Bi Xiao-Yun, Huang Shu, Chen Jing-Li, Wang Fang, Wang Yan, Guo Zi-Kuan
Nan-Fang Center of Biological Diagnosis and Therapy, Hospital of Developmental District of Guangzhou, Guangzhou 510730, Guangdong Province, China.
Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2014 Apr;22(2):491-5. doi: 10.7534/j.issn.1009-2137.2014.02.041.
The release of microvesicles(MV) is one of the critical mechanisms underlying the angiogenesis-promoting activity of mesenchymal stem cells(MSC). This study was aimed to explore the appropriate condition under which MSC releases MV. Bone marrow samples from 5 healthy adults were collected, and MSC were isolated, culture-expanded and identified. MSC at passage 5 were suspended in medium without or medium with 10% fetal(FCS) calf serum and seeded into culture dishes. The culture was separately maintained in hypoxia (1% oxygen) or normoxia (around 20% oxygen), and 20 dishes of cells (2×10(6)/dish) were used for each group. The supernatants were collected for MV harvesting. The cell number was counted with trypan blue exclusion test and the protein contents in the MV were determined. MV were identified by observation under an electron microscope. The surface markers on MV were analyzed by flow cytometry. MTT test was performed to observe the pro-proliferative activity of MV that were added into the culture of human umbilical cord vein endothelial cells at a concentration of 10 µg/ml. The results showed that the majority of MV released by MSC were with diameters of less than 100 nm, and MV took the featured membrane-like structure with a hypodense center. They expressed CD29, CD44, CD73 and CD105, while they were negative for CD31 and CD45. The increase multiples of the adherent trypan blue-resistant cells cultured in normoxia with serum, in normoxia without serum, in hypoxia with serum and hypoxia in the absence of serum were 4.05 ± 0.73, 1.77 ± 0.48, 5.80 ± 0.65 and 3.69 ± 0.85 respectively, and the estimated protein contents per 10(8) cells were 463.48 ± 138.74 µg, 1604.07 ± 445.28 µg, 2389.64 ± 476.75 µg and 3141.18 ± 353.01 µg. MTT test showed that MV collected from MSC in hypoxia seemed to promote the growth of endothelial cells more efficiently than those from cells in normoxia. It is concluded that hypoxia can enhance the release of microvesicles from MSC, and cultivation of MSC in hypoxia and medium without serum may provide an appropriate condition for MV harvesting.
微泡（MV）的释放是间充质干细胞（MSC）促血管生成活性的关键机制之一。本研究旨在探索MSC释放MV的适宜条件。采集5名健康成年人的骨髓样本，分离、培养扩增并鉴定MSC。将第5代MSC悬浮于不含或含10%胎牛血清（FCS）的培养基中，接种于培养皿。培养分别在低氧（1%氧气）或常氧（约20%氧气）条件下进行，每组使用20个细胞培养皿（2×10⁶/皿）。收集上清液用于收获MV。通过台盼蓝排斥试验计数细胞数量，并测定MV中的蛋白质含量。通过电子显微镜观察鉴定MV。通过流式细胞术分析MV表面标志物。进行MTT试验，观察以10μg/ml浓度加入人脐静脉内皮细胞培养物中的MV的促增殖活性。结果显示，MSC释放的大多数MV直径小于100nm，MV呈具有低密度中心的特征性膜样结构。它们表达CD29、CD44、CD73和CD105，而CD31和CD45呈阴性。在含血清常氧、无血清常氧、含血清低氧和无血清低氧条件下培养的抗台盼蓝贴壁细胞的增加倍数分别为4.05±0.73、1.77±0.48、5.80±0.65和3.69±0.85，每10⁸个细胞的估计蛋白质含量分别为463.48±138.74μg、1604.07±445.28μg、2389.64±476.75μg和3141.18±353.01μg。MTT试验表明，低氧条件下从MSC收集的MV似乎比常氧条件下的细胞来源的MV更有效地促进内皮细胞生长。结论是，低氧可增强MSC释放微泡，在低氧和无血清培养基中培养MSC可能为收获MV提供适宜条件。
