羟丙基甲基纤维素在脐血干细胞分离中应用
Application of hydroxypropyl methylcellulose in separation of human umbilical mesenchymal stem cells
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摘要: 目的 探讨羟丙基甲基纤维素在人脐血间充质干细胞(MSCs)分离中应用的可行性及分离效果。方法 对脐血样本先用生理盐水稀释,然后分别加入3种不同沉降因子(羟丙基甲基纤维素,明胶和羟乙基淀粉),血液、生理盐水、沉降因子比例为2:1:3,混匀后静置至界面分层清晰时记录时间;用Percoll液对上清液进一步分离,计数回收MSCs量与存活数;将MSCs进行培养、传代,传到第3、20代及液氮冻存6个月后复苏再传至35代时用流式细胞仪检测CD44、CD90、CD105,CD34、CD45表达。结果 羟丙基甲基纤维素沉降红细胞速度最快,仅需15 min,明胶和羟乙基淀粉均需要30 min左右,差异具有统计学意义(P<0.05);0.1%~0.3%羟丙基甲基纤维素对脐血MSCs回收量在(6.24~6.72)×106个/mL,高于4%~8%羟乙基淀粉对脐血MSCs回收量(5.12~6.88)×106个/mL),明显高于0.3%~0.6%明胶对脐血MSCs回收量(2.08~2.56)×106/mL),差异均具有统计学意义(P<0.05);羟丙基甲基纤维素回收MSCs的存活率最高,高于明胶和羟乙基淀粉,差异具有统计学意义(P<0.05);用羟丙基甲基纤维素分离、培养的MSCs传到第3、20代及液氮冻存6个月复苏后传到第35代,CD44、CD90、CD105表达均呈阳性,CD34、CD45表达呈阴性,符合干细胞表面标志特征。结论 羟丙基甲基纤维素可以用于脐带血MSCs分离(最适宜浓度为0.2%),效果优于常用的分离试剂明胶和羟乙基淀粉。Abstract: Objective To explore the feasibility and effectiveness of application of hydroxypropyl methylcellulose in the separation of human umbilical mesenchymal stem cells(MSCs).Methods Umbilical blood samples were collected from healthy full-term births(50-100 ml for each birth) at a municipal hospital in Shenyang city and treated with heparin.The experiment was performed within 6 hours of blood collection.The umbilical blood samples were firstly diluted with saline.Hydroxypropyl methylcellulose,gelatin,and hydroxyethyl starch were used as sedimentation factors in erythrocyte sedimentation.The erythrocyte sedimentation rate,the number of umbilical MSCs recovered,and the survival rate of MSCs were determined after the reagents were mixed at the ratio of 2 (blood sample):1(saline):3(sedimentation factor) and settled down for the occurrence of clear interface.The MSCs were further separated with Percoll solution.The MSCs were subcultured and the expressions of CD44,CD90,CD105,CD34,and CD45 were detected with flow cytometry for the 35th generation of MSCs subcultured with the revived cells of 3rd and 20th generation after 6 months' freezing with liquid nitrogen.Results The erythrocyte sedimentation time was significantly shorter using hydroxypropyl methylcellulose compared to that using gelatin or hydroxyethyl starch(15 minutes vs.30 minutes)(P<0.05).The recovery of umbilical MSCs was 6.24×106/ml-6.72×106/ml using hydroxypropyl methylcellulose and higher than that using hydroxyethyl starch(5.12×106/ml-6.88×106/ml) or gelatin (2.08×106/ml-2.56×106/ml),with significant differences(both P<0.05).With the application of hydroxypropyl methylcellulose,positive expressions of CD44,CD90,and CD105 and negative expressions of CD34 and CD45 were observed in 35th generation of MSCs and the expressions were consistent with those of the stem cells.Conclusion Hydroxypropyl methylcellulose can be used for umbilical blood stem cells separation at the appropriate concentration of 0.2%,with better result than that using gelatin or hydroxyethyl starch in cell separation.
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