Wei Xudong, He Jian, Gao Jiangxia, Chen Jing, Wang Jingyu
Lin Chuang Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2014 Nov;28(21):1636-41.
To investigate the self-renewal mechanism of CD133+ cancer stem cells from Hep-2 cell line.
The CD133+ cells were sorted by flow cytometry from Hep-2 cell line. Then the sorted CD133+ cells were cultured in RPMI1640. The ability of self-renewal of CD133+ cells were tested by MTT assay. mRNA and protein expression of self-renewal related genes were detected by western blot and RT- PCR.
(3.10 ± 0.21)% of Hep-2 cells expressed the membrane antigen CD133. CD133+ fraction was raised to (90.20 ± 5.51)% by flow cytometry. In vitro culture and growth curve showed CD133+ cells had more active proliferation ability than CD133- cells, which showed statistically significant difference between these two group (P < 0.01). RT- PCR and western blot results showed upregulated mRNA and protein expression of Fas, c-myc, survivin in CD133+ group (P < 0.01). In the same time, the ratio of Bcl-2/Bax gene expression was obviously increased in CD133+ group. Self-renewal related gene such as β-catenin, SHH, SMOH and Bmi-1,Gli-1 were all up-regulated in CD133+ group both in mRNA and protein. On the contrary, PTCH gene was down-regulated.
CD133 positive cells are a small proportion of a Hep-2 cell line. The results of this experiment verified that CD133 positive cells owned the properties of cancer stem cells. Upregulated anti-apoptotic gene is the foundatiom of self-renewal mechanism of CD133+ cells. Cancer stem cells related signal pathways such as Hedgehog, Wnt and Bmi-1 pathway are in state of activation. The identification of self-renewal mechanism about cancer stem cell provides a powerful tool to investigate the tumorigenic process in the larynx and to develop therapies targeting to these signal pathways.
研究人喉癌细胞系Hep-2中CD133+肿瘤干细胞的自我更新机制。
采用流式细胞术从Hep-2细胞系中分选CD133+细胞，将分选后的细胞接种于RPMI1640培养基中培养，采用MTT法检测CD133+细胞的自我更新能力，运用蛋白质免疫印迹法和逆转录聚合酶链反应检测自我更新相关基因的mRNA和蛋白表达。
Hep-2细胞中CD133膜抗原表达阳性率为(3.10±0.21)%，经流式细胞术分选后，CD133+细胞比例提高至(90.20±5.51)%。体外培养生长曲线显示，CD133+细胞较CD133-细胞具有更强的增殖活性，差异有统计学意义(P<0.01)。蛋白质免疫印迹法和逆转录聚合酶链反应结果显示，CD133+组Fas、c-myc、survivin的mRNA和蛋白表达上调(P<0.01)，同时Bcl-2/Bax基因表达比值明显升高。自我更新相关基因如β-连环蛋白、音猬因子(SHH)、SMOH、Bmi-1、Gli-1在CD133+组的mRNA和蛋白水平均上调，相反，PTCH基因表达下调。
CD133阳性细胞在Hep-2细胞系中占比少，本实验结果证实CD133阳性细胞具有肿瘤干细胞特性。抗凋亡基因上调是CD133+细胞自我更新机制的基础，Hedgehog、Wnt、Bmi-1等肿瘤干细胞相关信号通路处于激活状态。肿瘤干细胞自我更新机制的明确为深入研究喉癌的发生发展机制及针对这些信号通路的靶向治疗提供了有力工具。
