CAJ | 학술논문

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。
목적통과비교간암간세포적불동단세포극륭적체외분화능력，분석동일조직래원적종류간세포분화능력시부구유이질성，이진일보명학종류간세포적분화특성，위파향종류간세포적치료제공실험의거。 　　방법통과유한희석법대간암간세포진행단세포극륭배양，획득적단세포극륭통과 MTT 측정비교기증식능력。연후분별도선증식속도교쾌화교만적3개극륭，채용 RT-PCR 방법비교기간세포표지물표체수평。대간세포표지표체수평고적3개극륭（A2、A3화 B2）진행향성골、연골화지방방향적유도분화。채용실시형광정량 PCR 방법검측유도전후성골、연골화지방표지분자적표체。 　　결과획득적20개단세포극륭증식능력불동，병차증식능력쾌적극륭표체간세포표지물 CD133、Oct4、c-kit、SCF、nestin 비증식능력만적극륭강。향성골방향유도후，A2、A3화 B2극륭 I 형효원 mRNA 표체수평분별승고료（4.71±0.11）、（2.13±0.15）화（3.82±0.3）배；골개소mRNA 적표체수평분별승고（8.55±0.18）、（7.02±0.03）화（7.91±0.09）배，설명 A2극륭향성골세포분화능력최강。향연골방향유도후，B2분화능력최강，연골표지분자단백취당화 II 형효원적표체분별상조（25.01±0.19）배화（17.49±0.19）배，이재 A2미발생명현변화，A3지경미상조。향지방방향유도후， A2、A3화 B2극륭지련소mRNA 표체수평분별승고료（6.12±0.15）、（11.45±0.36）화（12.41±1.03）배，과양화물매체증식물격활수체γmRNA 적표체수평분별승고（4.92±0.02）、（9.54±0.18）화（8.96±0.11）배。 　　결론래원우동일조직적간암간세포재분화능력상구유이질성，저가능시도치종류조직이질성적원인지일，야제시파향종류간세포적치료수요연합용약。
Objective To investigate the differentiation heterogeneity of cancer stem cells (CSCs) by comparing differentiation potential of clonally expanded subpopulations derived from the same cancer tissue in vitro. Methods Single cell clones of CSCs were obtained by limited dilution method. The proliferation of these clones was evaluated by MTT assay. The expression of stem cell markers was detected by RT-PCR. Three clones (A2, A3 and B2) were induced to differentiate toward mesenchymal-like cell for 3 weeks. Real-time PCR was used to detect the mRNA levels of mesenchymal-specific markers. Results The proliferation of the obtained 20 clones was differed between each other, and the expression of stem cell markers CD133, Oct4, c-kit, stem cell factor and nestin was higher in highly proliferation of clones than in the low proliferation of ones. Upon induction toward osteoblast, the mRNA level of collagen type I in clone A2, A3 and B2 was up-regulated to (4.71 ± 0.11), (2.13 ± 0.15) and (3.82 ± 0.3) times respectively;and the mRNA level of osteocalcin was up-regulated to (8.55 ± 0.18), (7.02 ± 0.03) and (7.91 ± 0.09) times respectively, which showed that A2 has stronger potential toward osteoblast. Upon induction toward chondrocyte, clone B2 exhibited very strong differentiation potential as shown the mRNA level of aggrecan and collagen type II was up-regulated to (25.01 ± 0.19) and (17.49 ± 0.19) times. However, the expression of aggrecan and collagen type II was not increased in clone A2 and increased slightly in clone A3. Upon induction toward adipocyte, the mRNA level of adiponectin in clone A2, A3 and B2 was up-regulated to (6.12 ± 0.15), (11.45 ± 0.36) and (12.41 ± 1.03) times respectively;and the mRNA level of peroxisome proliferator-activated receptor (PPARγ) was up-regulated to (4.92 ± 0.02), (9.54 ± 0.18) and (8.96 ± 0.11) times respectively, which suggested that A3 and B2 had stronger differentiation potential toward adipocyte. Conclusions The CSCs derived from the same cancer tissue has heterogeneity in differentiation potential, which may be one reason of tumor heterogeneity. These results indicate that combination of drugs might be needed in targeting CSCs treatment in the future.