CoCl2缺氧处理对脐带间充质干细胞促血管生长因子表达的影响

目的: 探讨氯化钴(CoCl2)模拟缺氧预处理对人脐带间充质干细胞(UC-MSCs)中碱性成纤维生长因子(bFGF)和血管内皮生长因子(VEGF)基因表达及蛋白分泌的影响。方法: 分离、培养UC-MSCs。取第3代的UC-MSCs，运用含150 μmol/L的CoCl2培养液培养细胞72 h，用流式细胞仪检测缺氧对UC-MSCs表面标志物(CD34、CD45、CD90、CD105、CD29及CD49)的影响。依据CoCl2的浓度(0、50、100 μmol/L及150 μmol/L)将UC-MSCs分为4个组，即对照组、低、中、高CoCl2组，每个组处理24 h、48 h和72 h。用SYBRGREN荧光定量RT-PCR检测bFGF、VEGF mRNA的表达，用ELISA法检测bFGF和VEGF蛋白的表达。结果: 经含150 μmol/L CoCl2的培养液缺氧处理，没有改变细胞的形态和表面标记物。在缺氧培养时间相同的情况下，bFGF、VEGF基因的表达均随着CoCl2浓度的增加而增加，CoCl2为150μmol/L时达到峰值（P<0.05）。当CoCl2浓度为150μmol/L培养不同时间(24h、48 h及72 h)时，随着缺氧预处理时间的延长，bFGF、VEGF基因的表达随之增加，于48 h时达到峰值（P<0.05），bFGF、VEGF基因的表达分别为8.15±0.10和16.67±0.17，延长缺氧培养的时间基因的表达减小（P<0.05）；蛋白与基因表达的变化基本一致。在含150 μmol/L CoCl2的培养液培养48 h，bFGF和VEGF的表达达到顶峰（P<0.05），分别为(69.63±7.90) ng/L和(89.55±5.45) ng/L。结论: 适当浓度的CoCl2(<150 μmol/L)对UC-MSCs的生物学功能影响轻微。CoCl2可用于细胞的模拟缺氧，缺氧对UC-MSCs bFGF、VEGF的分泌具有时间和剂量依赖性，以含150 μmol/L CoCl2的培养液培养48 h，为促进bFGF、VEGF基因和其蛋白表达的最适条件。

Effect of hypoxia treatment by CoCl_2 on the expression of vascular growth factor in UC-MSCs

AIM: To study the effect of hypoxia induced by cobalt chloride (CoCl2) on the gene expression and protein secretion of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in human umbilical cord mesenchymal stem cells (UC-MSCs). METHODS: UC-MSCs were isolated and cultivated. The third passage cells were cultured in medium containing 150 μmol/L CoCl2 for 72 h. The effect of hypoxia treatment on UC-MSCs surface markers (CD34, CD45, CD90, CD105, CD29 and CD49) was detected by flow cytometry. UC-MSCs were divided into four groups according to the concentration of CoCl2 (0 μmol/L, 50 μmol/L, 100 μmol/L and 150 μmol/L) and each group was divided into three subgroups according to three different times of hypoxia treatment (24, 48 and 72 h). mRNA and protein expression of bFGF and VEGF in UC-MSCs were examined by SYBRGREN fluorescent quantitation and ELISA. RESULTS: Hypoxia treatment with CoCl2 had no effect on cell morphology and surface markers. Gene expression of bFGF and VEGF enhanced with the increase of CoCl2 concentration at the same anaerobic cultivating time and reached its peak when the CoCl2 concentration was 150 μmol/L. When the CoCl2 concentration was 150 μmol/L, the gene expression of bFGF and VEGF also increased with the extension of hypoxia incubation time in the three subgroups, and they reached their peak at 48 h, respectively, 8.15±0.10 and 16.67±0.17. But gene expression decreased with the extension of hypoxia incubation time. Change in protein secretion was consistent with that in gene expression. Protein secretion of bFGF and VEGF also reached its peak at the CoCl2 concentration of 150 μmol/L after 48 h hypoxia-precondition, respectively, (69.63±7.90)ng/L and (89.55±5.45)ng/L. CONCLUSION: The biological function of UC-MSCs is only mildly affected by the appropriate concentration of CoCl2 (<150 μmol/L). CoCl2 can be used to stimulate hypoxic conditions. The effect of hypoxia on the secretion of bFGF and VEGF is time- and dose-dependent. The ideal conditions to promote the expression of bFGF, VEGF gene and protein are 150 μmol/L of CoCl2 and 48 h.