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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/m L,高于4%~8%羟乙基淀粉对脐血MSCs回收量[(5.12~6.88)×106个/m L)],明显高于0.3%~0.6%明胶对脐血MSCs回收量[(2.08~2.56)×106/m L)],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。
Objective To investigate the feasibility and effect of hydroxypropyl methylcellulose in the separation of human umbilical cord blood mesenchymal stem cells (MSCs). Methods Umbilical cord blood samples were diluted with normal saline before adding 3 different sedimentation factors (hydroxypropyl methylcellulose, gelatin and hydroxyethyl starch) respectively. The ratio of blood, physiological saline and sedimentation factor was 2: 1: 3 , Mix and let stand until the interface stratified clear record time; the supernatant was further separated with Percoll solution, counting the amount of recovered MSCs and survival count; MSCs were cultured, passaged to the 3rd and 20th generation and liquid nitrogen After 6 months of cryopreservation, the expression of CD44, CD90, CD105, CD34 and CD45 were detected by flow cytometry after the resuscitation and then passage to the 35th generation. Results Hydroxypropyl methylcellulose precipitated erythrocytes fastest, only 15 min, gelatin and hydroxyethyl starch need about 30 min, the difference was statistically significant (P <0.05); 0.1% -0.3% hydroxypropyl The recovery of cord blood MSCs by methylcellulose was (6.24 ~ 6.72) × 106 cells / m L, higher than that of 4% ~ 8% hydroxyethyl starch ([5.12 ~ 6.88 × 106 cells / m L)] was significantly higher than that of 0.3% ~ 0.6% gelatin in cord blood MSCs recovery [(2.08 ~ 2.56) × 106 / m L)], the differences were statistically significant The survival rate of MSCs recovered by cellulose was the highest, higher than that of gelatin and hydroxyethyl starch (P <0.05). The MSCs isolated and cultured with hydroxypropyl methylcellulose reached the 3rd and 20th generation And liquid cryopreserved for 6 months, then reached the 35th generation. The expression of CD44, CD90 and CD105 were positive, and the expression of CD34 and CD45 was negative, which was in line with the characteristics of stem cell surface markers. Conclusion Hydroxypropyl methylcellulose can be used for cord blood MSCs isolation (the optimal concentration is 0.2%), which is better than the commonly used separation reagent gelatin and hydroxyethyl starch.