羟基喜树碱富集结肠肿瘤干细胞样群体的初步研究

背景:肿瘤干细胞是临床上肿瘤耐药的主要机制,识别并靶向肿瘤干细胞为肿瘤治疗的研究带来了新的思路。目的:探讨在体外以化疗药物富集结肠肿瘤干细胞样群体的可行性。方法:应用前期实验中以药物浓度递增诱导法建立的羟基喜树碱(HCPT)耐药人结肠癌细胞株SW1116/HCPT,免疫荧光标记干细胞表面抗原CD133,Hoechst33342/PI双染流式细胞仪检测、分选侧群(SP)细胞,实时PCR检测干细胞相关和多药耐药相关基因表达。结果:亲代细胞株SW1116中CD133阳性群体和SP群体分别仅占2%和1%,耐药细胞株SW1116/HCPT中则高达7%和61%,多药耐药基因MDR1抑制剂维拉帕米可使两组SP群体均下降至接近0水平。SW1116/HCPT细胞SP群体中干细胞相关基因CD133、Nanog和多药耐药相关基因MDR1、MRP1、MRP2、MRP6 mRNA表达水平较非SP群体显著上调(P0.05)。结论:HCPT药物浓度递增诱导法可成功富集结肠肿瘤干细胞样群体,其SP亚群中存在于细胞相关和多药耐药相关基因高表达。

Enrichment of Colon Tumor Stem Cell-like Population by Hydroxycamptothecin Chemotherapy in vitro; A Preliminary Study

Background： It has been reported that tumor stem cell is an important mechanism of drug resistance in cancer therapy. Identifying and targeting tumor stern cells offer a new approach to anticancer strategies. Aims： To investigate the feasibility of enriching colon tumor stem cell-like population by chemotherapy drug in vitro. Methods： A hydroxyeamptothecin （HCPT）-resistant human colon cancer cell line SW1116/HCPT established by HCFF stepwise concentration-increasing method as in previous study was employed. Stem cell surface antigen CD133 was labeled by immunofluorescence method; side population （SP） cells were assayed and sorted by Hoechst 33342/PI-mediated flow cytometry; and real time PCR was used to determine the expressions of stem cell-related and fnuhidrug resistance-related genes. Results： The percentages of CD133-positive cells and SP cells in parental cell line SWlll6 were 2% and 1%, respectively, while those in drug resistance cell line SWl116/HCPT were 7% and 61%, respectively. Addition of verapamil, an inhibitor of muhidrug resistance gene MDR1, could decrease the percentage of SP cells to 0% in both SWl116 and SWl I I6/HCPT cells. The mRNA expression levels of stem cell-related genes （CD133, Nanog） and multidrug resistance-related genes （MDR1, MRP1, MRP2, MRP6） were up-regulated in SP cells isolated from SWIll6/HCPT cell line when compared with those in non-SP cells （P〈0.05）. Conclusions： Colon tumor stem cell-like population can be enriched by HCPT stepwise concentration-increasing method in vitro. Its SB subpopulation is characterized by overexpressions of stem cell-related and muhidrug resistance-related genes