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目的研究融合型自杀基因Fcy∶∶Fur联合5-氟胞嘧啶(5-FC)对人卵巢癌细胞株(SKOV3)的杀伤作用。方法构建含有融合型自杀基因Fcy∶∶Fur的荧光真核表达质粒,转染SKOV3细胞,筛选稳定表达的细胞株,RT-PCR和Western blot检测Fcy∶∶Fur表达。以MTT法和FCM法检测5-FC对Fcy∶∶Fur转基因细胞的杀伤效应。结果测序鉴定证实插入片段正确。细胞转染24h后,60%转染细胞发出绿色荧光。经G418筛选,RT-PCR和Westernblot法检测到Fcy∶∶Fur表达。加入5-FC后,MTT法检测到明显杀伤效应,FCM法检测到显著凋亡峰。结论融合型自杀基因Fcy∶∶Fur联合5-FC对人卵巢癌细胞SKOV3有明显的杀伤作用。
Objective To study the killing effect of fusion suicide gene Fcy::Fur combined with 5-fluorocytosine (5-FC) on human ovarian cancer cell line SKOV3. Methods Fluorescent eukaryotic expression plasmid containing fusion suicide gene Fcy::Fur was constructed and transfected into SKOV3 cells. Stably expressing cell lines were screened and Fcy::Fur expression was detected by RT-PCR and Western blot. The killing effects of 5-FC on Fcy::Fur transgenic cells were detected by MTT assay and FCM assay. Results sequencing confirmed that the insert was correct. After 24 h of transfection, 60% of transfected cells fluoresced green. After G418 screening, Fcy::Fur expression was detected by RT-PCR and Western blot. After addition of 5-FC, obvious killing effect was detected by MTT method, and significant apoptosis peak was detected by FCM method. Conclusion The fusion suicide gene Fcy::Fur combined with 5-FC has obvious killing effect on human ovarian cancer cell line SKOV3.