Zhang Wanguang, Zhang Huilan
Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
J Huazhong Univ Sci Technolog Med Sci. 2006;26(5):520-3. doi: 10.1007/s11596-006-0508-5.
In order to construct plasmid of hypoxia-inducible factor-1alpha (HIF-1alpha), and transfect into human lung cancer cells A549, the change in sensitivity of lung cancer cells A549 to chemotherapy was observed. HIF-1alpha mRNA structure region was amplified by RT-PCR and inserted into plasmid pcDNA3. The expression plasmid pcDNA3/HIF-1alpha was transfected into A549 with Lipofec-tAMINE2000. The expression of HIF-1alpha protein was detected by Western blot. After A549 cells were transfected with HIF-1alpha prior to addition of 5-Fu, the growth activity was measured by growth curve, apoptosis was detected by flow cytometry at 48 h, and the levels of caspase3 and MDR-1 were determined by Western blot. The results showed that the constructed expression plasmid was analyzed with restriction enzymes and gel electrophoresis. Two DNA lanes at 2.55 kb and 5.4 kb respectively were found, which were consistent with that expected. The growth rate in 5-Fu group was significantly inhibited, and the apoptosis index and caspase3 activity were increased significantly as compared with control group. After HIF-1alpha being transfected into A549, the activity of MDR-1 was increased and the effect of 5-Fu was weakened. In conclusion, HIF-1alpha can promote chemoresistance by increasing the activation of MDRI and suppressing apoptosis during lung cancer cells A549 induced with 5-Fu.
为构建缺氧诱导因子-1α（HIF-1α）质粒，并将其转染至人肺癌细胞A549，观察肺癌细胞A549对化疗敏感性的变化。通过逆转录聚合酶链反应（RT-PCR）扩增HIF-1α mRNA结构区域，并将其插入质粒pcDNA3。用脂质体2000将表达质粒pcDNA3/HIF-1α转染至A549细胞。通过蛋白质免疫印迹法检测HIF-1α蛋白的表达。在加入5-氟尿嘧啶（5-Fu）之前，用HIF-1α转染A549细胞后，通过生长曲线测定生长活性，在48小时时通过流式细胞术检测细胞凋亡，并通过蛋白质免疫印迹法测定半胱天冬酶3（caspase3）和多药耐药蛋白1（MDR-1）的水平。结果显示，构建的表达质粒经限制性内切酶和凝胶电泳分析。分别在2.55 kb和5.4 kb处发现两条DNA条带，与预期一致。与对照组相比，5-Fu组的生长速率明显受到抑制，凋亡指数和caspase3活性显著增加。将HIF-1α转染至A549细胞后，MDR-1的活性增加，5-Fu的作用减弱。综上所述，HIF-1α可通过增加MDR1的激活并抑制5-Fu诱导的肺癌细胞A549凋亡来促进化疗耐药。
