舌鳞癌组织中肿瘤干细胞的分离培养及PIWIL4对其的影响

Tongue Squamous Cell Carcinoma Cancer Stem Cell Isolation and Culture and PIWIL4 Effects on it

目的从人舌鳞癌组织中分离培养肿瘤干细胞细胞(CSC),鉴定其生物学特性,并研究PIWIL4在CSC中的表达和意义。方法收集9例不同临床分期舌鳞癌患者组织标本,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CSC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PIWIL4 mRNA在CSC中的表达;裸鼠皮下接种CSC,观察其成瘤能力。结果成功的从人舌鳞癌组织分离培养获得可悬浮生长、稳定传代的CSC,CSC高表达CD133和CD44,裸鼠皮下接种1×104、1×105个CSC均可全部成瘤,PIWIL4 mRNA在CSC的表达也显著高于正常舌组织细胞。结论采用无血清悬浮培养法成功从人舌组织中分离获得CSC,具有肿瘤干细胞特性,能高度表达PIWIL4,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 （CSC） from human squamous cell carcinoma of tongue and identify their biological characteristics, investigate the expression and signiifcance of PIWIL4 in CSC. Methods Tumor samples were obtained from 9 patients of carcinoma of the tongue at stages T3-T4. The method of suspension culture in speciifc serum free medium was employed to isolate and obtain CSC spheres by using enzymatic digestion combined with primary culture. The expressions of cell surface markers CD133 and CD44 were detected by flow cytometry. CD133＋CD44＋cells were separated with magnetic activated cell sorting system. Semi-quantitative reverse transcription polymerase chain reactions were performed to detect the PIWIL4 mRNA expression in CSC. The tumor formation ability was evaluated by subcutaneous injection of CSC into nude mice. Results CSC were obtained from human squamous cell carcinoma of tongue and formed lfoating cell spheres under the suspension culture method with serum-free medium, and could propagate stably, which highly expressed CD133 and CD44 of CSC markers. And PIWIL4 mRNA expression level in CSC was signiifcantly higher than that in normal tongue tissues. Tumor formated in all mice after subcutaneous injection of 1×10^4 and 1×10^5 CSC and exhibited a high degree of similarity with the primary tumor in carcinoma of the tongue. Conclusion CSC can be isolated under the suspension culture method with serum-free medium, which possess the potentials of CSC and highly expressed PIWIL4.