摘要
目的探讨5-脱氧杂氮胞苷（5-Aza—CdR）对肝癌细胞分泌exosomes及其负载的肿瘤相关抗原和免疫相关分子含量的影响。方法采用离心超滤联合蔗糖密度梯度离心方法，分离和纯化经5-Aza—CdR处理和未处理的HepG2和Hep3B肝癌细胞释放的exosomes，并对exosomes进行计数和蛋白定量测定。采用免疫电镜和Western blot技术，观察exosomes表达HSP70、HLA—I和NY—ESO-1蛋白的变化；以逆转录聚合酶链反应（RT—PCR）检测5-Aza—CdR处理前后HepG2和Hep3B细胞野生型p53基因mRNA的表达。结果经5-Aza—CdR处理后，HepG2和Hep3B细胞p53基因mRNA表达较未处理组均明显增加，exosomes数量和蛋白含量均较未处理组显著增加（P〈0．05）。经免疫电镜和Western blot鉴定，exosomes上均附载有HSP70、HLA—I和NY—ESO—1蛋白，且两种细胞来源的exosomes经5-Aza—CdR处理后，HSP70、HLA—I和NY—ESO-1蛋白含量均明显增加。结论DNA甲基转移酶抑制剂5-Aza—CdR可使肝癌细胞分泌更多数量的exosomes，增加exosomes中的免疫相关分子含量，其机制可能与p53基因上调和DNA去甲基化有关。
Objective To investigate the effects of 5-Aza-deoxycytidine （5-Aza-CdR） on the amount of exosomes and immuno-associated proteins produced in hepatoma HepG2 and Hep3B cells. Methods Exosomes derived from HepG2 and Hep3B cells with or without treatment by 5-Aza-CdR were isolated and purified by combination of ultrafiltration centrifugation and sucrose gradient ultracentrifugation. The number of exosomes was counted under the electron microscope. The concentration of proteins in exosomes was detected by BCA. The expression of HSP70, HLA-I and NY-ESO-1 proteins in exosomes was assayed by Western blot and immuno-electron microscopy. The mRNA expression of p53 gene was observed by reverse transcription-polymerase chain reaction （RT-PCR）. Results The mRNA expression of p53 gene was increased in both hepatoma cell lines after treatment with 5-Aza-CdR. The number of exosomes and the concentration of total proteins in exosomes were significantly increased after treatment by 5-Aza-CdR （ P 〈 0.05 ）. The immuno-electron microscopy and Western blotting showed that after treatment with 5-Aza-CdR, the contents of HSP70, HLA-I and NY-ESO-1 proteins were increased in exosomes in both HepG2 and Hep3B hepatoma cells. Conclusion 5-Aza-CdR, an inhibitor of DNA methyhransferase, can increase the amount of exosomes and exosome-containning immuno-associated proteins secreted by hepatoma cells. It may be contributed by up-regulation of p53 gene and demethylation mechanism of 5-Aza-CdR.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2009年第7期495-499,共5页
Chinese Journal of Oncology
基金
首都医学发展科研基金（2005-3086）
