反义c-myc重组腺病毒的构建及其抗骨肉瘤细胞的作用

Construction of Antisense c-myc Recombinant Adenovirus and Its Anti-tumor Effects on Osteosarcoma Cell Lines MG-63 and U2OS

背景与目的:c-myc原癌基因在细胞的增殖调节中发挥重要作用,研究表明c-myc在骨肉瘤中常常扩增和过表达,而且具有促进细胞转化和诱导转移的特性。本文构建表达反义c-myc的重组腺病毒,并探讨其对不同p53基因型的骨肉瘤细胞系MG-63(P53缺失型)、U2OS(p53野生型)的影响。方法:应用基因重组技术构建表达反义c-myc的重组腺病毒(Ad-As-c-myc),并在体外转染MG-63、U2OS细胞,采用蛋白免疫印迹(Westernblot)、吖啶橙染色、RT-PCR、流式细胞仪(FCM)等方法检测或观察其对细胞c-mycmRNA表达、瘤细胞体外增殖、凋亡及细胞周期的影响。结果:成功构建Ad-As-c-myc,滴度可达2×109pfu/ml,体外转染MG-63、U2OS细胞后可明显抑制细胞的体外增殖,而且对携带野生型p53的U2OS更敏感。Ad-As-c-myc转染48h后可降低c-mycmRNA表达。吖啶橙染色及FCM检测证实,转染Ad-As-c-myc可诱导骨肉瘤细胞凋亡,细胞周期分析显示转染Ad-As-c-myc的MG-63细胞出现G2/M期阻滞,而U2OS细胞出现G1期阻滞。结论:腺病毒介导反义的c-myc能以p53依赖或非依赖性途径诱导骨肉瘤细胞凋亡,并抑制骨肉瘤细胞的增殖。

BACKGROUND & OBJECTIVE: c-myc, an oncogene, plays an important role in regulation of cell proliferation, and has been found to be amplified and overexpressed in osteosarcoma. Moreover, it can promote cell transformation, and induce metastasis. This study was to construct recombinant adenovirus encoding antisense c-myc, and to investigate its effects on osteosarcoma cell lines MG-63 (p53-deficient) and U2OS (with wild type p53). METHODS: Recombinant adenovirus Ad-As-c-myc encoding antisense c-myc was constructed by gene reconstruction technique, defined by polymerase chain reaction (PCR), and transfected into human osteosarcoma cell lines MG-63 and U2OS. Western blot, acridine orange staining, reverse transcription-PCR (RT-PCR), and flow cytometry (FCM) were used to detect expression of c-myc, proliferation, apoptosis, and cell cycle of MG-63 and U2OS cells. RESULTS: Ad-As-c-myc encoding antisense c-myc was obtained with the titer of 2×109 pfu/ml. Ad-As-c-myc significantly inhibited proliferation of both cell lines, while U2OS with wild type p53 was more susceptible to Ad-As-c-myc. Expression of c-myc mRNA was down-regulated in 2 cell lines 48 h after transfection of Ad-As-c-myc. Acridine orange staining and FCM analysis showed that Ad-As-c-myc induced apoptosis of both cell lines, cell cycle analysis showed obvious G2/M phase arrest in MG-63 cells,and G1 phase arrest in U2OS cells after transfection of Ad-As-c-myc. CONCLUSION: Ad-As-c-myc could induce apoptosis through both P53-dependent and P53-independent pathways, and inhibit proliferation of osteosarcoma cells.