腺病毒介导反义c-myc增强骨肉瘤细胞对顺铂化疗敏感性的实验研究

Adenovirus mediated antisense c-myc gene on the chemotherapy sensitivity of osteosarcoma cells to cisplatin

目的构建表达反义cmyc的重组腺病毒,探讨重组腺病毒介导反义cmyc转染的人骨肉瘤MG63细胞对顺铂化疗敏感性的影响。方法应用基因重组技术,将约720bp的人cmyccDNA反向克隆到腺病毒载体,经重组、扩增、病毒包装后构建表达反义cmyc的重组腺病毒(AdAscmyc),并在体外转染骨肉瘤MG63细胞,采用瑞士染色、吖啶橙染色、蛋白免疫印迹(WesternBlot)、细胞体外增殖抑制试验(MTT)、流式细胞仪(FCM)等观察细胞形态、检测cmyc蛋白表达、瘤细胞体外增殖抑制、凋亡及细胞周期,分析AdAscmyc体外转染的骨肉瘤MG63细胞对顺铂化疗敏感性。结果成功构建AdAscmyc,滴度可达2×109pfu/ml,体外转染MG63细胞48h后,可降低cmyc蛋白表达,并与浓度为2.0、5.0μg/ml的顺铂作用2h后,可抑制MG63细胞的体外增殖,抑制率分为33.4%、54.2%,与对照腺病毒(AdLacZ)转染组相比差异有统计学意义(P<0.05),FCM检测证实AdAscmyc的转染可诱导骨肉瘤细胞凋亡,且顺铂治疗后凋亡比例增加,细胞周期分析显示AdAscmyc转染的骨肉瘤细胞出现G2/M期阻滞。结论腺病毒介导反义cmyc能诱导骨肉瘤MG63细胞凋亡并增加MG63细胞对顺铂化疗敏感性。

Objective To construct the recombinant adenovirus encoding antisense c-myc fragment and to investigate its effect on the chemotherapy sensitivity of osteosarcoma MG-63 cells to cisplatin. Methods The recombinant adenovirus(Ad-Asc-myc) encoding antisense c-myc fragment was constructed by cloning c-myc cDNA of about 720 base pairs in a reverse direction into adenovirus vector, then undergoing recombination, amplifying and being complemented in vivo.The osteosarcoma MG-63 cells were transfected by the Ad-Asc-myc in vitro,and Wright staining,Acridine Orange staining,Western Blot,MTT,Flow Cytometry(FCM) were used to study cell morphology, expression of c-myc protein, tumor cell proliferation in vitro, apoptosis and cell cycle change. Results Ad-Asc-myc encoding antisense c-myc fragment was obtained with the titer of 2×10~9 pfu/ml. 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, 5.0ug/ml cisplatin for (2 h) could inhibit tumor cells proliferation in vitro by 33.4% and 54.2% respectively, which were significantly difference compared with control recombinant adenovirus(Ad-LacZ)groups(P<0.05). Acridine Orange staining and FCM analysis showed that Ad-Asc-myc could induce apoptosis of transfected cells, which was enhanced by the treatment of cisplatin cell. Cycle analysis showed that obvious G_2/M phase arrested in transfected cells. Conclusion Ad-Asc-myc increases the chemotherapy sensitivity of osteosarcoma MG-63 cells to cisplatin as well as induced apoptosis.