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目的:探讨急性单核细胞白血病THP-1细胞株中核因子-κB(NF-κB)信号通路的活化状态及其抑制剂pathenolide(PN)对细胞增殖、凋亡的影响。方法:THP-1细胞置于37℃、体积分数为5%CO2培养箱中培养。①收集处于对数增殖期的细胞,采用RT-PCR方法检测THP-1细胞中P65、IκBαmRNA的表达。②用细胞裂解液分别提取THP-1的细胞质和细胞核蛋白,采用Western Blotting法检测细胞质及细胞核中P65、IκBα蛋白的表达。③将处于对数增殖期的THP-1细胞分成10组[(空白对照孔、阴性对照孔及各实验孔(1、2、4、6、8、10、15、20μmol/LPN)],按12 h、24 h 2个检测点计算细胞增殖抑制率及增殖半数抑制浓度(IC50)的范围。④将处于对数增殖期的THP-1细胞分为3组(空白对照、PBS对照和6μmol/L PN处理组),按第0天至第6天设计,绘制细胞增殖曲线。⑤取处于对数增殖期的THP-1细胞,分5组(分别以0、2、4、6和8μmol/L的PN处理)培养,24 h后收获细胞,分别检测细胞周期和细胞早期凋亡率。结果:①RT-PCR结果示THP-1细胞中有P65、IκBαmRNA的表达,Western blot-ting结果示细胞质中均有P65、IκBα蛋白的表达,细胞核中有P65蛋白的表达。②PN对THP-1有抑制作用,其抑制率在一定范围内存在着剂量-效应关系,测定范围内最大抑制率可达(69.56±2.52)%;PN作用12 h、24 h的IC50分别为8~10μmol/L、6~8μmol/L。③以6μmol/L的PN处理后,THP-1细胞增殖曲线明显下降。④在2~6μmol/L范围内,随PN浓度增高,G0/G1期细胞构成增高;在2~8μmol/L范围内,S期细胞随PN浓度的增加而减少。0、2、4、6及8μmol/L PN组细胞早期凋亡率分别为(3.84±1.50)%、(4.67±1.97)%、(12.67±2.13)%、(17.72±2.78)%和(23.62±3.36)%,差异有统计学意义(F=36.280,P<0.001)。结论:THP-1细胞中有NF-κB通路的持续激活,PN对THP-1细胞有抑制增殖和促进凋亡的作用。
Objective: To investigate the activation of nuclear factor-κB (NF-κB) signaling pathway in acute monocytic leukemia THP-1 cells and the effect of its inhibitor pathenolide (PN) on cell proliferation and apoptosis. Methods: THP-1 cells were cultured in a 5% CO 2 incubator at 37 ℃. ① The cells in logarithmic proliferation phase were collected and the expression of P65, IκBαmRNA in THP-1 cells was detected by RT-PCR. ② The cytoplasm and nucleoprotein of THP-1 were extracted by cell lysate, and the expression of P65 and IκBα in cytoplasm and nucleus were detected by Western Blotting. (3) THP-1 cells in logarithmic phase were divided into 10 groups (blank control, negative control and experimental wells (1, 2, 4, 6, 8, 10, 15 and 20μmol / LPN) 12 h, 24 h 2 test points to calculate the cell proliferation inhibition rate and proliferation half inhibitory concentration (IC50) of the range.④ THP-1 cells in logarithmic proliferation phase were divided into 3 groups (blank control, PBS control and 6μmol / L PN treatment group), the cell proliferation curve was designed according to day 0 to day 6. THP-1 cells in logarithmic proliferation phase were divided into 5 groups (0, 2, 4, 6 and 8 μmol / L PN treatment), and the cells were harvested after 24 h.The cell cycle and the early apoptosis rate were detected respectively.Results: (1) The expression of P65 and IκBαmRNA in THP-1 cells was detected by RT-PCR and Western blotting showed that the cytoplasm P65 protein expression in the nucleus and P65 protein expression in the nucleus.PN has inhibitory effect on THP-1, and its inhibition rate in a certain range exists dose-effect relationship, the maximum inhibition rate in the determination range is up to ( 69.56 ± 2.52)%. The IC50 of PN at 12 h and 24 h was 8 ~ 10 μmol / L and 6 ~ 8 μmol / L, respectively. ③ After treatment with 6 μmol / L PN, the proliferation of THP-1 cells (P <0.05) .④In the range of 2 ~ 6μmol / L, the cells in G0 / G1 phase increased with the increase of PN concentration, while the cells in S phase decreased with the increase of PN concentration in the range of 2 ~ 8μmol / (4.84 ± 1.50)%, (4.67 ± 1.97)%, (12.67 ± 2.13)%, (17.72 ± 2.78)% and (23.62 ± 3.36)% respectively in the PN group at 4, 6 and 8μmol / %, The difference was statistically significant (F = 36.280, P <0.001) .Conclusion: THP-1 cells in a sustained activation of NF-κB pathway, PN on THP-1 cells inhibit proliferation and promote apoptosis.