人膀胱癌组织中肿瘤干细胞样细胞的分离培养及PIWIL2表达

Study of isolation,cultivation and identification of cancer stem-like cells from human bladder cancer tissues and PIWIL2 expression

目的从人膀胱移行细胞癌(BTCC)组织中分离培养肿瘤干细胞样细胞(CSLC),鉴定其生物学特性,并研究PIWIL2在CSLC中的表达和意义。方法收集12例不同临床分期BTCC患者组织标本,通过酶消化和原代培养相结合等方法处理,采用无血清悬浮培养法分离培养获得含CSLC的悬浮细胞球,流式细胞仪检测细胞表面分子标志CD133和CD44的表达,免疫磁珠分选系统分离CD133+CD44+细胞;采用半定量逆转录聚合酶链反应检测PIWIL2mRNA在CSLC中的表达;裸鼠皮下接种CS-LC,观察其成瘤能力。结果成功地从人膀胱癌组织分离培养获得可悬浮生长、稳定传代的CSLC,CSLC高表达CD133和CD44,裸鼠皮下接种1×104、1×105个CSLC均可全部成瘤,PIWIL2mRNA在CSLC的表达显著高于正常膀胱组织细胞。结论采用无血清悬浮培养法成功从人膀胱癌组织中分离获得CSLC,具有肿瘤干细胞特性,能高度表达PIWIL2,为靶向肿瘤干细胞的治疗提供了实验依据。

Objective To isolate and cultivate cancer stem-like cells（CSLC） from human bladder transitional cell carcinoma（BTCC） and identify their biological characteristics,and to investigate the expression and significance of PIWIL2 in CSLC.Methods Tumor samples were obtained from 12 patients with T3-T4 BTCC.The method of suspension culture in specific serum free medium was employed to isolate and obtain CSLC spheres by using enzymatic digestion combined with primary culture.The expressions of cell surface markers CD133 and CD44 were detected with flow cytometry.CD133＋CD44＋ cells were separated with magnetic activated cell sorting system.PIWIL2 mRNA expression in CSLC was detected with semi-quantitative reverse transcription polymerase chain reaction.The tumor formation ability was evaluated with subcutaneous injection of CSLC into nude mice.Results CSLCs were obtained from BTCC and floating cell spheres were formed under the suspension culture method with serum-free medium,which could propagate stably.The CSLCs highly expressed CD133 and CD44 of CSC markers.PIWIL2 mRNA expression was significantly higherin CSLC than in normal bladder tissues.Tumor formed in all mice after subcutaneous injection of 1×104 and 1×105 CSLCs and exhibited a high degree of similarity with the primary tumor in BTCC.Conclusion CSLC can be isolated from the suspension culture method combined with serum-free medium,which possess the potentials of CSC and highly expresses PIWIL2.