摘要
目的观察DNA甲基转移酶抑制剂5-氮杂-2’-脱氧胞苷（5-AZa—CdR）对人肺腺癌A549细胞的增殖、凋亡的影响及其对A549细胞中Apaf-1基因表达的影响。方法不同浓度的5-Aza—CdR处理A549细胞后，采用噻唑蓝（MTT）比色法检测细胞生长抑制率；采用流式细胞术检测处理72h后的细胞周期及凋亡情况；逆转录-聚合酶链反应（RT—PCR）法观察Apaf-1基因及甲基转移酶的mRNA水平；Westernblot法检测Apaf-1蛋白的表达变化。结果各浓度的5-Aza—CdR处理A549细胞后，A549细胞的生长均呈抑制状态，有时间和剂量依赖性。而流式细胞仪分析表明，各浓度的5-Aza—CdR作用72h后，A549细胞的G0／G1期比例增加，S期比例明显减少，细胞凋亡率明显增加。RT—PCR结果显示，5-Aza—CdR处理组中DNA甲基转移酶的mRNA表达明显受抑，而Apaf-I基因的mRNA表达却明显增加。Westernblot结果显示Apaf-1基因的蛋白表达明显增加，而阴性对照组中未观察到上述变化。结论5-Aza—CdR可抑制A549细胞的生长，并通过抑制DNA甲基转移酶的表达而使Apaf-1基凶在人肺腺癌细胞株A549中重新表达。
Objective To explore the effect of DNA methyltransferase inhibitor 5-Aza-2＇-deoxycytidine （5-Aza-CdR） on the growth of human lung cancer cell line A549 and study the effect of 5-Aza- CdH on the expression of Apaf-l gene in A549 cell line. Methods A549 cells were treated with 5-AzaCdR at different concentrations. The growth inhibition rate of A549 cells was detected by MTT assay. Changes in cell cycle distribution and apoptosis rate were detected by flow cytometry 72 h after treatment with 5- Aza-CdR. The expression of methyhransferase mRNA and Apaf-1 mRNA was observed by RT-PCR, and the expression of Apaf-1 protein detected by Western blot respectively. Results The growth inhibition rate of A549 cells was increased in a concentration-and time-independent manner. The proportion of apoptotic cells and the cells in G1 phase were significantly increased 72 h after treatment with 5-Aza-CdR,while the cells in S phase were reduced. The expression of Apaf-1 mRNA and protein was increased, while the expression of methyhransferase mRNA decreased after treatment with 5-Aza-CdR. Conclusion 5-Aza-CdR could reduce the growth of tumor cells by inhibiting DNA methltransferase and reactivating tumor suppressor Apaf-1 gene in A549 cell line.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2009年第3期324-326,共3页
Chinese Journal of Experimental Surgery
基金
基金项目：国家自然科学基金资助项目（30570961）
