pNEgr-mIL-12真核表达重组质粒的构建及在COS-7细胞和黑色素瘤B16细胞中的表达

目的:构建含辐射敏感启动子的pNEgr-mIL-12真核表达载体,转染哺乳动物细胞后经不同剂量X射线照射,检测mIL-12的表达.方法:限制性内切酶酶切构建pNEgr-mIL-12表达载体;脂质体转染法转染COS-7细胞和B16细胞;ELISA法检测mIL-12p70表达.结果:①pNEgrmIL-12重组质粒转染瞬时表达细胞COS-7细胞,经不同剂量X射线照射后,照射组mIL-12表达量比未照射组明显增高(P＜0.05,P＜0.001),约为未照组的1.5～2.5倍;在2.0 Gy照后表达增高最为明显,表达于照后4 h达峰值,在观察的72h内保持于较高水平;②转染pNEgr-mIL-12的B16细胞于2.0 Gy照后增高明显,为未照射组的1.5倍;并且经2.0 Gy X射线照射后随时间延长表达水平逐渐增高;低至0.05 Gy的剂量对两种细胞亦均有激活作用.结论:pNEgr-mIL-12重组质粒具有辐射激活下游基因表达的功能,且经不同剂量X射线照射后表达均有增强,尤以2.0 Gy照射组作用明显,但在低剂量50、75、100 mGy照后表达水平增高与对照组差异均有显著性.

Construction of pNEgr-mIL-12 recombinant plasmid and its expression in COS-7 and B16 melanoma cells

Objective:pNEgr mIL 12 recombinant plasmid was constructed and its expression of mIL 12p70 in the supernatant of cultured COS 7 and B16 melanoma cells was detected after X irradiation in different doses. Methods:pNEgr mIL 12 recombinant plasmid was constructed with pNG mIL 12 and T Egr 1 ; Liposome transfection method was used to transfect the cells with pNEgr mIL 12; ELISA assay was applied to detect the expression of mIL 12p70. Results:The expression of mIL 12p70 in the supernatant of COS 7 and B16 melanoma cells was highest after irradiation with 2 0 Gy, and doses as low as 0 05 Gy also showed a stimulatory effect. Time course studies showed that the expression of mIL 12p70 in the supernatant of COS 7 cells (transient expression cells) reached its peak 4h after irradiation with sustained high level thereafter for at least 72 h and a progressive increase in expression of mIL 12p70 in the supernatant of B16 melanoma cells (stable expression cells) was observed over the study period of 72 h. Conclusion:X irradiation could stimulate pNEgr mIL 12 recombinant plasmid with the induction of the expression of its downstream genes The expression of mIL 12p70 was highest after 2 0 Gy X irradiation and low dose irradiation could also activate the Egr 1 promoter and induce the expression mIL 12p70. The implications of these observations in clinical tumor gene irradiation therapy were discussed.