无血清悬浮法培养结肠癌HT29细胞系及肿瘤干细胞样亚群筛选

目的：研究无血清培养基悬浮培养人结肠癌细胞系HT29，筛选并鉴定HT29结肠癌细胞系中肿瘤干细胞相关亚群。方法：通过无血清培养基筛选结肠癌细胞系肿瘤于细胞相关亚群。应用克隆形成实验、表面标志检测、双苯酰亚胺（Hoechst）33342染色检测来确定培养细胞中肿瘤十细胞比例及其培养后的肿瘤干细胞含量变化。结果：结肠癌细胞系HT29中约50％的肿瘤细胞在无血清培养基中能够存活、增殖，形成自南飘浮的细胞球。细胞球可连续传代，若重新接种于含血清培养基中可重新贴壁分化，分化后细胞与培养储存的HT29细胞形态无明显差异。流式细胞仪检测表面标志，HT29细胞系中CD44＋细胞含量为（44．18±2．18）％．而细胞球中CD44‘细胞含量为（83．41±11．21）％：双苯酰亚胺33342染色检测提示HT29中侧群（sidepopulation，sp）细胞含量为（3．82±0．08）％．而细胞球中含量明显增高。结论：结肠癌细胞系HT29可在含生长因子的无血清培养基中悬浮生长，并维持细胞系。该细胞系中含有一定比例的具有增殖和自我更新能力的结肠癌干细胞样细胞亚群。表面标志以及双苯酰亚胺33342检测差异提示细胞球中仅部分细胞具有干细胞的功能。

Preliminary study on serum free floating-culture system and screening of stem cell like subpopulation in human colon cancer cell line HT29

Objective To investigate the serum free floating culture system for colon cancer cell line HT29 and the screening and identification of subpopulation with cancer stem cell-like properties. Methods Cancer stem cell-like cell subpopulation of colon cancer line HT29 was screened and obtained through floating culture in serum free medium （SFM）. Clone formation test, surface marker detection and Hoechst 33342 staining test were used to assess the proportion of cancer stem cell-like cell in cultured cells and the post-culture change in contents of cancer stem cell-like cell. Results About 50% of HT29 cells could survive, proliferate and assemble into cell spheres freely suspended in SFM. Cell spheres could pass several generations, and cell adherence and differentiation could be resumed again if re-inoculated into serum supplemented medium; no significant difference in morphology was found between the differentiated cell and culture stored HT29 cell. Cytoflowmetry showed that the proportion of CD44＋ cell in HT29 cell line was （44.18±2.18）%, while that in cell spheres was （83.41±11.21）%. Hoechst 33342 staining test showed that the side population （SP） in HT29 cell line was （3.82±0.08）% and that in cell spheres was markedly increased. Conclusions Human colon cancer cell line HT29 could be floating cultured in serum free medium containing growth factors and maintain the cell line. The HT29 cell line contained a subpopulation of colon cancer stem cell-like cell which had the capability of proliferation and self-renewal. Surface marker and Hoechst 33342 test showed that only a portion of the cells in cell spheres had stem cell-like function.