Sherif Rania Naiem, Abdellatif Hussein, Hazem Noha, Ebrahim Neven A, Saleh Dalia, Shiha Gamal, Eltahry Huda, Botros Kamal G, Gabr Omar M
Department of Human Anatomy and Embryology, University of Mansoura, Faculty of Medicine, Mansoura, Egypt.
Department of Human Anatomy and Embryology, University of Mansoura, Faculty of Medicine, Mansoura, Egypt; Department of Anatomy, College of Medicine, University of Bisha, Bisha, Saudi Arabia.
Tissue Cell. 2018 Feb;50:125-132. doi: 10.1016/j.tice.2018.01.002. Epub 2018 Jan 8.
Hepatocellular carcinoma (HCC) is the most frequent primary liver malignancy. Chronic liver injuries as chronic hepatitis C and hepatitis B viruses, aflatoxins consumption and nonalcoholic fatty liver disease are well-established causes of HCC. HCC is associated with a series of molecular changes, as alternation in glypican-3, P53 expression and Wnt/β-catenin pathway. Hepatic cancer progenitor cells could contribute to HCC development. This research aimed to study the effectiveness of human CD34+ hematopoietic stem cell on Wnt4 and P53 genes expression, histopathological grading and hepatic progenitor cells percentage in HCC rat model.
HCC was induced in the experimental group of outbred Sprague Dawley rats by administration of 50 mg/L N-nitroso-Di-Ethylamine (DEN) in drinking water for 15 weeks. Forty-six animals were used in total, they were initially subdivided into two groups; control (n = 6) and experimental (n = 40), the latter consisting of 4 DEN-unaffected, 6 DEN-lethalities and 30 surviving DEN-animals with elevated AFP. These 30 animals with elevated AFP were then subdivided into a new HCC control group (n = 15) and the stem cell treated group (n = 15). The latter group was injected with CD34 human hematopoietic stem cell (1 × 10 cells/rat) in the rat's tail vein. Cyclosporine A (10 mg/kg) was injected intraperitoneal, starting 24 h before human stem cell transplantation. After 20 weeks passing since the beginning of the experiment, all rats were sacrificed and liver specimens were subjected to histopathological examination, RT-PCR in order to examine Wnt4 and P53 gene expression and flow cytometry to measure hepatic progenitor OV6 positive cells percentage.
The saline-treated HCC group (with prior 15 week DEN exposure) showed higher levels of wnt4 and p53 gene expression (1.59 and 1.36 fold, respectively) and increased percentage in OV6+ progenitor cells (+4.9% in absolute terms) compared to saline-treated controls (p < 0.01, ANOVA). Compared with the saline HCC-group, transplantation with CD34+ human hematopoietic stem cells produced a further increase in the levels of wnt4 (+19.4%) and p53 gene expression (+53%), a 2-fold increase in the percentage of cancer progenitor cells and increased HCC pathology grading (all p < 0.01). The positive correlation between p53 and HCC grade (Spearman rho +0.73, p < 0.05) and negative correlation between wnt and OV6% levels (rho -0.65, p < 0.05) in the saline-HCC group were not observed in the CD34+ HCC group.
Human CD34 cells transplantation has a deteriorating effect on HCC.
肝细胞癌（HCC）是最常见的原发性肝脏恶性肿瘤。慢性丙型肝炎和乙型肝炎病毒、黄曲霉毒素摄入以及非酒精性脂肪性肝病等慢性肝损伤是HCC的公认病因。HCC与一系列分子变化相关，如磷脂酰肌醇蛋白聚糖-3的改变、P53表达以及Wnt/β-连环蛋白信号通路的变化。肝癌祖细胞可能在HCC的发展中发挥作用。本研究旨在探讨人CD34+造血干细胞对HCC大鼠模型中Wnt4和P53基因表达、组织病理学分级以及肝祖细胞百分比的影响。
在远交群斯普拉格-道利大鼠的实验组中，通过在饮用水中给予50mg/L的N-亚硝基二乙胺（DEN）持续15周来诱导HCC。总共使用了46只动物，最初分为两组：对照组（n = 6）和实验组（n = 40），后者包括4只未受DEN影响的动物、6只因DEN死亡的动物以及30只甲胎蛋白（AFP）升高的存活DEN处理动物。然后将这30只AFP升高的动物进一步分为新的HCC对照组（n = 15）和干细胞治疗组（n = 15）。后一组通过大鼠尾静脉注射CD34人造血干细胞（1×10个细胞/只大鼠）。从人干细胞移植前24小时开始，腹腔注射环孢素A（10mg/kg）。自实验开始20周后，处死所有大鼠，取肝脏标本进行组织病理学检查、逆转录聚合酶链反应（RT-PCR）以检测Wnt4和P53基因表达，以及流式细胞术以测量肝祖细胞OV6阳性细胞百分比。
与生理盐水处理的对照组相比，经15周DEN暴露后用生理盐水处理的HCC组显示出更高水平的wnt4和p53基因表达（分别为1.59倍和1.36倍），且OV6+祖细胞百分比增加（绝对值增加4.9%）（p < 0.01，方差分析）。与生理盐水处理的HCC组相比，移植CD34+人造血干细胞使wnt4水平进一步升高（+19.4%）和p53基因表达升高（+53%），癌祖细胞百分比增加2倍，且HCC病理分级增加（所有p < 0.01）。在CD34+ HCC组中未观察到生理盐水-HCC组中p53与HCC分级之间的正相关（Spearman秩相关系数+0.73，p < 0.05）以及wnt与OV6%水平之间的负相关（秩相关系数-0.65，p < 0.05）。
人CD34细胞移植对HCC有恶化作用。
