Armengol Carolina, Tarafa Gemma, Boix Loreto, Solé Manel, Queralt Rosa, Costa Dolors, Bachs Oriol, Bruix Jordi, Capellá Gabriel
BCLC Group, Liver Unit, Digestive Disease Institute, Hospital Clínic, Institut d'Investigació Biomèdica August Pi i Sunyer (IDIBAPS), Barcelona, Spain.
Clin Cancer Res. 2004 Mar 15;10(6):2150-7. doi: 10.1158/1078-0432.ccr-03-1028.
To allow the longitudinal investigation of molecular events associated with the progression of human hepatocellular carcinoma (HCC), we sought to develop a murine model by orthotopic implantation of tumor fragments obtained from patients diagnosed at early stage.
Tumor pieces (2 x 2 mm) were implanted on the liver surface of nu/nu mice. After xenograft growing, subsequent passages were performed to achieve long-term implant viability. Isolation of tumoral hepatocytes was done to establish new cell lines. HCC characteristics, proliferation rate, apoptotic index (terminal deoxynucleotidyl transferase-mediated nick end labeling), and expression of cell-cycle regulators (cyclins E and A, p21(Cip1), p27(Kip1), p16(INK4a), pRb, and p53) were assessed by Western Blot and immunohistochemistry, to correlate them with tumor progression.
Five (50%) of the 10 primary HCCs resulted in small slow-growing liver implants. Three of them are viable after 48 months, whereas the remaining two survived for 15 and 13 months. Xenografts throughout passages exhibited a more aggressive phenotype with a poorer degree of differentiation, intense proliferation, moderate apoptosis, cell-cycle deregulation, p53 alterations, microvascular invasion, and dissemination. In one single passage, we observed critical growth delay, which was associated with significant p27(kip1) overexpression. We established the anchor-free growing BCLC-9 cell line from one xenograft. This has gains of chromosomes 7, 5p, 6q, and 9q, is hepatitis B virus-DNA positive, does not secrete alpha-fetoprotein, and has TP53 missense mutations in codons 192 and 242.
The orthotopic implantation of early HCC fragments in nude mice provides a useful model to investigate the mechanisms of human HCC evolution and to establish new cell lines.
为了能够纵向研究与人类肝细胞癌（HCC）进展相关的分子事件，我们试图通过原位植入从早期诊断患者获取的肿瘤碎片来建立一种小鼠模型。
将肿瘤块（2×2毫米）植入裸鼠的肝脏表面。异种移植物生长后，进行后续传代以实现长期植入存活。分离肿瘤肝细胞以建立新的细胞系。通过蛋白质免疫印迹法和免疫组织化学评估HCC特征、增殖率、凋亡指数（末端脱氧核苷酸转移酶介导的缺口末端标记）以及细胞周期调节因子（细胞周期蛋白E和A、p21（Cip1）、p27（Kip1）、p16（INK4a）、视网膜母细胞瘤蛋白（pRb）和p53）的表达，以将它们与肿瘤进展相关联。
10例原发性HCC中有5例（50%）形成了生长缓慢的小肝脏植入物。其中3例在48个月后仍存活，而其余2例分别存活了15个月和13个月。传代后的异种移植物表现出更具侵袭性的表型，分化程度较差、增殖强烈、凋亡适度、细胞周期失调、p53改变、微血管侵犯和播散。在一次传代中，我们观察到关键的生长延迟，这与p27（kip1）的显著过表达相关。我们从一个异种移植物中建立了无锚定生长的BCLC - 9细胞系。该细胞系有7号、5p、6q和9q染色体增加，乙肝病毒DNA阳性，不分泌甲胎蛋白，并且在密码子192和242处有TP53错义突变。
将早期HCC碎片原位植入裸鼠提供了一个有用的模型，用于研究人类HCC演变的机制并建立新的细胞系。
