喉癌细胞CD44^＋CD133^＋高致瘤亚群的生物学特性

Biological characteristics of highly tumorigenic CD44 ^＋ CD133 ^＋ subpopulation of laryngeal carcinoma cells

目的筛选喉癌细胞中的高致瘤亚群，探讨其作为肿瘤干细胞的生物学特性。方法原代培养喉癌细胞，采用流式细胞仪分选CIM4^＋、CD133^＋、CD44^＋CD133^＋、CD44^＋CD133^-细胞，并分别以1×10^6、1×10^5、1×10^4、1×10^3个／只接种于裸小鼠左腋皮下，观察各亚群成瘤时间、成瘤率、平均瘤重及肿瘤体积，筛选出在极低细胞浓度下的高致瘤亚群。采用Boyden小室体外侵袭实验，检测各亚群细胞的侵袭能力。采用免疫细胞化学染色检测各亚群细胞中干细胞抗原-1（SCA-1）及整合素β1的表达。采用逆转录聚合酶链反应（RT—PCR）和Western blot法，检测各亚群细胞中Bmi-1基因的表达。结果CIM4^＋CD133^＋细胞以1×10^3个／只接种于裸小鼠，可以成瘤。当各亚群细胞以1×10^6个／只接种于裸小鼠4周后，CD44^＋CD133^＋细胞接种组裸小鼠的肿瘤体积和重量分别为（7726．81±196．93）mm^3和（5．51±0．12）g，均大于CIM^＋、CD44^＋CD133^-及未分选细胞接种组（均P〈0．05），而与CD133^＋细胞接种组裸小鼠的肿瘤体积和重量差异无统计学意义（P〉0．05）。CD44^＋ CD133^＋细胞24h的侵袭细胞数为83．62±1．61，显著高于其他各亚群喉癌细胞。CD44^＋CD133^＋细胞高表达整合素β1和SCA-1。Bmi-1mRNA在CD44^＋CD133^＋细胞中的表达水平为0．951±0．112，显著高于CD44^＋CD133^-细胞和未分选的喉癌细胞（0．532±0．214和0．268±0．193，均P〈0．01）；Bmi-1蛋白在CD133^＋和CIM4^＋CD133^＋细胞中高表达，而在CD44^＋、CIM4^＋CD133^-和未分选的喉癌细胞中仅有少量表达。结论CD44^＋CD133^＋喉癌细胞具有高致瘤性及肿瘤干细胞的生物学特性，可能是喉癌恶性增殖的根源，并有望成为喉癌治疗的新靶点。

Objective To separate the cell subpopulation with high tumorigenic ability and study the biological characteristics of this subpopulation in laryngeal carcinoma cells. Methods Human laryngeal carcinoma cells were obtained by primary tissue culture technique. CD44 and CD133 molecules were used as markers to isolate the CIM4 ^＋ , CD133 ^＋ , CD44 ^＋ CD133 ^＋ and CD44 ^＋ CD133^- cell subpopulations from the laryngeal carcinoma ceils by flow cytometry. A nude mouse tumor xenograft model was developed for the study of the tumorigenic effects of the different cell populations. 1 × 106, 1×10^5, 1×10^4 and 1 ×10^3 cells were injected into the left flank of the mice, respectively. The mice were observed for palpable tumor formation and were sacrificed at 4 weeks later to assess the tumor formation rate, tumor volume and tumor weight. Boyden chamber migration assay was used to determine the migration ability and immunochemistry was used to detect the expression of stem cell antigen SCA-1 and β1-integrin. Semi-quantities RT-PCR and Western blot analysis were performed to detect the expression level of Bmi-1 in the different cell subpopulations. Results The growth of subcutaneous tumors in nude mice showed that a tumor can be generated with 1 × 10^3 CD44^＋ CD133^＋ cells. When the same dose of 1 ×10^6 CD44^＋ CD133^＋ cells was injected into the mice, both the average weight and volume of the tumors were significantly higher than those generated from other cell subpopulations. Boyden chamber migration assay showed that the invasion ability of CD44^＋ CD133 ^＋ cells was significantly higher than that of other cell subsets. The results of immunochemical analysis showed an abundant expression of stem cell antigen SCA-1 and β1-integrin in the CD44 ^＋ CD133^＋ cells. Semi-quantitative RT-PCR and Western blot analysis provided strong evidence that the Bmi-1 expression in CD44 ＋ CD133 ^＋ and CD133 ＋ cells was very signifieantly higher than that in CD44^＋ , CD44^＋ CD133^- and control cells （P 〈 0.01 ）. Conclusion Our findings demonstrate that CD44 ^＋ CD133 ^＋ subset cells in laryngeal carcinoma posses some biological characteristics of tumor stem cells, which may be the original cells of laryngeal carcinoma and may become a new target of tumor therapy.