还原性谷胱甘肽对兔骨髓间充质干细胞体外冷藏保存活性保护作用研究

目的观察冷藏保存对兔骨髓间充质干细胞生长活性的影响及还原性谷胱甘肽对其可能存在的保护作用。方法日本雄性大耳白兔12只，取出兔骨髓后，采用Percoll非连续性密度梯度离心法分离出单个核细胞，取原代培养后细胞经免疫磁珠分析和流式细胞术鉴定后进行传代培养。第3代传代培养细胞分别在体外4℃冷藏后分为对照组(0 h)和实验组。实验组分为冷藏6 h(6 h组)，12 h(12 h组)和24 h(24 h组)。部分冷藏12 h单个核细胞分别加入0.5 mmol/L和1.0 mmol/L还原性谷胱甘肽(GSH)共培养，分别为12 h+0.5 mmol/L GSH组和12 h+1.0 mmol/L GSH组。对各组进行8天生长曲线的绘制并完成活性的测定。采用SPSS 17.0软件进行配对 t检验分析，P ＜0.05为差异有统计学意义。结果对照组分离出单个核细胞12天可铺满瓶底。细胞原代培养后，流式细胞仪显示经免疫磁珠分选的细胞CD29阳性率达91.7％，表达CD29，CD44，CD90，不表达CD19，CD45，CD34。生长曲线显示，6 h组，12 h+0.5 mmol/L GSH组，12 h+1.0 mmol/L GSH组与对照组的细胞增殖能力差异均无统计学意义(均 P ＞0.05)。统计学显示12 h组和24 h组的细胞增殖能力与对照组，6 h组，12 h+0.5 mmol/L GSH组及12 h+1.0 mmol/L GSH组的细胞相比差异有统计学意义(均 P ＜0.01)；24 h组的细胞增殖能力与12 h组的细胞相比差异有统计学意义( P ＜0.01)。结论Percoll非连续性密度梯度离心法对于兔骨髓的骨髓间充质干细胞分离起着良好的效果。体外4℃冷藏保存对骨髓间充质干细胞的生长活性起着抑制效果，且随着冷藏时间的延长而更加明显。还原性谷胱甘肽对冷藏保存后的骨髓间充质干细胞起着保护活性的作用。

Reduced Glutathione Protect Rat Bone MarrowMesenchymal Stem Cells from Refrigeration

Objective To observe the impact of refrigeration on the viability of rabbit bone marrow derived mesenchymal stem cells (BM-MSCs) and detect feasible protective method．Methods The rabbit MSCs were isolated，cultured and purified by discontinuous Percoll density gradient centrifugation．The isolated mononuclear cells (MNCs) were labelled by CD29 antibody，separated by immunomagnetic separation columns (IMSC) and identified by flow cytometry when cell surface markers were labeled．After 3 generationsubculture，MNCs were refrigerated at 4℃ for 0 h，6 h，12 h and 24 h，and they were divided into 0 h group，6 h group，12 h group and 24 h group．0.5 mmol/L and 1.0 mmol/L reduced glutathione (GSH) were added in 12 h group MNCs and divided into 12 h plus 0.5 mmol/L GSH group and 12 h plus 1.0 mmol/L GSH group．All morphology were observed，the growth curves were drawn．The SPSS 17.0 software was used to analyse the date and P <0.05 was considered statisticalsignificantly．Results Cells of control group were spindle-shaped and spreadto whole bottom in 12 days．Flow cytometry showed MNCs were 91.7% positive for CD29 when separated by IMSC．The separated MNCs were negative for CD19，CD45 and CD34，but positive for CD44 and CD90．Growth curves showed that no significant difference in cell proliferation between 6 h group，12 h+0.5 mmol/L GSH group，12 h+1.0 mmol/L GSH group and control group ( P ＞0.05)．There was significant difference of proliferative ability when cells in 12 h group and 24 h group cpmpared with control group，6 h group，12 h+0.5 mmol/L GSH group and 12 h+1.0 mmol/L GSH group ( P ＜0.01)．There was significant difference of proliferative ability when group 24 h compared with group 12 h ( P ＜0.01)．Conclusion The rabbit BM-MSCs could be effectively isolated and purified by discontinuous Percoll density gradient centrifugation．Refrigeration had a inhibitive activity in BMSCs in vitro．GSH can protect BMSCs proliferative activity in refrigeration．