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背景:通过体外培养人角膜缘干细胞并进行传代、建系研究可为角膜缘干细胞移植的基础与临床研究提供足够的细胞储备。目的:探讨一种体外培养人角膜缘干细胞传代、建系的方法。设计:随机对照观察。单位:赣南医学院。材料:实验于2003-06/2004-04在赣南医学院科研中心与中山大学医学院眼科医院国家重点实验室完成。新鲜人角膜缘组织块分别取自2名健康人角膜组织供体,均经过供体者知情同意。RPMI-1640(SigmaR8755,含L-谷氨酰胺),200g/L胎牛血清(Gibco16140-071)。DMEM培养基、硫酸软骨素、人表皮生长因子购自美国Sigma公司,HEPES、DMSO购自美国Gibco公司,100%甘油购自上海运佳黄浦制药有限公司,戊二醛购自德国E.Merk公司,乙醇、盐酸、丙酮、甲醛等购自北京化学试剂公司,0.25%胰蛋白酶液购自上海新华制药厂,上述试剂均为分析纯级。方法:人角膜缘深部色素区组织块经消化后,分别在含有RPMI-1640、200g/L胎牛血清的培养瓶与以羊膜细胞外基质为培养载体的培养皿中进行体外培养。光镜及扫描电镜观察原代及传代培养细胞的生长情况;采用台盼蓝排斥实验计算冻存细胞逐代冻存处理后的复苏率。主要观察指标:①人角膜缘干细胞体外原代及传代培养观察结果。②细胞冻存复苏率。结果:①原代培养结果:经PRMI-1640培养基中培养1d后,倒置相差显微镜下可见培养瓶中细胞多已贴壁,均匀稀疏排列成单层并贴于培养瓶底部。②传代培养结果:传第2代时添加人表皮生长因子后,细胞分散成单层,贴壁生长旺盛。所有细胞传至第30代后形态的变异性增加明显,细胞体积明显增大,形态呈圆形或不规则圆形,密集成群。在培养液中传33代后,细胞仍然保持旺盛的分化、增殖能力,并且更适宜在羊膜细胞外基质上生长。③细胞冻存复苏率:冻存细胞的复苏率为82.2%。结论:将人角膜缘干细胞经体外培养33代并逐代冻存后初步建立了人角膜缘干细胞系,人角膜缘干细胞更适合在含有羊膜细胞外基质为底物的培养基中生长。
BACKGROUND: By establishing human limbal stem cells in vitro and passaged, the establishment of a pedigree provides sufficient cell reserve for basic and clinical studies of limbal stem cell transplantation. Objective: To explore a method of passage and establishment of human limbal stem cells in vitro. Design: Randomized controlled observation. Unit: Gannan Medical College. MATERIALS: Experiments were performed at the Research Center of Gannan Medical College and State Key Laboratory of Ophthalmology, Sun Yat-sen Medical College from June 2003 to April 2004. Fresh corneal limbal tissue blocks were taken from two healthy human corneal tissue donors, after informed consent of the donor. RPMI-1640 (Sigma R8755 with L-glutamine), 200 g / L fetal bovine serum (Gibco16140-071). DMEM medium, chondroitin sulfate, human epidermal growth factor were purchased from Sigma, USA, HEPES and DMSO were purchased from Gibco, USA. 100% glycerol was purchased from Shanghai Yunjia Huangpu Pharmaceutical Co., Ltd., glutaraldehyde was purchased from E. Merk , Ethanol, hydrochloric acid, acetone, formaldehyde and other purchased from Beijing Chemical Reagent Company, 0.25% trypsin solution was purchased from Shanghai Xinhua Pharmaceutical Factory, the above reagents were of analytical grade. Methods: After deep digestion of corneal limbal tissue sections, the cells were cultured in culture flasks containing RPMI-1640, 200g / L fetal bovine serum and petri dishes cultured with amniotic extracellular matrix respectively. Light and scanning electron microscopy were used to observe the growth of primary and subcultured cells. The survival rate of cryopreserved cells was calculated by trypan blue exclusion experiment. MAIN OUTCOME MEASURES: ① The observation of primary and subcultured human limbal stem cells in vitro. ② cell cryogenic recovery rate. Results: ① Primary culture results: After cultured for 1 day in PRMI-1640 medium, cells in culture flasks were adherent, evenly and sparsely arranged into a single layer and affixed to the bottom of the culture flask under inverted phase contrast microscope. ② subculturing results: When the second generation of human epidermal growth factor added, the cells dispersed into a single layer, adherent growth and vigorous. After all the cells reached the 30th generation, their morphological variability increased obviously, the cell volume increased obviously, the shape was round or irregular circle, densely clustered. After passage for 33 generations in culture medium, the cells still maintained strong differentiation and proliferation capacity and were more suitable for growth on the amniotic extracellular matrix. ③ cell cryogenic recovery rate: the survival rate of cryopreserved cells was 82.2%. CONCLUSION: Human limbal stem cell line was initially established after subcultured for generations 33 and cryopreserved. Human limbal stem cells are more suitable for growth in medium containing amnion extracellular matrix.