黑素瘤MIA启动子介导的CD/TK双自杀基因重组腺病毒系统选择性杀伤黑素瘤细胞

Adenovirus-mediated CD/TK fusion gene system driven by MIA promoter selectively kills malignant melanoma cells

目的:探讨黑素瘤抑制蛋白(melanoma inhibitory activity,MIA)启动子介导的CD/TK双自杀基因重组腺病毒系统,对黑素瘤细胞A375的选择性杀伤作用。方法:构建腺病毒穿梭质粒pAdrMIA-CD/TK,在293细胞内包装、扩增、纯化后,体外转染人表皮黑素细胞HeMa-LP、黑素瘤细胞A375和人宫颈癌细胞HeLa,RT-PCR检测基因CD和TK片段,加入前体药物5-氟胞嘧啶(5-FC)和/或丙氧鸟苷(GCV),用MTT法测定该体系对细胞株的杀伤效应。结果:制备的病毒滴度为1×1011pfu/L,用100m.o.i.重组腺病毒感染细胞后,发现目的基因CD和TK片段可在黑素瘤细胞中表达,转染目的基因的黑素瘤细胞A375对5-FC和GCV具有一定的敏感性,细胞存活率较对照组显著下降(P<0.05),联合应用两种药物对A375细胞的杀伤作用较单用一种显著增强(P<0.05)。对人表皮黑素细胞HEMa-LP和人宫颈癌细胞HeLa无显著杀伤作用。结论:构建的黑素瘤MIA启动子介导的CD/TK双自杀基因重组腺病毒系统,在体外可选择性杀伤黑素瘤细胞A375。

Objective： To determine the selectivdy killing effect of adenovirus- mediated double suicide gene driven by melanoma inhibitory activity （MIA） prometer on malignant melanoma A375 cells. Methods： The plasmid pAdrMIA- CD/TK was constructed and transfected into 293 packaging cells for amplification of adenovirus, which was used to infect the human epidermal melanocytes （adult） HEMa - LP cells, the malignant melanoma A375 cells and the human cervical carcinoma HeLa cells. CD and TK gene were examined by RT- PCR. The cells were treated with 5 - flurocytosine （5 - FC） and ganciclovir （GCV） after infection. The killing effect of the fusion gene system on cells was evaluated by MTr. Results： The titer of the recombinant adenovirus was 1 ×10^11 pfu/L. After the A375 cells were infected with recombinant adenovirus （ 100 m. o. i. ）, it was shown by RT - PCR that CD gene and TK gene were expression in the infected cells. The infected cells became sensitive to both of 5 - FC and GCV （ P 〈 0.05）. The killing effect of the CD/TK fusion gene on the infected cells was much stronger than that of either 5 - FC or GC alone（ P 〈0.05）. Conclusion： Adenovirus- mediated CD/TK fusion gene system driven by MIA promoter can selectively kill malignant melanoma cells A375 in vitro.