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目的探讨DNA甲基化转移酶抑制剂5-氮-2′-脱氧胞苷(5-aza-dC)联合组蛋白去乙酰化抑制剂曲古抑菌素A(TSA)对人肺腺癌细胞株A549增殖和凋亡的影响。方法人肺腺癌细胞株A549细胞用含10%胎牛血清的DMEM培养液培养。实验分4组,5-aza-dC组:细胞接种20h后,加入5μmol/L的5-aza-dC;TSA组:细胞接种20h后,加入300nmol/LTSA;5-aza-dC+TSA组:细胞接种20h后,加入5μmol/L5-aza-dC,24h后再加入300nmol/L TSA;对照组:DMEM培养液中加入等量的二甲基亚砜(DMSO)。MTT比色法测定各处理因素对A549细胞生长的影响;Annexin V/PI双染法测定各组细胞培养72h后的凋亡情况;应用RT-PCR检测各组细胞培养72h后细胞CCAAT增强子启动蛋白α(C/EBPα)mRNA的表达。结果 (1)5-aza-dC及TSA呈时间依赖性地抑制A549细胞的生长,而在同一时间不同组别比较联合用药组细胞生长抑制率较单用药组明显增加[12h:35.51%vs21.63%,19.59%;24h:43.19%vs32.39%,30.60%;36h:54.85%vs38.83%,37.35%;48h:60.69%vs45.79%,43.84%;60h:68.92%vs50.60%,50.20%;72h:74.54%vs59.23%,57.82,P<0.05];(2)5-aza-dC和TSA能够诱导A549细胞凋亡,且联合用药组A549细胞凋亡率(75.35±1.46)%明显高于5-aza-dC(49.76±1.32)%(P<0.05)和TSA组(50.88±1.06)%(P<0.05);(3)5-aza-dC和TSA能够上调C/EBPαmRNA的表达,联合用药组中C/EBPαmRNA表达量(0.581±0.024)明显高于5-aza-dC组(0.402±0.016)(P<0.05)和TSA组(0.394±0.031)(P<0.05),有统计学意义;(4)5-aza-dC组与TSA组在抑制A549细胞生长、诱导细胞凋亡、增强C/EBPαmRNA的表达方面比较,差别均无统计学意义(P>0.05)。结论与单用5-aza-dC或TSA相比,联合应用两种药物能够更好的抑制人肺腺癌A549细胞的增殖、诱导细胞凋亡和增强C/EBPα基因mRNA的表达。
Objective To investigate the effect of 5-aza-dC combined with histone deacetylation inhibitor trichostatin A (TSA), a DNA methyltransferase inhibitor, on human lung adenocarcinoma cells Effect of strain A549 on proliferation and apoptosis. Methods Human lung adenocarcinoma cell line A549 cells were cultured in DMEM containing 10% fetal bovine serum. 5-aza-dC group: 5μmol / L 5-aza-dC was added after the cells were inoculated for 20h; TSA group: 300nmol / L TSA was added 20h after the cells were inoculated; Cells were inoculated with 5μmol / L 5-aza-dC for 20h and then added with 300nmol / L TSA for 24 hours. The control group: DMEM medium was added with DMSO. MTT assay was used to determine the effect of various treatment factors on the growth of A549 cells; Annexin V / PI double staining method was used to determine the apoptosis of the cells in each group after 72 hours; The CCAAT enhancer promoter Protein α (C / EBPα) mRNA expression. Results (1) 5-aza-dC and TSA inhibited the growth of A549 cells in a time-dependent manner. However, the cell growth inhibition rates of the 5-aza-dC and TSA groups at different time points were significantly increased compared with those of the single drug group [12h: 35.51% vs21. 63%, 19.59%; 24h: 43.19% vs32.39%, 30.60%; 36h: 54.85% vs38.83%, 37.35%; 48h: 60.69% vs45.79%, 43.84%; 60h: 68.92% vs50.60% , Respectively. The apoptotic rates of A549 cells in combination group were significantly higher than those in the control group (P <0.05); 50.20%; 72h: 74.54% vs59.23%, 57.82, P <0.05] ) Was significantly higher than that of 5-aza-dC (49.76 ± 1.32)% (P <0.05) and TSA group (50.88 ± 1.06)% (P <0.05) (0.581 ± 0.024) in combination group was significantly higher than that in 5-aza-dC group (0.402 ± 0.016) and TSA group (0.394 ± 0.031) (P <0.05) (P <0.05). (5) There was no significant difference between 5-aza-dC group and TSA group in inhibiting A549 cell growth, inducing apoptosis and enhancing C / EBPαmRNA expression. Conclusions Compared with 5-aza-dC alone or TSA, the combination of these two drugs can better inhibit the proliferation of human lung adenocarcinoma A549 cells, induce apoptosis and enhance the expression of C / EBPα gene mRNA.