Yang Di-sheng, Xie Xian-kuan, Ye Zhao-ming, Tao Hui-min
Department of Orthopaedics, The Second Hospital Affiliated Zhejiang University College of Medicine, Hangzhou 310009, China.
Zhonghua Wai Ke Za Zhi. 2005 Jun 15;43(12):799-802.
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.
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.
Ad-Asc-myc encoding antisense c-myc fragment was obtained with the titer of 2 x 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, 5.0 microg/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 G2/M phase arrested in transfected cells.
Ad-Asc-myc increases the chemotherapy sensitivity of osteosarcoma MG-63 cells to cisplatin as well as induced apoptosis.
构建携带反义c-myc片段的重组腺病毒，并研究其对骨肉瘤MG-63细胞顺铂化疗敏感性的影响。
将约720个碱基对的c-myc cDNA反向克隆至腺病毒载体，经重组、扩增及体内包装构建携带反义c-myc片段的重组腺病毒（Ad-Asc-myc）。体外将Ad-Asc-myc转染骨肉瘤MG-63细胞，采用瑞氏染色、吖啶橙染色、蛋白质免疫印迹法（Western Blot）、噻唑蓝比色法（MTT）、流式细胞术（FCM）观察细胞形态、c-myc蛋白表达、体外肿瘤细胞增殖、凋亡及细胞周期变化。
获得携带反义c-myc片段的重组腺病毒Ad-Asc-myc，病毒滴度为2×10(9) pfu/ml。Ad-Asc-myc转染MG-63细胞48 h后可下调c-myc蛋白表达，联合2.0、5.0 μg/ml顺铂作用2 h，体外抑制肿瘤细胞增殖率分别为33.4%和54.2%，与对照重组腺病毒（Ad-LacZ）组比较差异有统计学意义（P < 0.05）。吖啶橙染色及FCM分析显示Ad-Asc-myc可诱导转染细胞凋亡，顺铂作用可增强凋亡效果。细胞周期分析显示转染细胞出现明显的G2/M期阻滞。
Ad-Asc-myc可提高骨肉瘤MG-63细胞对顺铂的化疗敏感性并诱导其凋亡。
