| 罗云桃,王一玮,贺思佳,赵明慧,赵煜翠,黄 倩.同源肿瘤克隆细胞蛋白表达和侵袭能力的异质性研究[J].,2019,19(23):4401-4406 |
| 同源肿瘤克隆细胞蛋白表达和侵袭能力的异质性研究 |
| Research on Heterogeneity of Protein Expression and Invasion among Homologous Tumor Clones |
| 投稿时间：2019-05-23 修订日期：2019-06-18 |
| DOI：10.13241/j.cnki.pmb.2019.23.001 |
| 中文关键词: 肿瘤异质性 单克隆 同源 |
| 英文关键词: Tumor heterogeneity Monoclonal Homologous |
| 基金项目:国家自然科学基金项目（NSFC81572951；NSFC81120108017） |
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| 中文摘要: |
| 摘要 目的：比较同源肿瘤细胞来源的不同单克隆表型差异。方法：采用极限稀释法，在悬浮培养条件下获取HCT116结肠癌细胞系的单个细胞，对每孔含单个的细胞进行扩增培养，获得子代单克隆，并以同样方法继续挑取单克隆，连续获得子三代克隆。根据单克隆形态特点，选取第三代的三株代表性的单克隆，采用Western blot和免疫荧光法比较其SOX2、EpCAM和Vimentin蛋白表达差异。采用放疗观察三株单克隆的Vimentin蛋白的动态变化，研究其放疗干预的时间异质性，Transwell体外侵袭实验比较克隆侵袭力的差异。结果：三株由单细胞扩增培养的同源第三代子克隆依然存在明显生物学差异。形态有明显区别的球形与不规则的克隆形态。不规则形态克隆更表现为SOX2低表达及Vimentin的高表达。并且在单个细胞水平上，同个单克隆群体内也存在个体细胞间蛋白的表达差异（Vimentin; EpCAM）。通过观察放疗前后Vimentin蛋白在不同时间点上的荧光强度，发现肿瘤单克隆细胞存在时间异质性。Transwell体外侵袭实验也显示三个同源克隆间存在明显的差异性。结论：同源的、连续单细胞扩增获得的第三代单克隆依然存在明显生物学差异，提示肿瘤内部异质性是其固有特征，并且在治疗干预下，也会引起肿瘤时间异质性的产生。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the phenotypic differences among homologous tumor clones derived from a single cancer cell. Methods: Using limited dilution method, the HCT116 colon cancer cells from the suspended culture condition were dissociated into single-cell suspension and seeded in ultra low attachment plates with average one cell per well. Within several weeks, the progeny clones derived from a single cell were obtained and expanded. By the same limited dilution method, the second and third generations of sub-monoclones, derived from one of their ex-generation clones respectively, were also acquired and expanded. Then, we chose the three representative clones of the third generation based on their distinct morphology for the further experiment. The expression of SOX2, EpCAM and Vimentin were detected via western blot or immunofluorescence. Through X-ray irradiation, the temporal heterogeneity of the three representative clones was evaluated by their dynamic alteration of Vimentin expression before and after treatment. The invasive ability of the three clones was also compared by transwell assay. Results: The considerable diversity of biological phenotype (eg: morphology; protein expression; invasive ability) was still present among the three clones, even they were the homologous progeny of third generation derived from a single HCT116 cancer cell. Their morphology was obvious differences (irregular or well-rounded shape). The protein expression of the irregular clone (expression of SOX2low and Vimentin high) was also different from other clones. Furthermore, the distinct protein expression was present at the single-cellular level (Vimentin; EpCAM), even they were among the same clone expanded from a single cancer cell. When the three homologous clones were treated with radiation, the temporal heterogeneity of Vimentin protein expression was present. The distinct invasive ability was also observed among the three clones. Conclusion: There was still obvious biological heterogeneity among the third-generation clones, even they derived from consecutive single-cell expansion. It provided the evidence to consider that the heterogeneity is the tumor inherent characteristics, and the influence of treatment intervention will also induce the temporal heterogeneity in the tumor cells. |
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