摘要
目的建立无血清悬浮培养分离人胰腺癌细胞系PANC1干细胞球的方法。方法采用无血清悬浮法培养PANC1细胞，显微镜下观察干细胞球形成率；流式细胞仪检测细胞CD133表达和细胞周期；以含10％FBS培养基诱导干细胞球细胞分化，荧光显微镜下观察细胞形态及CK18的表达；干细胞球细胞接种NOD／SCID小鼠皮下，观察其成瘤能力。结果在无血清悬浮培养条件下存活的PANC1细胞形成干细胞球，体外连续传代培养20代始终保持4‰～5‰的干细胞球形成率。干细胞球细胞CD133表达率（5．91±0．7）％，G0／G1期细胞占（80．99±2．60）％，与原代PANC1细胞的（1．44±0．52）％和（69．01±5．03）％相差显著（P〈0．05）。将干细胞球细胞置含血清培养基中培养，细胞逐渐恢复原代细胞形态，并表达上皮标志蛋白CK18，2×10^3的干细胞即在NOD／SCID小鼠皮下成瘤。结论无血清悬浮法培养PANC1细胞可分离出肿瘤干细胞，其具有自我更新、多向分化和成瘤能力。
Objective To establish the method of suspension culture for stem cells from human pancreatic cancer cell line PANC1. Methods PANC1 cells were cultured in serum-free medium under floating-culture system. Tumor cell spheres were observed by optical microscope. Expression of CD133 and cell cycle were detected by flow cytometry. Cancer stem cells were induced to differentiate with 10% FBS, and expression of CK18, was evaluated with immunofluorescencc microscope. Spheres cells were injected into the subcutaneous space of NOD/SCID rat and tumor formation was monitored weekly. Results PANC1 cells could form the stem cells spheres, and the rate of sphere formation was stable between 4‰ and 5‰ after 20 passages in vitro. The expression of CD133 （5.91 ± 0.7% ） and proportion of G0/G1 phase cell （80.99 ± 2.60% ） was significantly increased in spheres ceils compared with parental PANC1 cells （ 1.44 ±0. 52% and 69.01 ± 5.03% ）, and the difference was statistically significant （P 〈 0.05 ）. When these spheres cells were cultured in media with serum, these cells gradually returned to the status of parental cells and expressed CK18, 2 × 10^3 sphere cells injection could initiate tumor formation in NOD/SCID rat. Conclusions Tumor spheres stem cells could be generated under serum-free floating-culture system. The sphere ceils possessed the capacities of self renew, differentiation, and tumorigenic potential.
出处
《中华胰腺病杂志》
CAS
2009年第5期315-317,共3页
Chinese Journal of Pancreatology
基金
广东省自然科学基金（8151008901000139）
关键词
胰腺肿瘤
干细胞
培养基
无血清
悬浮法
Pancreatic neoplasms
Stem cells
Culture media, Serum-free
Floating-culturel system
