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背景:膀胱癌干细胞参与促进膀胱癌的术后复发及耐药性,而多项研究表明角蛋白6B参与多个不同类型肿瘤的发生和进展,并与肿瘤的预后密切相关.目的:观察角蛋白6B在CD44+膀胱癌干细胞中的表达差异及其对膀胱癌干细胞增殖、迁移及自我更新的影响,并进一步探讨角蛋白6B表达对膀胱癌患者预后的影响.方法:①使用免疫磁珠分选法分选出人CD44+5637膀胱癌干细胞,使用RT-PCR检测分选后的相关干性基因SOX2,OCT4和NANOG的表达,采用体外肿瘤球形成实验进一步验证CD44+细胞具有肿瘤干细胞的自我更新能力,RT-PCR检测CD44+膀胱癌干细胞中的角蛋白6B的表达情况;②将CD44+5637膀胱癌干细胞分为2组,其中角蛋白6B沉默组将角蛋白6B小干扰RNA转染至分选后的CD44+膀胱癌干细胞,未转染的CD44+膀胱癌干细胞作为空白对照组,转染2 d后收集细胞,分别用平板克隆实验、划痕实验和体外肿瘤球形成实验检测角蛋白6B沉默后膀胱癌干细胞的增殖,迁移和自我更新能力;③采用免疫组织化学染色检测膀胱癌患者24例膀胱肿瘤组织切片的角蛋白6B和CD44v6的表达;④利用ONCOMINE数据库比较膀胱肿瘤组织中角蛋白6B的表达情况对患者总生存率的影响.结果 与结论:①分选后的CD44+细胞中相关干性基因(SOX2,OCT4,NANOG)和角蛋白6B表达高于CD44-细胞(P<0.05),其细胞增殖,迁移和体外成球能力显著升高(P<0.05),角蛋白6B小干扰RNA可下调角蛋白6B在CD44+膀胱癌干细胞中的表达(P<0.05);②与空白对照组相比,角蛋白6B小干扰RNA转染后的CD44+膀胱癌干细胞增殖和迁移能力显著降低(P<0.05),肿瘤球的数量明显降低(P<0.05),而Notch1和Hes1 mRNA表达升高(P<0.05);③角蛋白6B与CD44v6在患者膀胱癌组织中的表达差异有显著性意义(P=0.006),其中角蛋白6B高表达的膀胱癌患者总生存率低于角蛋白6B低表达的患者;④上述数据证实,角蛋白6B可在CD44+膀胱癌干细胞中可高度表达,并且促进膀胱癌干细胞的增殖、迁移和自我更新,角蛋白6B高表达有助于膀胱癌患者生存情况的改善.“,”BACKGROUND: Bladder cancer stem cells could promote the recurrence and drug resistance of bladder cancer. Numerous studies have shown that keratin 6B (KRT6B) is involved in the production and progression of tumors, and is closely related to the prognosis of tumors. OBJECTIVE: To observe the expression of keratin 6B in CD44+ bladder cancer stem cells and to show the influence of keratin 6B on proliferation, migration, and self-renewal of bladder cancer stem cells, and to further explore the effect of keratin 6B expression on the prognosis of bladder cancer patients. METHODS: (1) CD44+ 5637 bladder cancer stem cells were isolated by magnetic active cell sorting. Cancer stem cell-related gene expression of SOX2, OCT4, and NANOG was detected via real-time polymerase chain reaction. The spheroid formation assay was used to detect the ability of self-renewal of cancer stem cells in CD44+ cells. Keratin 6B expression was detected in CD44+ bladder cancer stem cells by real-time polymerase chain reaction. (2) The CD44+5637 bladder cancer stem cells were divided into two groups. In the keratin 6B siRNA group, keratin 6B small interfering RNA was transfected into CD44+ bladder cancer stem cells. Untransfected CD44+ bladder cancer stem cells were used as the black control group. Cells were collected at 2 days post-transfection. The proliferation, migration, and self-renewal capacity of keratin 6B siRNA CD44+ bladder cancer stem cells were detected by the colony and wound healing assay and spheroid formation respectively. (3) Totally 24 bladder cancer tissues were used by immunohistochemistry to analyze the expression of CD44v6 and keratin 6B. (4) ONCOMINE database was used to analyze the effect of keratin 6B expression on the overall survival of bladder cancer. RESULTS AND CONCLUSION: (1) Cancer stem cell-related genes (SOX2, OCT4, NANOG) and keratin 6B expression was higher in CD44+ cells isolated by magnetic active cell sorting compared with CD44- cells (P < 0.05). Cell proliferation, migration, and in vitro spheroid formation were significantly increased (P < 0.05). Keratin 6B small interfering RNA down-regulated the expression of keratin 6B in CD44+ bladder cancer stem cells (P < 0.05). (2) Compared with the blank control group, the proliferation and migration of CD44+ bladder cancer stem cells after transfection of keratin 6B small interfering RNA (P < 0.05), and the number of tumorsphere significantly diminished (P < 0.05); the expression of Notch1 and Hes1 mRNA increased (P < 0.05). (3) Keratin 6B and CD44v6 were significantly different in bladder cancer tissue (P=0.006). The overall survival rate of bladder cancer patients with high expression of keratin 6B was lower than that of patients with low expression of keratin 6B. (4) The results showed that keratin 6B was highly expressed in CD44+ bladder cancer stem cells, and could promote the proliferation, migration, and self-renewal capacity of bladder cancer stem cells. The high expression of keratin 6B contributes to improving the survival of bladder cancer patients.