肝癌细胞条件培养液诱导新生儿脐带血贴壁细胞的转化

Transformation of Attached Cells Derived from Fetal Umbilical Cord Blood Induced by Conditional Medium of Hepatoma Cells

背景与目的:脐带血干细胞为早期未分化细胞,对外界环境的影响十分敏感,在致癌因素作用下脐带血干细胞可能发生恶性转化。本研究旨在探讨脐带血干细胞对致癌因子的敏感性。方法:应用淋巴细胞分离液从新生儿脐带血中分离出有核细胞后进行细胞培养,当细胞贴壁后,应用含有10%HepG2肝癌细胞条件培养液的培养液继续培养,获得了一个新的细胞系,将其命名为H-UCB。应用电镜、流式细胞仪和Westernblot等检测其特征,应用软琼脂实验检测其克隆形成率。结果:在培养到第三代以后H-UCB细胞增殖能力明显增强。形态学观察显示,细胞形态呈成纤维细胞样和肝细胞样,核浆比例增大。电镜观察,细胞表面有较多微绒毛,核大并且形态不规则,胞浆内有空泡,有胞吞胞饮现象。染色体分析显示,细胞的染色体众数在50～70条之间,为异倍体。流式细胞仪检测显示,细胞增殖周期为22.9h,染色体数目为二倍体和四倍体之间。Westernblot检测结果显示,c-Myc蛋白、核增殖相关蛋白(PCNA)的表达水平较高。经免疫荧光染色后进行流式细胞仪检测,显示H-UCB细胞表面标志物CD105、CD34、CD106阳性率分别为79.0%、1.2%、12.2%,而对照组HepG2细胞的CD105、CD34、CD106的阳性反应率分别为15.0%、9.8%、1.4%。软琼脂克隆形成率为(13.2±2.6)%。结论:从新生儿脐带血分离获得的有核细胞在肝癌细胞条件培养液作用下发生了恶性转化,具有肿瘤细胞的特征,属内皮细胞来源。

BACKGROUND ＆ OBJECTIVE： Stem cells derived from fetal umbilical cord blood are of undifferentiated at early stage. They are sensitive to stimulations from the environment, and may be transformed under the effects of carcinogenic factors. This study was to explore the sensitivity of stem cells derived from fetal umbilical cord blood to carcinogenic factors. METHODS： Mononuclear cells were isolated from fetal umbilical cord blood, and the attached cells were cultured in the medium containing 10% conditional medium of HepG2 hepatoma cells. A new cell line was gained, termed H-UCB. The biological features of H-UCB cells were detected by electron microscopy, karyotype analysis, cell cytometry, Western blot, and colony formation assay. RESULTS： H-UCB cells proliferated faster after passage 3. The cells were fibroblast-like and hepatocyte-like, with the ratio of nucleus to cytoplasm increased. Under electron microscope, many microvilli on the surface and numbers of vacuoles in the cytoplasm of the cells were observed, the nuclei were large and irregular, endocytosis phenomena were noticed. Karyotype analysis indicated that the cells were heteroploid, and the number of chromosomes was between 50 and 70. Flow cytometry data indicated that the proliferation period was 22.9 h, and the karyotype was between diploid and tetraploid. Western blot showed that c-Myc protein and proliferating cell nuclear antigen （PCNA） were overexpressed in H-UCB cells. According to flow cytometry, the positive rates of surface markers of H-UCB cells were 79.0% for CD105, 1.2% for CD34, and 12.2% for CD106; those of control HepG2 cells were 15.0% for CO105, 9.8% for CD34, and 1.4% for CD106. The colony formation rate of H-UCB cells in soft agar was （13.2±2.6）%. CONCLUSION： H-UCB cells are derived from endothelial cells, and are transformed as malignant cells with tumor cell characteristics.