人喉癌细胞系Hep-2 中CD133 阳性肿瘤干细胞的分离及意义

目的:研究喉癌细胞系Hep-2中CD133的表达;比较CD133~+细胞、未分选细胞、CD133~-细胞的体外增殖、克隆形成能力及其在裸鼠体内的成瘤能力;探讨喉癌肝细胞对化疗药物顺铂(cisplatin,DDP)的抵抗作用。方法:采用流式细胞仪检测CD133在Hep-2细胞系中的表达;免疫磁珠分选技术纯化CD133阳性肿瘤细胞;使用四甲基偶氮唑蓝(MTT)法和平板克隆形成实验检测分选所得各细胞亚群细胞以及未分选细胞的体外增殖能力和克隆形成能力;将CD133阳性肿瘤细胞和CD133阴性肿瘤细胞以一定的数量级注入重症联合免疫缺陷小鼠腹部皮下,比较其成瘤差异性;此外,使用DDP干预分选所得各细胞亚群细胞,检测比较CD133阳性肿瘤细胞和CD133阴性肿瘤细胞的体外增殖能力与体内成瘤能力。结果:流式细胞仪示CD133在Hep-2细胞系中呈微量恒定表达,表达概率为40.12±1.32%;CD133阳性肿瘤细胞的体外增殖能力显著强于CD133阴性肿瘤细胞的增殖能力(P0.05),且其克隆形成能力也强于CD133阴性肿瘤细胞;体内成瘤实验结果显示CD133阳性肿瘤细胞较CD133阴性细胞、未分选细胞在重症联合免疫缺陷小鼠体内具有更强的成瘤性(P0.05);在DDP的干预下,相对于CD133阴性肿瘤细胞,CD133阳性肿瘤细胞表现出更强的抵抗力。结论:喉癌Hep-2细胞系中,CD133阳性癌细胞具有强的体外增殖能力、体内成瘤能力且对化疗药物具有较强的抵抗性,可作为喉癌肿瘤干细胞的标志之一。

Identification and Characterization of CD133 Positive Cells fromHep-2 Human Laryngeal Cancer Cell Line

Objective:To making further research and exploration on drugs to laryngeal carcinoma stem cells (CSCs), we detected the expression of CD133 in Hep-2 Human Laryngeal Cancer Cell Line, and then compared the in vitro proliferation abilities, clone formation abilities and in vivo tumorigenesis abilities of CD133 positive cells and CD133 negative cells. Furthermore, we compared the biological characteristics of CD133 positive/negative cells with or without cisplatin intervention.Methods:Flow cytometry (FCM) was used to detect the expression of CD133 in Hep-2 cell line. The MACS technology was applied to purify CD133 positive cells, after that, cells from each sub-population and unsorted cells were injected subcutaneously into the left abdomen of nude mice to compare their tumor-forming ability. Afterwards, the proliferation ability and tumor forming ability of different subpopulations cells under cisplatin (DDP) treatment were detected by applying MTT methods and in vivo tumorigenesis experiment.Results:Upon flow cytometry analysis, CD133 was expressed constantly on 40.12± 1.32%in Hep-2 cell line. Cell proliferation, colony formation ability were higher in CD133 positive cells compared to CD133 negative cells, and the in vivo tumorigenesis experiment showed the same results as in vitro assay (the differences among each group were significant, P<0.05). Both two subpopulations cells were sensitive to DDP, among which, the effect of DPP on proliferation ability and tumor forming ability of CD133 positive cells was more obviously than that of CD133 negative cells.Conclusion:CD133 positive cells have properties of higher proliferation, colony formation abilities, tumorigenesis and stronger drugs resistance in Hep-2 cell line indicating CD133 could be one of the markers to characterize laryngeal cancer stemcells.