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目的通过比较肝癌干细胞的不同单细胞克隆的体外分化能力,分析同一组织来源的肿瘤干细胞分化能力是否具有异质性,以进一步明确肿瘤干细胞的分化特性,为靶向肿瘤干细胞的治疗提供实验依据。方法通过有限稀释法对肝癌干细胞进行单细胞克隆培养,获得的单细胞克隆通过MTT测定比较其增殖能力。然后分别挑选增殖速度较快和较慢的3个克隆,采用RT-PCR方法比较其干细胞标志物表达水平。对干细胞标志表达水平高的3个克隆(A2、A3和B2)进行向成骨、软骨和脂肪方向的诱导分化。采用实时荧光定量PCR方法检测诱导前后成骨、软骨和脂肪标志分子的表达。结果获得的20个单细胞克隆增殖能力不同,并且增殖能力快的克隆表达干细胞标志物CD133、Oct4、c-kit、SCF、nestin比增殖能力慢的克隆强。向成骨方向诱导后,A2、A3和B2克隆I型胶原mRNA表达水平分别升高了(4.71±0.11)、(2.13±0.15)和(3.82±0.3)倍;骨钙素mRNA的表达水平分别升高(8.55±0.18)、(7.02±0.03)和(7.91±0.09)倍,说明A2克隆向成骨细胞分化能力最强。向软骨方向诱导后,B2分化能力最强,软骨标志分子蛋白聚糖和II型胶原的表达分别上调(25.01±0.19)倍和(17.49±0.19)倍,而在A2未发生明显变化,A3只轻微上调。向脂肪方向诱导后,A2、A3和B2克隆脂联素mRNA表达水平分别升高了(6.12±0.15)、(11.45±0.36)和(12.41±1.03)倍,过氧化物酶体增殖物激活受体γmRNA的表达水平分别升高(4.92±0.02)、(9.54±0.18)和(8.96±0.11)倍。结论来源于同一组织的肝癌干细胞在分化能力上具有异质性,这可能是导致肿瘤组织异质性的原因之一,也提示靶向肿瘤干细胞的治疗需要联合用药。
OBJECTIVE: To compare the differentiating ability of different single cell clones of hepatocellular carcinoma stem cells in vitro and analyze the heterogeneity of differentiating ability of tumor stem cells in the same tissue so as to further clarify the differentiation characteristics of tumor stem cells and provide experimental evidence for targeting cancer stem cells. Methods Single cell clone culture of hepatocellular carcinoma stem cells was carried out by limited dilution method. The single cell clone obtained was compared by MTT assay to compare its proliferation ability. Three clones with faster and slower proliferation were selected, and their expression levels of stem cell markers were compared by RT-PCR. Three clones with high expression of stem cell markers (A2, A3 and B2) were induced to differentiate into osteoblasts, cartilages and fat. Real-time fluorescent quantitative PCR was used to detect the expression of osteogenic, cartilage and fat markers before and after induction. The results showed that the proliferation of 20 single-cell clones was different, and the clones with high proliferative capacity expressed CD133, Oct4, c-kit, SCF and nestin more rapidly than those with slow proliferative capacity. The mRNA expression of type I collagen in A2, A3 and B2 clones was increased by 4.71 ± 0.11, 2.13 ± 0.15 and 3.82 ± 0.3, respectively, after osteogenic induction. The expression of osteocalcin mRNA was (8.55 ± 0.18), (7.02 ± 0.03) and (7.91 ± 0.09) fold respectively, indicating that A2 clone has the strongest ability to differentiate into osteoblasts. After being induced to cartilage, B2 had the strongest differentiation ability. The expression of cartilage marker proteoglycan and type II collagen were up-regulated by (25.01 ± 0.19) times and (17.49 ± 0.19) -fold respectively, while there was no obvious change in A2. Slight increase. After inducing to adipose, mRNA expression of adiponectin A2, A3 and B2 increased by (6.12 ± 0.15), (11.45 ± 0.36) and (12.41 ± 1.03) folds respectively, while peroxisome proliferator-activated receptor The expression of body γmRNA increased by 4.92 ± 0.02, 9.54 ± 0.18 and 8.96 ± 0.11, respectively. Conclusions Hepatoma stem cells derived from the same tissue are heterogeneous in differentiation ability, which may be one of the causes of heterogeneity of tumor tissues. It also suggests that the treatment of targeted cancer stem cells requires combination therapy.