葡萄糖对人脐带间充质干细胞生长与代谢的影响

Effects of glucose on growth and metabolism of human umbilical cord mesenchymal stem cells

背景:目前对人脐带间充质干细胞的研究主要集中于细胞生物学特性与组织分化等领域的研究,对细胞代谢途径调控的报道还很少。目的:考察了不同起始浓度的葡萄糖对人脐带间充质干细胞生长和代谢特征的影响。设计、时间及地点:对比观察实验,于2008-02/06在北京工业大学生命科学与生物工程学院病毒与药理研究室完成。材料:采集产妇遗弃健康足月分娩胎儿脐带4份。方法:①从人脐带组织Wharton’sJelly中成功分离出人脐带间充质干细胞,采用流式细胞仪对分离培养的第3代人脐带间充质干细胞免疫分型进行分析。②将细胞以2.5×107L-1接种于24孔板中,在含有不同起始葡萄糖浓度(0.31,2.78,6.12,21.58mmol/L)、体积分数为0.10FBS的DMEM培养基中培养,每隔24h取样计数。③将细胞以2.5×108L-1的密度接种于25cm2方瓶中培养96h后,收集不同葡萄糖浓度作用后的细胞,采用流式细胞仪进行细胞周期分析。④将细胞以2.5×108L-1密度接种于含有不同起始葡萄糖浓度DMEM培养基75cm2方瓶中,培养96h后,离心收集上清和细胞,分别用于细胞代谢物检测与酶活性分析。主要观察指标:①人脐带间充质干细胞细胞形态和表面抗原表达。②细胞生长情况。③细胞周期检测。④细胞胞内外营养物、代谢副产物及酶活性的测定分析。结果:①采用流式细胞仪对人脐带间充质干细胞进检测,阳性表达的有CD13,CD29,CD44,CD105,CD147,CD90,CD71,表达率均高于90%,而造血干细胞特有标记CD34,CD45及白细胞抗原HLA-DR、CD38呈阴性表达。②葡萄糖能促进人脐带间充质干细胞的生长,但当培养基中缺失葡萄糖时,细胞也可维持生长,在120h细胞达到最高密度11.6×107L-1。③细胞群体在G0/G1期的分布比例显著高于S期和G2/M期,随着葡萄糖浓度的增加,细胞群体处于G0/G1期的比例逐渐降低,S期相应增加。④当葡萄糖浓度为21.58mmol/L时,己糖激酶与丙酮酸激酶活性分别比缺失葡萄糖时增加了39.7%和64.3%,而谷氨酰胺、丝氨酸、亮氨酸等氨基酸的比消耗速率却随着葡萄糖浓度的增加而显著降低。结论:当培养基中缺失葡萄糖时,细胞可利用氨基酸等营养物维持生长;随着葡萄糖浓度的增加,细胞周期分布和葡萄糖代谢途径关键酶活发生改变,从而影响了人脐带间充质干细胞的生长与代谢特征。

BACKGROUND： Currently, the researches of Umbilical cord mesenchymal stem cells （UMSCs） are focus on cytobiology characteristics and tissue differentiation. However, the report on the regulation of cell metabolism is quite few. OBJECTIVE： To investigate the effects of different concentrations of glucose on growth and metabolism of UMSCs cells. DESIGN, TIME AND SETTING： The controlled experiment was performed at the Laboratory of Virulogy and Pharmacology Affiliated to College of Life Science and Bioengineering, Beijing University of Technology from February to June 2008. MATERIALS： Four human umbilical cords were obtained from full-term deliveries after the consent of each mother. METHODS： ①UMSCs were successfully isolated from the Wharton＇s Jelly of the umbilical cord. After the third passage, the expression of cell membrane proteins markers was analyzed by flow cytometry. ②Cells were seeded in 24-well plates at 2.5 × 10^7/L, and cultured in DMEM medium containing 0.10 fetal bovine serum with different concentrations （0.31, 2.78, 6.12, 21.58 mmol/L） of glucose. The number of cells per well were counted every day. ③Cells were plated onto 25 cm^2 flasks with different concentrations of glucose at 2.5 × 10^8/L, and collected after 96 hours for the cell cycle analysis with a flow cytometry. ④Cells were cultured in 75 cm^2 flasks with different concentrations of glucose at 2.5× 10^8/L. After 96 hours, media and cells were collected respectively to determinate of nutrient and byproduct concentrations, and the key metabolic enzymes activities. MAIN OUTCOME MEASURES： ①Morphology and surface antigens of human UMSCs; ②Cell proliferation assay; ③Cell cycle analysis; ④Determinations of cell nutrient, metabolites and enzymes activities. RESULTS： ①Using flow cytometric analysis, UMSCs was found to be positive for the CD13, CD29, CD44, CD105, CD147, CD90 CD71, and the expression rates were higher than 90%, but did not express hematopoietic lineage markers CD34, CD45, CD38, and HLA-DR. ②The growth of UMSCs was improved by glucose. Cells could maintain in the media without glucose, and a maximum cell density, 11.6 × 10^7/L, was obtained at 120 hours. ③The numbers of UMSCs cells in G0/G1 phase were significantly higher than those in S phase, and G2/M phase. The numbers of cells in G0/G1 phase decreased, and increased accordingly in S phase with the increase of initial glucose concentrations. ④When the concentration of glucose was 21.58 mmol/L, the activities of hexokinase and pyruvate kinase increased by 39.7% and 64.3%, respectively. However, the specific consumption rates of glutamine, serine and leucine were markedly decreased with the increased glucose concentration. CONCLUSION： UMSCs cells could maintain in the media without glucose. Cell cycle and the key metabolic enzymes activities in the glycolysis pathway were changed with the increase of initial glucose concentrations, resulting that the growth and metabolism characteristics of UMSCs were affected.