论文部分内容阅读
目的研究5-氟尿嘧啶(5-Fluorouracil,5-FU)筛选残存结肠癌细胞(drug surviving cells,DSCs)是否具有癌症干细胞(cancer stem cells,CSCs)属性及其增殖过程中免疫表型的改变。方法应用5-FU对体外培养的结肠癌细胞株SW480进行处理,收集残存DSCs,检测其细胞周期、克隆形成率、CD133阳性率及在SCID小鼠体内的致瘤率;在无血清培养基(serum-free medium,SFM)中培养DSCs,观察干细胞球的形成及CD133表达改变;机械分离干细胞球所得第2代DSCs在含血清培养基(serum-supplied medium,SSM)中培养,利用免疫荧光及AB/PAS特染技术检测癌细胞CD133、Oct4、VEGF、CD34、CK8/18的表达改变及黏液分泌情况。结果 5-FU作用72 h后继续SSM中培养1周,SW480细胞大量死亡,少量残存的DSCs以G0～G1期细胞为主,克隆形成率及SCID小鼠体内致瘤率明显高于未经处理的SW480细胞(P<0.05);DSCs中CD133+细胞数明显高于未经处理组(P<0.05),VEGF+细胞数明显减少(P<0.05);DSCs在SFM中经过3周连续培养能够形成干细胞球,细胞球内高表达CD133,VEGF表达也轻度增高(P<0.05)。第2代DSCs在SSM内生长时CD133、Oct4表达逐渐降低,而VEGF、CD34、CK8/18表达逐渐增高,并开始分泌黏液。结论 5-FU处理能富集CD133+结肠癌细胞,富集的残存癌细胞具有癌症干细胞属性,其在SSM内增殖过程中逐渐出现了免疫表型改变。
Objective To investigate whether 5-fluorouracil (5-FU) screening of survivors of colon cancer cells (DSCs) possesses the properties of cancer stem cells (CSCs) and their immunophenotypic changes during proliferation. Methods The colon cancer cell line SW480 cultured in vitro was treated with 5-FU. The residual DSCs were collected and the cell cycle, clonogenic rate, positive rate of CD133 and tumorigenicity in SCID mice were detected. In the serum-free medium (DSM) were cultured in serum-free medium (SFM) to observe the formation of stem cell spheres and the expression of CD133. The second generation DSCs obtained by mechanical separation of stem cells were cultured in serum-supplied medium (SSM) The expression of CD133, Oct4, VEGF, CD34 and CK8 / 18 in cancer cells and mucus secretion were detected by AB / PAS special staining technique. Results After 72 h of 5-FU treatment, the cells were continuously cultured in SSM for one week, and a large number of SW480 cells died. A small amount of residual DSCs were mainly G0-G1 phase cells. The clonogenic rate and the tumorigenicity rate of SCID mice were significantly higher than those of untreated Of SW480 cells (P <0.05). The number of CD133 + cells in DSCs was significantly higher than that in untreated group (P <0.05), and the number of VEGF + cells was significantly decreased (P <0.05). DSCs were able to form stem cells CD133 was highly expressed in the sphere and sphere, and the expression of VEGF was slightly increased (P <0.05). The expression of CD133 and Oct4 in the second generation of DSCs grew slowly in SSM, while the expression of VEGF, CD34 and CK8 / 18 gradually increased and began to secrete mucus. Conclusions 5-FU treatment can enrich CD133 + colon cancer cells. The enriched residual cancer cells have cancer stem cell properties, and their immunophenotypic changes gradually appear during the proliferation of SSM.