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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14d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14d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5×108L-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5d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48h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2,3,4,5d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。
BACKGROUND: Pancreatic stem cells have the characteristics of dividing and highly differentiated, and can be successfully isolated and cultured in vitro. However, how to effectively expand pancreatic stem cells in vitro is an urgent problem to be solved. OBJECTIVE: To subculture mouse pancreatic stem cells in vitro under the condition of fetal rat fibroblast feeder layer. DESIGN, TIME AND SETTING: The cytology in vitro observation was performed at the cell sterile culture room of Guangxi Medical College of Traditional Chinese Medicine from July 2007 to January 2008. MATERIALS: Twenty newborn SPF Kunming mice and Kunming mice of 14d pregnant were purchased from Experimental Animal Center of Guangxi College of Traditional Chinese Medicine. Methods: Pancreatic tissues from SPF Kunming mice were digested by type Ⅴ collagenase and completely undigested. After collecting the supernatant fluid containing cells, the cells were centrifuged and the supernatant was discarded. The cells were treated with keratinocyte growth factor, Fibroblast growth factor-free serum-free, low-glucose DMEM medium was grown in poly-lysine-treated 24-well plates. Pregnant 14d Kunming mice, fetus fetus removed to remove the head and internal organs, the trunk and extremities using tissue culture trypsin digestion fibroblasts, adjust the density of 5 × 108L-1 seeded in 24-well plates, To the third generation mitomycin appropriate treatment of feeder cells. Primary cultured 5d pancreatic stem cells were seeded at a density of 30 cells / cm2 into feeder cells, and passage continued when pancreatic stem cells covered 80% of the bottom area of the pores. MAIN OUTCOME MEASURES: Cell morphological changes were observed under an inverted microscope. Primary cultured cells were stained with alkaline phosphatase for 48 hours. The expression of nestin in pancreatic stem cell-specific markers was identified by immunohistochemical staining. The expression of alkaline phosphatase, nestin and pancreaticoduodenal homeobox 1 gene was detected at 2, 3, 4 and 5 days after passage. RESULTS: Primary cultured cells derived from pancreatic tissue showed some large, round and single nuclei. The cytoplasm showed strong refraction and a large proportion of nuclear cytoplasm. The cells grew in the wall and were positive for alkaline phosphatase staining Pancreatic stem cell-specific molecular markers nestin, and has an active ability to divide and proliferate. Under the conditions of mouse embryonic fibroblast feeder layer, the pancreatic stem cells were subcultured in vitro and could be transmitted serially to the third generation. The characteristics of large, round, single nucleus, large proportion of nuclear cytoplasm and proliferative ability of each generation of pancreatic stem cells , After the passage of time points alkaline phosphatase, nestin staining were positive, pancreatic duodenal homeobox gene 1 protein staining was negative, can remain undifferentiated state. CONCLUSION: Mouse embryonic fibroblasts can be used to secrete the necessary factors for the growth of pancreatic stem cells and inhibit the autologous differentiation of pancreatic stem cells, which can be persistently transmitted to the third passage in vitro after being treated with mitomycin C Better growth characteristics, high proliferative capacity and undifferentiated state.