能在B16细胞中表达,MTT法观察到表达产物对B16有明显的细胞增殖抑制作用,经Hoechst33258染色、AO/EB染色和流式细胞分析观察到细胞核浓缩、深染、细胞膜改变等凋亡特征,凋亡指数明显高于对照组。结论:GLS活性肽和小鼠IL-12基因的共表达产物对黑色素瘤细胞B16具有明显的增殖抑制作用及促凋亡作用。

Objective： To dectect the effect of granulysin and IL-12 genes＇ expression products on proliferation and apoptosis of melanoma B 16 cell in vitro. Methods： Co-expression plasmid including granulysin peptide and murine interleukin 12 （raiL-12） genes was transfected into melanoma B16 cell with Lipofectamin TM2000 and its expression products were detected by RT-PCR. Growth suppression was detected with MTT colorimetric assay, and cell apoptotic alterations were evaluated by Hoechst 33258 staining, AO/EB staining,and Annexin V-FITC flow cytometry （FCM）. Results： GLS peptite and IL-12 genes could be expressed in B16 cells. Expression products inhibited the proliferation of melanoma cells under MTI＂ observaton. Cells apoptosis with nuclear chromatin condensation, fragmentation and cell membrane change were observed under Hoecbst 33258 staining and AO/EB staining. FCM analysis showed the apoptotic rates in test group was 21.02% ,which was higher than that in control in control group （ 15.57% ）.Conclusion： Expression products of granulysin and mIL-12 genes can not only inhibit proliferation but also induce apoptosis of murine melanoma cell line B16 in vitro.