摘要
目的通过三种分离方法分离脊椎术中出血标本中的间充质干细胞并进行培养,比较得到的单核细胞(Mononuclearcell,MNC)数、细胞贴壁情况、原代培养时间、传代至第三代后得到的细胞总数,探索针对脊椎术中出血来源间充质干细胞理想的分离培养方法。为进一步实验及应用提供实验基础。方法以12例胸腰椎骨折病人为实验对象。分别取术中脊椎松质骨出血标本30ml随机分为3等份,标记为A、B、C三组。A组采用密度梯度法,B组采用甲基纤维素(Methylcellulose,MC)沉降法,C组采用密度梯度结合甲基纤维素的两步法分离MNC,用含10%胎牛血清的低糖DMEM培养基培养。72小时全量换液,以后3-5天按时换液,细胞达到汇合状态后进行胰蛋白酶消化传代。比较三种分离方法分离的MNC数、第三天贴壁细胞数、原代培养时间、第三代细胞数。倒置显微镜观察细胞形态及生长状态。流式细胞仪检测细胞表面标志鉴定细胞。结果用3种分离方法提取MNC数分别为：A组(6.99±0.77)×106,B组(11.5±1.70)×106,C组(8.20±0.96)×106,三组之间差异有统计学意义(P<0.05)。培养第三天观察细胞贴壁数分别为：A组11.40±2.37,B组为10.10±1.91,A组与B组两者之间的差异没有统计学意义(P>0.05)；C组24±3.27,与其它两组比较有统计学差异(P<0.05)比较三组原代培养时间,A组为28.20±1.69天,B组为28.40±2.12天, A组与B组之间的差异没有统计学意义(P>0.05)；C组为20.40±1.17天,与其它两组比较,差别有统计学意义(P<0.05)。传代培养3代后获得的细胞数,A组为(6.39±0.30)×106,B组为(5.54±0.42)×106, A组与B组比较没有统计学差异(P>0.05)；C组为(8.43±0.30)×106,与其它两组比较,差别有统计学意义(P<0.05),三组培养细胞在倒置显微镜下观察,细胞形态一致,均为贴壁生长的长梭形或成纤维细胞样。流式细胞仪检测细胞表面标志,初次分离的细胞均有CD44表达阳性,CD34、CD45阳性率低,经过培养传代后均高表达CD29、CD44、CD90,而低表达CD14、CD34、CD45。结论脊椎术中松质骨出血标本中存在间充质干细胞,但数量较少,培养所得细胞经鉴定为间充质干细胞；甲基纤维素沉降结合密度梯度两步分离方法比其他两种方法有更好的培养效率,是分离单核细胞的较好方法；脊椎术中出血分离的间充质干细胞可以在体外进行有效的培养扩增,具有广泛的应用前景。
Objective This study is compare the effect with the different separation methods of mesenchymal stem cells (MSCs) in bleeding of thoracolumbar vertebral operation. The aim is to explore for the ideal method of MSCs separating from bleeding of spinal operation. Methods 12 patients whith thoracolumbar fracture were included in this experiment. The 30ml blood specimens from bleeding of cancellous bone in thoracolumbar vertebral operation of every patient were divided into three equal parts randomly and marked as A, B,C three groups respectively with different isolating methods:A group by density gradient seperation, B group with methyl cellulose (MC) precipitate seperation, C group by two-step seperation,which combined density gradient separation with MC precipitate seperation. The obtained MNC were cultured in low level sugar DMEM medium containing 10% fetal bovine serum. All medium was changed wholely after 72 hours and changed partly medium on time after 3-5 days. MNC were trypsined and passaged when these cells reached confluence state. MNC number of adherent cells on the third day, the primary culture time, the third generation of cells with three methods of separation wre compared. MNC morphology and their growth behavior were observed with inverted microscope. Cell surface markers of these cells were identified with flow cytometry. Results MNC number with three kinds of separation method were (6.99±0.77)×106 in A group,(11.5±1.70)×106 in B group and (8.20±0.96)×106 in C group. There is significant differences between the three groups (P<0.05). Adherent cells number on the third day were 11.40±2.37 in A group,10.10±1.91 in B group and 24±3.27 in C group. There was no significant differences between A group and B group (P>0.05), but there is significant differences between C group and anohter two groups (P<0.05).The primary culture time were 28.20±1.69 days in A group,28.40±2.12 days in B group and 20.40±1.17 days in C group. There is no significant differences between A group and B group (P>0.05), but there is significant differences between C group and anohter two groups (P<0.05). Subcultured third generations of cells were (6.39±0.30)×106 in A group, (5.54±0.42)×106 in B group and (8.43±0.30)×106 in C group. There is no significant differences between A group and B group (P>0.05), but there is significant differences between C group and anohter two groups (P<0.05). The cell morphology of three groups were the same feature:adherent longer spindle or fibroblastoid under inverted microscope. Cell surface markers were identified by flow cytometry. Cells were first isolated have CD44 expression always, however, positive rate of CD34, CD45 was lower. These MSCs after passage culture have highly CD29, CD44, CD90 expression, and lower expression of CD14, CD34, CD45. Conclusion There are cell identified as MSCs in the bleeding samples of thoracolumbar vertebral cancellous operation, but the number is very fewer. Two-step separation is a better separation of monocytes method. MSCs extract from bleeding in vertebral operation can be cultured in vitro amplification effectively and will have a wide range of application.
引文
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