| 周鲁林,张 文,段新春.3D培养体系富集外周血中的循环肿瘤干细胞的研究[J].中国肿瘤,2019,28(5):381-386. |
| 3D培养体系富集外周血中的循环肿瘤干细胞的研究 |
| Establishment of 3D Culture System to Enrich Circulating Tumor Stem Cells from Peripheral Blood |
| 投稿时间：2018-10-10 |
| DOI：10.11735/j.issn.1004-0242.2019.05.A011 |
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| 中文关键词: 循环肿瘤细胞 循环肿瘤干细胞 3D培养 类器官 |
| 英文关键词:circulating tumor cells circulating cancer stem cells 3D culture organoid |
| 基金项目:国家自然科学基金项目（81472013）；国家重点研发计划项目（2017YFC1308700、2017YFC1308702）；中国医学科学院医学与健康科技创新工程（2017-I2M-1-005） |
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| 中文摘要: |
| 摘 要：［目的］ 探究建立3D培养体系富集外周血中的循环肿瘤干细胞。［方法］ 应用阴性筛选的方法富集外周血中的循环肿瘤细胞(circulating tumor cells，CTCs)，随后利用3D培养体系将富集得到的细胞进行培养。采用免疫荧光、定量RT-PCR对3D培养的前列腺癌LNCaP细胞系、肺癌H2347细胞系的干性表型特征进行分析。使用肺癌患者的血液样本分离CTCs进行3D培养验证该方法的可行性。 ［结果］ 在光镜下可观察到，在3D培养体系中从外周血分离的前列腺癌LNCaP细胞增殖形成肿瘤细胞微球。免疫荧光结果显示，在3D培养条件下成团生长的前列腺癌LNCaP细胞表达EpCAM和CD133。在3D无血清培养条件下，LNCaP细胞系成球率为18.67%±3.06%。前列腺癌LNCaP细胞在3D培养体系中与普通2D培养相比，干性相关基因CD133、CD44和ABCG2表达水平明显增高，3D培养的肺癌H2347细胞与普通2D培养相比，干性相关基因CD133表达水平明显升高。肺腺癌患者肺静脉血分离CTCs 3D培养，可形成多个微型肿瘤细胞微球（称为肺癌类器官）。在光镜下观察，同一个患者培养获得的肺癌类器官呈现不同的形态结构特征。［结论］ 利用3D培养体系能够培养外周血中的CTCs，并富集得到CD133+的肿瘤干细胞。肺癌患者的CTCs 3D培养能够形成肺癌类器官，并能够展现CTCs的异质性。 |
| 英文摘要: |
| Abstract：［Purpose］ To establish a 3D culture system for enriching circulating tumor stem cells in peripheral blood. ［Methods］ The circulating tumor cells(CTCs) in the blood were enriched by a negative selection method，then enriched cells were cultured in a 3D culture system. Quantitative RT-PCR and immunofluorescence were used to assess the stemness phenotype of 3D cultured cancer cells. The feasibility of the 3D culture system was further verified by using blood samples of lung cancer patients.［Results］ In the 3D culture system，the prostate cancer LNCaP cells isolated from the blood multiplied and formed tumorspheres. The results of immunofluorescence showed that EpCAM and CD133 were expressed in LNCaP cultured in 3D conditions. In the serum-free 3D culture system，the sphere forming rate of LNCaP cell lines was 18.67%±3.06%. The expression levels of stemness-related genes CD133，CD44 and ABCG2 in prostate cancer cells from 3D cultured system were significantly higher than those from monolayer culture. The expression level of CD133 in 3D-cultured lung cancer cell was significantly higher than that in monolayer culture. The CTCs which were isolated from pulmonary venous blood of patients with lung adenocarcinoma were expanded in the 3D culture system and formed multiple tumorspheres（organoids）. The morphology of the lung cancer organoids from one patient was apparently different under the light microscope. ［Conclusion］ The 3D culture system can be used to expand CTCs and enrich CD133+ cancer stem cells. The CTCs in lung cancer patients formed lung cancer organoids in the 3D culture system，which showed the heterogeneity of CTCs. |
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