反义c-myc对增强顺铂引起骨肉瘤细胞凋亡作用的实验研究

Antisense c-myc sensitizes osteosarcoma cells to cisplatin-induced apoptosis

目的:通过反义基因治疗降低人骨肉瘤MG-63细胞c-myc的表达,并探讨顺铂对c-myc低表达的人骨肉瘤MG-63细胞凋亡作用的影响。方法:以腺病毒为载体,构建表达反义c-myc的重组腺病毒(Ad-Asc-myc),并在体外转染骨肉瘤MG-63细胞,降低MG-63细胞c-myc的表达,与不同浓度的顺铂作用后,采用MTT、蛋白免疫印迹(Westernblot)、逆转录-聚合酶链反应(RT-PCR)、流式细胞仪(FCM)、透射电镜等检测顺铂对骨肉瘤MG-63细胞体外增殖抑制、凋亡相关基因的表达和凋亡作用。结果:构建的Ad-Asc-myc体外转染MG-63细胞48h后,可明显降低c-Myc蛋白表达,并与浓度为2.0mg/L的顺铂作用2h后,对MG-63细胞的体外增殖抑制率可达38.0%;转染的细胞Bcl-2表达降低,Bax表达增加,而E2F-1的表达无变化;FCM、电镜等显示Ad-Asc-myc转染后可诱导骨肉瘤细胞凋亡,并增加顺铂对骨肉瘤细胞的凋亡作用。结论:降低c-myc表达能诱导骨肉瘤MG-63细胞凋亡并增加顺铂对MG-63细胞的促凋亡作用。

AIM: To down-regulate expression of c-myc through antisense therapy and to investigate its effect on the sensitivity of osteosarcoma MG-63 cells to cisplatin-induced apoptosis. METHODS: The recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment was constructed and transfected into osteosarcoma MG-63 cells in vitro in order to down-regulate the expression of c-myc, and the change in the sensitivity to cisplatin-induced apoptosis was observed. MTT, Western blot, RT-PCR, flow cytometry (FCM) and electron microscope were used to evaluate tumor cell proliferation in vitro, genes expression related to apoptosis regulation and effects on the sensitivity of osteosarcoma MG-63 cells to cisplatin-induced apoptosis. RESULTS: Ad-Asc-myc down-regulated the expression of c-myc protein after transfected MG-63 cells for 48 h, combined with the treatment of 2.0 mg/L cisplatin for 2 h inhibited tumor cell proliferation in vitro by 38.0%. RT-PCR revealed that Ad-Asc-myc down-regulated the expression of Bcl-2 and up-regulated the expression of Bax. No appreciable change was observed in the expression of E_2F-1. FCM showed that Ad-Asc-myc induced apoptosis in intransfected cells, and rendered it more sensitive to cisplatin. CONCLUSION: Antisense c-myc is able per se to induce apoptosis and sensitize osteosarcoma cells to cisplatin-induced apoptosis.