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纸质出版:2014
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孟兰,程民,沈国栋,徐婷娟,刘林青,唐杨琛,韩晓青,沈干,胡世莲.5-氮杂-2-脱氧胞苷和曲古抑菌素A对胃癌MGMT基因表达及NF-κB活性的影响[J].陆军军医大学学报(原第三军医大学学报),2014,36(13):1370-1375.
Meng Lan,Cheng Min,Shen Guodong,Xu Tingjuan,Liu Linqing,Tang Yangchen,Han Xiaoqing,Shen Gan,Hu Shilian.Effects of 5-Aza-dC combined with trichostatin A on MGMT expression and NF-κB activity in gastric carcinoma cells[J].J Amry Med Univ (J Third Mil Med Univ),2014,36(13):1370-1375.
孟兰,程民,沈国栋,徐婷娟,刘林青,唐杨琛,韩晓青,沈干,胡世莲.5-氮杂-2-脱氧胞苷和曲古抑菌素A对胃癌MGMT基因表达及NF-κB活性的影响[J].陆军军医大学学报(原第三军医大学学报),2014,36(13):1370-1375. DOI:
Meng Lan,Cheng Min,Shen Guodong,Xu Tingjuan,Liu Linqing,Tang Yangchen,Han Xiaoqing,Shen Gan,Hu Shilian.Effects of 5-Aza-dC combined with trichostatin A on MGMT expression and NF-κB activity in gastric carcinoma cells[J].J Amry Med Univ (J Third Mil Med Univ),2014,36(13):1370-1375. DOI:
目的 探讨5-氮杂-2-脱氧胞苷(5-Aza-2′-deoxycytidine,5-Aza-dC)和曲古抑菌素A(trichostatin A,TSA)对不同分化程度的人胃癌MKN-45和MGC-803细胞系增殖、凋亡,抑癌基因O
6
-甲基鸟嘌呤-DNA甲基转移酶(O
6
-Methylguanine-DNA Methyltransferase
MGMT)甲基化水平及其核转录因子NF-κB磷酸化程度影响。 方法 利用CCK-8、流式细胞术、MSP、RT-PCR、Western blot等方法检测5-Aza-dC(10 μmol/L)、TSA(1 μmol/L)单独或联合刺激后,MKN-45和MGC-803细胞增殖、凋亡,MGMT甲基化、mRNA、蛋白表达水平及NF-κB p65蛋白的磷酸化程度。 结果 ①5-Aza-dC、TSA单独或联合使用均可抑制MKN-45和MGC-803细胞的生长,与单药组相比,联合用药组抑制作用更明显(
P
0.05);②5-Aza-dC及TSA均可促进MKN-45和MGC-803细胞的凋亡,联合用药组凋亡率[(8.43±1.80)%和(21.75±3.06)%
]
均明显高于5-Aza-dc组[(7.33±1.18)% 、(15.22±1.03)%
]
和TSA组[(3.53±1.18)% 、 (12.66±1.10)%
]
(
P
0.05);③5-Aza-dC、TSA单独或联合刺激均可逆转MGMT基因甲基化,使其mRNA及蛋白表达水平上调,联合用药作用更加明显(
P
0.05);④NF-κB活性高表达的MKN-45和MGC-803经5-Aza-dC和TSA处理后细胞的NF-κB活性降低,且联合用药组[(12.81±4.18)%和(9.08±2.66)%
]
抑制程度较5-Aza-dc组[(39.13±3.38)%、(34.90±4.89)%
]
和TSA组[(27.55±5.00)%、(22.11±3.07)%
]
更加明显(
P
0.05)。 结论 5-Aza-dC与TSA均可抑制胃癌细胞生长和促进细胞凋亡,逆转MGMT基因甲基化反应,提高基因表达和抑制NF-κB的活性,二药联合作用提示效果更加明显。
Objective To determine the effects of 5-Aza-2′-deoxycytidine (5-
Aza-dC) and trichostatin A (TSA) alone
or in combination on the proliferation
apoptosis
the methylation level of O
6
-methylguanine-DNA methyltransferase (MGMT) and the phosphorylation of NF-κB in human gastric carcinoma cell lines with different differentiation grades (MKN-45 and MGC-803). Methods After MKN-45 and MGC-803 cells were treated by 5-Aza-dC
TSA and combination respectively
the cell viability was assayed by CCK-8 kit
and apoptosis by Annexin V-FITC/PI staining. The methylation level of MGMT promoter was determined by methylation specific PCR (MSP)
and mRNA expression by RT-PCR. Western blotting was employed to detect the expression of MGMT
NF-κB p65 and p-NF-κB p65 in the 2 cell lines. Results 5-Aza-dC or TSA monotherapy and combination significantly inhibited cell growth in a dose- and time- dependent manner in MKN45 and MGC-803 cells
and the combination treatment was more significant in the inhibition (
P
0.05). The monotherapies and combination treatment both improved cell apoptosis
and the combination group [(8.43±1.80)% and (21.75±1.06)%
]
had higher apoptotic rate than the 5-Aza-dC [(7.33±1.18)% and (15.22±1.03)%
]
and TSA [(3.53±1.18)% and (12.66±1.10)%
]
monotherapies(
P
0.05). The monotherapies and combination treatment all reversed MGMT promoter methylation
and increased its mRNA and protein levels
but the combination treatment showed more significant effects (
P
0.05). The expression levels of NF-κB p65 were significantly reduced in the MKN-45 and MGC-803 cells after monotherapies and combination treatment
and the combination treatment [(12.81±4.18)% and (9.08±2.66)%
]
showed stronger inhibitory effects than the 5-Aza-dC [(39.13±3.38)% and (34.90±4.89)%
]
and TSA [(27.55±5.00)% and (22.11±3.07)%
]
monotherapies(
P
0.05). Conclusion Both 5-Aza-dC and TSA inhibit the proliferation
induce the apoptosis
and enhance MGMT expression by changing the methylation and decreasing NF-κB activity in human gastric carcinoma MKN-45
and MGC-803 cells. And the combination treatment shows more significant effects than either of the 2 drugs.
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