人骨髓间充质干细胞释放膜微囊条件探索

Exploration of Conditions for Releasing Microvesicle from Human Bone Marrow Mesenchymal Stem Cells

间充质干细胞(MSC)释放膜微囊(MV)是其促血管再生的重要机制。本研究旨在探索人骨髓MSC释放MV的适宜条件。收集5份健康人骨髓标本,分离培养并鉴定MSC。将第5代MSC分别悬浮于含10%胎牛血清或无血清的培养液中,按2×106/皿接种于直径为150 mm的培养皿中,在低氧(1%氧浓度)或常氧条件下培养72h,每组20皿。将培养上清高速离心收获MV。计数培养后贴壁细胞,并测定MV蛋白质含量。电子显微镜观察MV的形态以鉴定MV。流式细胞术分析MV的表面标志。在脐静脉内皮细胞培养体系中,加入不同来源的MV(10μg/ml),培养72 h后利用MTT实验观察细胞增殖活性。结果表明:多数MSC释放的MV直径<100 nm,在电子显微镜下呈特征性的中空膜样结构。MV表达CD29、CD44、CD73和CD105,不表达CD31和CD45。在常氧含血清、常氧无血清、低氧含血清和低氧无血清条件下,台盼蓝抵抗的贴壁细胞扩增倍数分别为4.1、1.8、5.8和3.7,计算108细胞释放的MV蛋白含量分别为463.48±138.74、1604.07±445.28、2389.64±476.75和3141.18±353.01μg。MTT实验表明,低氧条件下获得的MV具有更好的促内皮细胞增殖作用。结论:低氧可促进MSC释放MV,低氧和无血清培养MSC可能是制备MSC-MV的适宜条件。

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 × 106/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 108 cells were 463.48± 138. 74 μg, 1604. 07 ±445.28 μg, 2389. 64 ±476. 75μg and 3141.18 ±353.01 μg. MTT test showed that MV collected from MSC in hypoxia seemed to promote the growth of endothelial ceUs more efficiently than those from ceils 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.