胰腺癌细胞株PANC-1中肿瘤干细胞生物学行为的研究

Analysis of biological behavior of tumor stem cell of pancreatic adenocarcinoma cell line PANC-1

目的研究人胰腺癌细胞株PANC-1中肿瘤干细胞的生物学特性。方法将PANC-1细胞进行培养,以CD44和CD24作为细胞表面标志。利用流式细胞仪,从该细胞株中分离出细胞亚群。将所得到的有CD44-CD24-,CD44+CD24+表面标志的PANC-1细胞和未分选的PANC-1细胞置于含有10ng/mL成纤维细胞生长因子、20ng/mL上皮生长因子和ITS(胰岛素、转铁蛋白和硒混合溶液)无血清DMEM/F12(1:1)培养基中,以MTT法检测不同亚群细胞体外增殖活性;通过裸鼠体内接种成瘤实验,鉴定各亚群细胞体内增殖能力;通过ABC法检测成瘤组织和PANC-1细胞株中CD44和CD24的表达。结果PANC-1细胞系中CD44+和CD24+的表达分别为5.1%～17.5%和21.8%～70.1%,CD44+CD24+的表达为0.9%～3.5%;与CD44-CD24-亚群比较,CD44+CD24+亚群生长缓慢,第7天才出现指数生长的趋势,增殖能力较低,倍增时间更长,而前者第5天已出现指数生长的趋势。裸鼠皮下植入5×103个CD44+CD24+亚群细胞,4周即可见明显的新生肿瘤块(2/8),而植入1×105个D44-CD24-亚群细胞,12周也难形成种植瘤。前者体外成瘤能力比后者至少强20～50倍(P<0.05或P<0.01);所形成的肿瘤和PANC-1细胞系无组织学差异。结论CD44和CD24可作为分选PANC-1中肿瘤干细胞的表面标志;分选出的CD44+CD24+亚群细胞具有肿瘤干细胞的初步特征。

Objective To explore the method of isolation and biological analysis of tumor stem cell of pancreatic adenocarcinoma cell line PANC-I. Methods PANC-I cells were cultured. Subpopulation cells which have properties of tumor stem cells were isolated according to the cell surface markers CD44 and CD24 by flow cytometry from pancreatic adenocareinoma cell line PANC-1. The obtained CD44ˉ CD24ˉ , CD44^＋ CD24^＋ and unsorted PANC-1 cells were cuhured in serum-free medium （SFM）. The SFM was DMEM-F12 supplemented with I0 ng/mL fibroblast growth factor, 20 ng/mL epidermal growth factor, 5 kg/mL insulin, 2.75 mg/mL transferrin, 2.75 ng/mL selenium （ insulin-transferrin-selenium solution ） , penicillin （ 1 × 10^5 U/L） and streptomycin （100 mg/L）. Then the proliferative capability of these cells in vitro was estimated by MTT method. The tumor growth from different subpopulation cells inoculated into nude mice was studied, and expression of CD44 and CD24 of the CD44^＋ CD24^＋ cells -formed nodules and PANC-1 cells were detected by avidin-biotin-peroxidase complex （ABC） immunohistochemical staining. Results Expressed the cell surface marker CD44, in 5. 1% to 17.5% of sorted PANC-1 cells, 21.8% to 70. 1% expressed CD24, and only 0. 9% to 3. 5% of cells were CD44^＋ CD24^＋. Compared with CD44ˉ CD24ˉ cells, CD44^＋ CD24^＋ cells had a lower growth rate in vitro. For the former, index growth trend appeared at 5th day, while the latter appeared at 7 th day. The sorted cell population with the highest tumorigenic potential were those cells expressing CD44 and CD24. After implantation of 1×10^5 CD44ˉ CD24ˉ cells in nude mice, no tumor growth was evident at 12 weeks. In contrast, nude mice implanted with 5 × 10^3 CD44^＋ CD24^＋ ceils had large tumors evident at 4 weeks （2/8） , and at least a 20 -50-fold increase in tumorigenic potential（ P 〈 0.05 or P 〈 0.01 ）. There was no obvious histological difference between the cells of the CD44^＋ CD24^＋ cells-formed nodules and PANC-1 cells. Conclusions CD44 and CD24 may be used as the cell surface markers for isolation of pancreatic cancer strum cells from pancreatic adenocarcinoma cell line PANC-1. Subpopulation cells CD44^＋ CD24^＋ have properties of tumor stem cells.