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目的:观察和厚朴酚(HNK)联合青蒿素(ART)对人鼻咽癌细胞(CNE-2)细胞周期的调控作用,探讨其可能的机制。方法:CNE-2细胞经胸腺嘧啶核苷(TdR,终浓度为2 mmol.L-1)处理16 h,2次PBS洗脱后用RMPI-1640培养基10 h,再次加入终浓度为2 mmol.L-1的TdR处理16 h,PBS洗脱后收集细胞用于下一步的实验研究。HNK(7.5 mg.L-1),ART(7.5 mg.L-1)单独及联合作用于已同步化的CNE-2细胞24 h,同时设立对照组,流式细胞仪检测细胞周期分布的改变;RT-PCR检测cyclin D1,cyclin E1,p27 mRNA表达。结果:CNE-2细胞经胸腺嘧啶核苷2次处理后,流式细胞仪检测显示,G1,S,G2期细胞比例分别为(72.64±5.01)%,(24.60±4.35)%,(0.00)%,表明细胞处于G1/S交界处。HNK+ART组、HNK组、ART组的G1期细胞比例分别为(90.78±9.49)%,(67.86±1.59)%,(66.77±2.25)%,提示HNK+ART组较HNK组或ART组显著地将CNE-2细胞阻滞在G1期(P<0.05)。RT-PCR检测显示HNK+ART组、HNK组、ART组的cyclin D1mRNA表达分别为(0.237±0.014),(0.328±0.002)及(0.368±0.007);cyclin E1 mRNA的表达各组依次是(0.445±0.027),(0.572±0.005)及(0.542±0.006);p27 mRNA的表达各组分别是(1.069±0.046),(0.543±0.022)及(0.388±0.004),与单药组比较,HNK+ART组显著下调cyclin D1,cyclin E1 mRNA的表达(P<0.05),显著上调p27 mRNA的表达(P<0.05)。结论:HNK+ART应用以协同方式诱导CNE-2细胞阻滞于G1期,这可能与通过下调cyclin D1及cyckin E1的mRNA表达及上调p27 mRNA表达有关。
OBJECTIVE: To observe the regulatory effect of honokiol (HNK) and artemisinin (ART) on the cell cycle of human nasopharyngeal carcinoma (CNE-2) cells and to explore its possible mechanism. METHODS: CNE-2 cells were treated with thymidine (TdR, final concentration of 2 mmol.L-1) for 16 h, washed twice with PBS and then incubated with RMPI-1640 medium for 10 h. The final concentration of CNE- L-1 TdR treatment for 16 h, after PBS elution cells were collected for the next experimental study. The synchronized CNE-2 cells were treated with HNK (7.5 mg.L-1) and ART (7.5 mg.L-1) separately and in combination for 24 h. At the same time, the control group was established. The cell cycle distribution was detected by flow cytometry The expression of cyclin D1, cyclin E1 and p27 mRNA was detected by RT-PCR. Results: Flow cytometry showed that the percentage of cells in G1, S, G2 phase was (72.64 ± 5.01)%, (24.60 ± 4.35)%, (0.00) respectively after treated twice with thymidine in CNE- %, Indicating that the cells are at the G1 / S junction. The percentage of G1 phase cells in HNK + ART, HNK and ART groups was (90.78 ± 9.49)%, (67.86 ± 1.59)% and (66.77 ± 2.25)%, respectively, suggesting that HNK + CNE-2 cells were arrested in G1 phase (P <0.05). The expression of cyclin D1 mRNA in HNK + ART, HNK and ART groups was (0.237 ± 0.014), (0.328 ± 0.002) and (0.368 ± 0.007) ± 0.027), (0.572 ± 0.005) and (0.542 ± 0.006) respectively. The expression of p27 mRNA was (1.069 ± 0.046), (0.543 ± 0.022) and (0.388 ± 0.004) ART group significantly downregulated the expression of cyclin D1 and cyclin E1 mRNA (P <0.05), and significantly up-regulated the expression of p27 mRNA (P <0.05). CONCLUSION: HNK + ART can induce CNE-2 cells to arrest in G1 phase synergistically, which may be related to the down-regulation of mRNA expression of cyclin D1 and cyckin E1 and up-regulation of p27 mRNA expression.