文档介绍：5-Aza-CdR对人食管癌细胞株生物学行为及TFPI-2 mRNA表达的影响
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5-Aza-CdR对人食管癌细胞株生物学行为及TFPI-2 mRNA表达的影响
作者:杜雅冰,邵应举,樊青霞,孙桢,王琳,赵培荣作者单位:(郑州大学第一附属医院肿瘤内科,河南郑州 450002)
【摘要】目的探讨5-氮杂-2-脱氧胞苷(5-Aza-2-deoxycytidine,5-Aza-CdR)干预对食管鳞癌细胞株Eca9706生长增殖及其组织因子途径抑制物2(tissue factor pathway inhibitor-2,TFPI-2)基因表达的影响。方法选取食管鳞癌细胞株Eca9706,并用不同浓度的5-Aza-CdR处理该细胞株。MTT法检测干预前后细胞增长率的变化,FCM法检测干预前后细胞凋亡率的变化,RT-PCR技术检测干预前后Eca9706细胞TFPI-2基因mRNA的表达,免疫组化检测干预前后Eca9706细胞TFPI-2蛋白的表达。结果食管鳞癌细胞株Eca9706存在TFPI-2基因超甲基化状态,经5-Aza-CdR处理后,超甲基化状态解除,细胞增殖受到抑制,细胞凋亡率明显提高(P<);细胞中TFPI-2蛋白表达明显增强,同时mRNA的表达水平也较处理前明显提高(P<)。结论食管癌细胞系TFPI-2基因超甲基化可抑制其mRNA表达,当其超甲基化解除后,细胞增殖受到抑制,同时细胞凋亡率相应提高。
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【关键词】食管癌;组织因子途径抑制物-2;5-氮杂-2-脱氧胞苷;甲基化;免疫组化;逆转录聚合酶链反应;凋亡
ABSTRACT: Objective To explore the effects of 5-Aza-2-deoxycytidine (5-Aza-CdR) intervention on the growth and proliferation of Eca9706 cell line and protein expression of tissue factor pathway inhibitor-2 (TFPI-2). Methods Eca9706 cell line was treated by 5-azaCdR of different concentrations; MTT, flow cytometry, immunohistochemistry and RT-PCR were used to determine the cell growth, apoptosis, and the expression of TFPI-2 gene and its protein. Results Eca9706 cell line had hypermethylation of TFPI-2. After demethylation by 5-Aza-CdR treatment, the proliferation of Eca9706 was inhibited, the apoptosis rate was also increased significantly in the concentration-dependent manner. RT-PCR detected that mRNA expression of TFPI-2 gene in Eca9706 cell line recovered significantly (P<). Conclusion 5-Aza-CdR can slow the growth of Eca9706 cell, increase the apoptosis rate, reactivate the TFPI-2 gene transcription and protein expression by demethylation.
KEY WORDS: esophageal carcinoma; tissue factor pathway inhibitor-2; 5-Aza-CdR; methylation; immunohistochemistry; RT-PCR; apoptosis
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组织因子途径移植物2(tissue factor pathway inhibitor, TFPI-2)是丝氨酸蛋白酶抑制物,在调控肿瘤细胞浸润转移方面起着重要作用。目前,关于TFPI-2在食管癌中的基因表达情况的分析研究尚少。本研究旨在探讨TFPI-2基因的失活机制,并寻找食管癌治疗的新靶点。
1 材料与方法
材料
RPMI-1640培养基购自北京索莱宝生物科技有限公司;标准胎牛血清购自天津市灏洋生物制品科技有限公司;