摘要
目的 研究甲胎蛋白（AFP）对肝癌细胞肿瘤坏死因子相关凋亡诱导配体（TRAIL）受体-2（DR5）表达及其在肝癌细胞耐受TRAIL中的作用.方法 用Western blot法分析全反式维甲酸（ATRA）处理人肝癌细胞株Bel 7402细胞24 h后DR5表达的变化;免疫共沉淀（Co-IP）技术研究AFP与维甲酸受体（RAR）-β相互作用;激光共聚焦显微镜观察AFP与RAR-β的细胞共定位; RNA干扰技术抑制Bel 7402细胞AFP表达,再用ATRA处理24h后检测细胞内DR5表达的变化;用pcDNA3.1质粒和人AFP基因连接构建表达AFP的载体（称为pcDNA3.1-afp）,然后转染到不表达AFP的人肝癌细胞株HLE细胞;细胞生长状况用四甲基偶氮唑盐法检测.组间比较用f检验进行统计学分析.结果 Bel 7402和HLE细胞低表达DR5,ATRA（160μmol/L）处理24h后能促进肝癌细胞DR5表达; Co-IP技术研究显示AFP能与RAR-β结合;共聚焦显微镜观察发现AFP与RAR-β共定位于细胞质;干扰AFP表达后,Bel 7402细胞的DR5表达明显提高,抑制AFP表达后,ATRA能显著促进Bel 7402细胞内DR5的表达,并增加Bel 7402细胞对TRAIL的敏感性;转染pcDNA3.1-afp载体后,HLE细胞内的AFP能与RAR-β结合,并发现pcDNA3.1-afp载体能对抗TRAIL诱导HLE细胞凋亡.结论 Bel 7402细胞内表达的AFP具有抑制DR5表达的生物学功能;AFP可能通过抑制RAR-β入核调节DR5的表达;细胞内高表达的AFP是导致Bel 7402细胞耐受TRAIL诱导细胞凋亡的重要原因.
Objective To explore the mechanism ofAlpha-fetoprotein （AFP） effects on hepetocellular cancinoma cells （HCC） resistances apoptosis induced by tumor necrosis factor-related apoptosis inducingligand （TRAIL）. Methods The expressed alteration of TRAIL recepator-2 （DR5） after the human hepatoma cells line Bel 7402 （AFP-producing） and HLE cells （non-AFP producing） were treated with all trans retinoic acid （ATRA） were determined by Western blot; Interaction of AFP with RAR-beta was analyzed by co-immunoprecipitation （Co-IP）; Laser confocal microscopy was used to observe co-localization of AFP and RAR-beta; Short small RNA interfering （RNAi） was applied to knock down the expression of AFP in Bel 7402 cells; The full AFP gene cDNA was inserted into pcDNA3.1 vector and constructed the expressed vector of AFP （named pcDNA3, 1-afp）; The growth of hepatoma cells was analyzed by MTT. Results Bel 7402 and HLE cells expressed DR5, lowed dosage of ATRA （40 μmol/L） had no influence on the expression of DR5 in Bel 7402 cells, but ATRA （160 μ mol/L） could inhibit the expression of AFP and promote the expression of DR5 significantly; Co-IP indicated that AFP had a property for interacting with RAR-beta; The results also demonstrated AFP co-localization with PAR-beta in cytoplasm of Bel 7202 cells; The expression of DR5 was enhanced while the expression of AFP was knocked down by RNAi. pcDNA3.1-afp vector was transfected into HLE cells, the growth of HLE cells were stimulated and TRAIL cytotoxicity of HLE cells were reduced. But when the expression of AFP was knocked down the sensitivity of Bel 7402 cells to TRAIL was enhanced. Conclusions These data provided that AFP had a capability to interact with RAR-beta and suppressed the expression of DR5. AFP could play pivotal role on hepatoma cells resistanceinduced apoptosis by TRAIL.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2010年第10期745-750,共6页
Chinese Journal of Hepatology
基金
基金项目：国家自然科学基金（30760090
30960153
31060164）
海南省自然科学基金（309034）
海南省卫生厅基金（2009-50）
关键词
癌
肝细胞
甲胎蛋白
TRAIL受体2
细胞凋亡
Carcinoma, hepatocellular, Alpha-fetoprotein
TRAIL receptor-2
Apoptosis
