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中国癌症防治杂志

中国癌症防治杂志 ›› 2015, Vol. 7 ›› Issue (1): 6-12.doi: 10.3969/j.issn.1674-5671.2015.01.02

• 基础研究 • 上一篇    下一篇

肝细胞癌合并门静脉癌栓细胞的分离、培养及其特征的研究

  

  1. 广西医科大学附属肿瘤医院肝胆外科；广西医科大学研究生学院
  • 出版日期:2015-02-25 发布日期:2015-02-28
  • 通讯作者: 周韶璋。E-mail：179913337@qq.com
  • 基金资助:

    国家自然科学基金资助项目（81160262）

Isolation， culture and characterization of portal venous tumor thrombus cells from patients with hepatocellular carcinoma （HCC） with or without portal vein tumor thrombosis

  • Online:2015-02-25 Published:2015-02-28

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摘要：

目的 分别选取肝细胞癌（hepatocellular carcinoma，HCC）合并门静脉癌栓（portal  vein tumor thrombus，PVTT）和HCC无PVTT的原发灶组织进行原代细胞分离、培养，并将培养稳定的细胞继续体外培养并行体外实验，初步对同一患者的HCC有无PVTT的特征进行研究。方法 取人HCC合并PVTT和HCC无PVTT的新鲜手术组织标本，利用酶消化法进行肿瘤细胞原代短暂培养并稳定传代。将得到稳定培养的细胞分为HCC无PVTT细胞组、HCC合并PVTT细胞组和PVTT细胞组，分别简称为HCC-PVTT（-）细胞组、HCC-PVTT（+）细胞组和PVTT细胞组。以流式细胞术检测仪检测CD90+细胞含量，采用qRT-PCR、Western blot法检测细胞培养后的HCC干细胞相关基因及其蛋白的表达。用Transwell小室检测细胞的侵袭性。结果 成功培养出原代的HCC细胞和原代PVTT细胞，并可稳定地传代。细胞表面标志物的干细胞含量、干细胞相关基因及其蛋白表达量及侵袭性均表现为PVTT细胞组> HCC-PVTT（+）细胞组，HCC-PVTT（+）细胞组> HCC-PVTT（-）细胞组，差异均有统计学意义（P均<0.05）。结论 HCC及PVTT组织中均存在CD90+肝癌干细胞，干细胞干性越强的原发灶具有更强的侵袭和转移能力。

关键词: 肝肿瘤, 肿瘤干细胞, 人门静脉癌栓细胞, CD90, SALL4, CK19

Abstract:

Objective To explore the stem cell-like properties of primary cultures of hepatocellular carcinoma （HCC） cells and portal vein tumor thrombus（PVTT） cells. Methods Fresh hepatocellular carcinoma（HCC） with portal vein tumor thrombosisand（PVTT）or not were used for short-term primary culture and stability batches by utilizing enzymatic digestion. We would be classify the stability of the cultured cells as HCC without PVTT, the T-primary tumor of HCC with PVTT and PVTT,called section of HCC-PVTT（-）,HCC-PVTT（+）and PVTT. Flow cytometry detect the content of CD90+ and the expressions of relative gene and protein from HCC with PVTT or not，which were tested by qRT-PCR and Western blot methods. Testing the cells of invasion by Transwell chamber. Results We successfully cultured primary HCC cells and PVTT cells. Flow cytometry revealed the presence of a CD90+ subpopulation in both types of cultures. The different cultures varied in invasiveness as follows：PVTT cells>HCC-PVTT（+） cells>HCC-PVTT（+） cells>HCC-PVTT（-）cells（P<0.05）. Conclusions Stem cell-like populations exist in HCC and PVTT tissue，and their stem cell characteristics correlate positively with tumor invasion and metastasis.

Key words: Liver neoplasm, Cancer stem cells, Portal vein tumor thrombus cells, CD90, SALL4, CK19