| 潘韵芝,马 赛,曹凯悦.高表达ANXA2肝癌干细胞外泌体对肝癌细胞恶性生物学特性的调控作用[J].中国肿瘤,2019,28(4):308-314. |
| 高表达ANXA2肝癌干细胞外泌体对肝癌细胞恶性生物学特性的调控作用 |
| Exosomal ANXA2 of Cancer Stem Cell Regulates Biological Behavior of Liver Cancer |
| 投稿时间：2018-03-14 |
| DOI：10.11735/j.issn.1004-0242.2019.04.A011 |
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| 中文关键词: 肿瘤干细胞 外泌体 ANXA2 自我更新 侵袭 转移 |
| 英文关键词:cancer stem cell exosomes ANXA2 self-renewal capacity invasion migration ability |
| 基金项目:苏州市感染性疾病临床医学中心项目（SZZX201508） |
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| 中文摘要: |
| 摘 要：［目的］ 探讨肝癌干细胞来源外泌体中差异表达的蛋白质，重点分析外泌体ANXA2分子对肝癌恶性生物学特性（自我更新、侵袭和转移能力）的影响。［方法］ 采用无血清悬浮培养法和干细胞标志物流式分选术分离人肝癌干细胞；分别采用干细胞球形成实验、体外侵袭转移能力鉴定分离所得肝癌干细胞；采用蛋白质谱分析肝癌干细胞来源外泌体中的差异表达蛋白质；siRNA靶向抑制ANXA2表达后，分别进行肝癌细胞球形成实验、体外侵袭与迁移实验检测外泌体ANXA2对人肝癌细胞自我更新、侵袭、转移能力的影响；收集肝癌患者和正常人血浆，通过凝胶排阻层析联合CD63免疫磁珠分离纯化外泌体，Western blot检测外泌体中ANXA2表达情况，结合临床病理资料，综合分析外泌体ANXA2的临床相关性。［结果］ 成功分离得到肝癌干细胞CD133+ SK-Hep-1，其具有典型的肿瘤干细胞生物学特性。与CD133- SK-Hep-1细胞外泌体相比，CD133+ SK-Hep-1肝癌干细胞来源外泌体中具有多种差异表达蛋白，其中外泌体ANXA2表达明显上调。siRNA靶向抑制肝癌干细胞ANXA2表达后，其外泌体中ANXA2表达也下降。分别采用PBS、siRNA靶向抑制肝癌干细胞ANXA2后外泌体（Exo-LCSCs-siANXA2）、肿瘤干细胞来源外泌体（Exo-LCSCs）刺激肝癌细胞系HepG2、SNU398，其自我更新、侵袭和转移能力依次增强。Western blot结果显示，肝癌患者血浆外泌体ANXA2表达明显高于健康人血浆；肝癌患者血浆外泌体ANXA2表达与患者年龄、性别无明显相关性，而与肿瘤TNM分期和转移显著相关。［结论］ 高表达ANXA2的肝癌干细胞的外泌体对肝癌细胞恶性生物学特性具有调控作用。 |
| 英文摘要: |
| Abstract：［Purpose］ To investigate the expression of exosomal proteins in liver cancer stem cells(CSCs) and the effect of exosomal protein ANXA2 on biological behavior of liver cancer. ［Methods］ The serum-free suspension culture and flow cytometry sorting technology were used to isolate CSCs from liver cancer. Biological characteristics of liver CSCs were detected by sphere colony formation assay，invasion and migration assay in vitro. Protein mass spectrometry was used to analyze the differentially expressed proteins in CSCs-derived exosomes. After transfection with ANXA2 siRNA，the changes of biological behaviors of liver cancer were examined by sphere colony formation assay，invasion and migration assay. Exosomes were isolated from plasma of liver cancer patients and healthy subjects by gel exclusion chromatography combined with CD63 immunomagnetic beads method. Western blot was used to detect exosomal-ANXA2 expression，and the association of exosomal-ANXA2 expression with clinicopathological features of liver cancer was analyzed. ［Results］ Liver CSCs were successfully isolated by using the serum-free suspension culture and fluorescence-activated cell sorting. Functional studies revealed that CD133+SK-Hep-1 had cancer stem cell-like characteristics. Protein mass spectrometry analysis illustrated that the expression of exosomal-ANXA2 was significantly up-regulated in CD133+SK-Hep-1 cells. After inhibition of ANXA2 in CD133+SK-Hep-1 cells，the expression of exosomal-ANXA2 was down-regulated. The self renewal，invasive and metastatic ability of liver cancer HepG2 and SNU398 cells were increased in PBS，exo-LCSCs-siANXA2 and exo-LCSCs groups. Western blot results showed that the expression of exosomal-ANXA2 in liver cancer patients was significantly higher than that in healthy subjects. No significant association between exosomal-ANXA2 expression and age，sex of patients was detected in 50 liver cancer patients. However，the levels of exosomal-ANXA2 were significantly correlated with tumor differentiation，TNM stage and depth of invasion. ［Conclusion］ The study indicates that exosomes of cancer stem cells with high expression of ANXA2 regulate biological characteristics of liver cancer. |
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