小鼠胰腺干细胞饲养层条件下的连续传代培养

背景：胰腺干细胞具有分裂与高度分化潜能的特性，可以在体外进行成功分离和培养，但体外如何对其进行有效扩增是亟待解决的问题。 目的：在胎鼠成纤维细胞饲养层条件下体外传代培养小鼠胰腺干细胞。 设计、时间及地点：细胞学体外观察，于2007-07/2008-01在广西中医学院基础医学院细胞无菌培养室完成。 材料：SPF级新生昆明小鼠20只，孕14 d昆明小鼠多只，均购自广西中医学院实验动物中心。 方法：取SPF级新生昆明小鼠的胰腺组织，Ⅴ型胶原酶消化，未消化完全的组织块自然沉降后，收集上层的含细胞离散液，离心弃上清，加入含角质细胞生长因子、碱性成纤维细胞生长因子的无血清低糖DMEM培养基，在经多聚赖氨酸溶液处理的24孔板中培养。取孕14 d昆明小鼠，剖腹取胎鼠，去除头部及内脏，将躯干及四肢采用组织块胰蛋白酶消化法分离培养成纤维细胞，调整密度为5×108 L-1接种于24孔板中，传至第3代经丝裂霉素适当处理制备饲养层细胞。取原代培养5 d的胰腺干细胞，按30个/cm2密度种入铺有饲养层细胞的孔板中，当胰腺干细胞铺满小孔底部面积的80%时继续传代。 主要观察指标：倒置显微镜下观察细胞形态变化，原代培养48 h对细胞进行碱性磷酸酶染色，通过免疫组织化学染色鉴定胰腺干细胞特异分子标志物巢蛋白的表达。分别于传代后2，3，4，5 d检测碱性磷酸酶、巢蛋白和胰十二指肠同源盒基因1的表达。 结果：原代培养的来源于胰腺组织的细胞中，可见一些大、圆、单个核的细胞，胞浆折光性强，核浆比例大，呈附壁生长，碱性磷酸酶染色呈阳性，表达胰腺干细胞特异性分子标志巢蛋白，且具有活跃的分裂增殖能力。在小鼠胚胎成纤维细胞饲养层条件下对胰腺干细胞进行体外传代培养，可连续传至第3代，各代胰腺干细胞仍保持大、圆、单个核、核浆比例大及增殖能力强等特性，传代后各时间点碱性磷酸酶、巢蛋白染色均呈阳性，胰十二指肠同源盒基因1蛋白染色呈阴性反应，可保持未分化状态。 结论：以小鼠胚胎成纤维细胞作为饲养层，经丝裂霉素C处理后可分泌供胰腺干细胞生长所需的因子，并抑制胰腺干细胞的自主分化，体外连续传至第3代仍能够保持较好的生长特性、高度增殖能力及未分化状态。

In vitro serial passage culture of mouse pancreatic stem cells under the condition of embryonic fibroblast feeder layers

BACKGROUND: Pancreatic stem cells exhibit the capacities to division and high differentiation and can be successfully isolated and cultured in vitro. But how to perform effective in vitro proliferation remains to be solved. OBJECTIVE: To in vitro subculture mouse pancreatic stem cells under the condition of embryonic fibroblast feeder layers. DESIGN, TIME AND SETTING: A cytological, in vitro observation was performed at the Room for Cell Sterile Culture, School of Basic Medical Sciences, Guangxi Traditional Chinese Medical University between July 2007 and January 2008. MATERIALS: Twenty newborn Kunming mice of specific pathogen free and of either gender, as well as some 14-day-pregnant Kunming mice, were purchased from Laboratory Animal Center, Guangxi Traditional Chinese Medical University. METHODS: The pancreas were obtained from newborn Kunming mice and digested with V type collagenase. When tissue blocks that were not completely digested settled naturally, the cell suspension on the upper layer was collected and centrifuged. After supernatant removal and addition of serum-free low glucose dulbeccos modified eagles medium containing keratinocyte growth factors and basic fibroblast growth factors, cell suspension was cultured in polylysine-treated 24-well plate. Mouse embryo was taken from 14-day-pregnant Kunming mouse. After head and internal organs removal, the body and four limbs were isolated and cultured for obtaining fibroblasts by trypsin digestion. Following density adjustment, cells were inoculated into a 24-well plate at a density of 5×108 /L. After 3 passages, mitomycin C treatment was conducted to prepare embryonic fibroblast feeder layer. Pancreatic stem cells primarily cultured for 5 days were inoculated into feeder layer cells-containing 24-well plate at a density of 30 cells per square centimeter. When cells spread about 80% of the whole bottom, passage could be continued. MAIN OUTCOME MEASURES: Cell morphology was observed under an inverted microscope. After 48 hours of primary culture, cells were dyed with alkaline phosphatase(ALP), and Nestin expression was identified by immunohistochemistry. ALP, Nestin, pancreatic duodenal homeobox-1(PDX-1) detections were performed at 2, 3, 4, and 5 days after passage. RESULTS: Among the cells derived from pancreas by primary culture, some large, round cells with single nucleus could be found, which exhibited strong endochylema refraction and high karyoplasmic ratio, grew adhering to the wall, were immunoreactive for ALP and Nestin, and simultaneously showed strong division and proliferation capacity. Under the condition of mouse embryonic fibroblast feeder layer, pancreatic stem cells could be serially passaged to the third generation. Each generation of cells still maintained their properties including large, round appearance with single nucleus, high karyoplasmic ratio, and proliferative capability. At each time point after passage, the cells were positive for ALP and Nestin staining but negative for PDX-1 staining, and maintained undifferentiated state. CONCLUSION: After mitomycin treatment, mouse embryonic fibroblast feeder layer can secrete the factors that promote the growth of pancreatic stem cells but inhibit the self-differentiation. After in vitro serial passages, the third generation of pancreatic stem cells can maintain their properties including good growth characteristics, high proliferation capacity, and undifferentiated state.