[1]卢洁),王春美),盛光耀)#.THP1细胞中核因子κB信号通路的活化状态及其抑制剂pathenolide对细胞增殖、凋亡的影响[J].郑州大学学报(医学版),2010,(02):255-258.
 LU Jie),WANG Chunmei),SHENG Guangyao).Activation condition of NFκB pathway in THP1 cells and the efficacy of pathernolide on cell’s proliferation and apoptosis[J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2010,(02):255-258.
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THP1细胞中核因子κB信号通路的活化状态及其抑制剂pathenolide对细胞增殖、凋亡的影响()
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《郑州大学学报(医学版)》[ISSN:1671-6825/CN:41-1340/R]

卷:
期数:
2010年02期
页码:
255-258
栏目:
论著
出版日期:
2010-03-30

文章信息/Info

Title:
Activation condition of NFκB pathway in THP1 cells and the efficacy of pathernolide on cell’s proliferation and apoptosis
作者:
卢洁1)王春美2)盛光耀2)#
1)郑州大学公共卫生学院卫生统计学教研室4500012)郑州大学第一附属医院儿科郑州450052
Author(s):
LU Jie1) WANG Chunmei2) SHENG Guangyao2)
1)Department of Epidemiology and Biostatistics,College of Public Health,Zhengzhou University,Zhengzhou 4500012)Department of Paediatrics,the First Affiliated Hospital,Zhengzhou University,Zhengzhou 450052
关键词:
急性髓细胞白血病核因子κBpathenolide细胞增殖细胞凋亡
Keywords:
acute myelocytic leukemiaNFκBpathenolideproliferationapoptosis
分类号:
R392.4
文献标志码:
A
摘要:
探讨急性单核细胞白血病THP1细胞株中核因子κB(NFκB)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:THP1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RTPCR方法检测THP1细胞中P65、IκBαmRNA的表达。②用细胞裂解液分别提取THP1的细胞质和细胞核蛋白,采用WesternBlotting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THP1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12h、24h2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THP1细胞分为3组(空白对照、PBS对照和6μmol/LPN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THP1细胞,分5组(分别以0、2、4、6和8μmol/L的PN处理)培养,24h后收获细胞,分别检测细胞周期和细胞早期凋亡率。结果:①RTPCR结果示THP1细胞中有P65、IκBαmRNA的表达,Westernblotting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THP1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12h、24h的IC50分别为8~10μmol/L、6~8μmol/L。③以6μmol/L的PN处理后,THP1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/LPN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:THP1细胞中有NFκB通路的持续激活,PN对THP1细胞有抑制增殖和促进凋亡的作用。
Abstract:
To explore the activation condition of NFκB pathway in acute monocytic leukemia cell lines THP1, and the efficacy of pathenolide(PN), the inhibitor of NFκB, on proliferation and apoptosis of THP1 cells.Methods:THP1 cell was cultured in medium DMEM at 37 ℃ in the presence of 5% CO2. ①Cells in exponential growth stage were harvested.Total RNA was extracted from cells with Trizol reagent, and semiquantitative RTPCR was used to examine the P65, IκBα mRNA. ②The total cellular protein and the nuclear protein were extracted using Nuclear and Cytoplasmic Extraction Reagents Kit.Western blotting was used to detect their protein expression in cytoplasm extracts or nuclear extracts, respectively. ③Cells in 96well plates were allocated into 10 groups(the blank control, the negative control and the tested wells(1,2,4,6,8,10,15,20 μmol/L PN),cell proliferation was determined using Cell Counting Kit8;percentages of proliferation inhibition and 50%inhibiting concentration (IC50) were calculated at 12 h,24 h checktimes.④Cells were allocated into 3 groups (the blank control, the negative control, the group treated with 6 μmol/L PN), and cultured for 6 days, then cell growth curves were made. ⑤Cells were allocated into 5 groups and treated with 0, 2, 4, 6, and 8 μmol/L PN respectively,Flow cytometry was used to determine the distribution of cell cycle. Annexin VFITC staining was employed to analyze cell apoptosis.Results: ①RTPCR showed that P65 and IκBα mRNA expressed in THP1 cells.Western blotting showed that both P65 and IκBα protein expressed in cytoplasm, and some p65 protein expressed in nuclear. ②Growth inhibition was showed in THP1 cells treated with PN in a concentrationdependant manner and the highest inhibiting rate was (69.56±2.52)%;the IC50 of PN on THP1 for 12 h,24 h were 8~10 μmol/L,6~8 μmol/L,respectively.③The growth curve of cells treated with 6 μmol/L of PN was lower than that of controls.④The constituent ratio of cells in G0/G1 stage increased with the increasing of PN concentrations from 2 μmol/L to 6 μmol/L, which was up to (63.07±3.91)% in 6 μmol/L group. On the other hand,the constituent ratio of cells in S stage decreased which the increasing of PN concentrations,which was (23.55±4.34)% in 8 μmol/L group. ⑤The early apoptosis rates of the blank control, 2 μmol/L PNgroup,4 μmol/L PNgroup,6 μmol/L PNgroup and 8 μmol/L PNgroup were(3.84±1.50)%,(4.67±1.97)%,(12.67±2.13)%,(17.72±2.78)% and (23.62±3.36)% respectively, and the difference was significante(F=36.280,P<0.001).Conclusion: Constitutively activated NFκB signaling pathway exists in the THP1 cells. PN, a NFκB inhibitor, effectively inhibits cell proliferation and induces apoptosis.

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备注/Memo

备注/Memo:
#通讯作者,男,1957年生,硕士,教授,研究方向:儿科血液病学,Email:shenggy666@yahoo.com.cn
更新日期/Last Update: 2010-04-29