两种体外分离成人骨髓间充质干细胞方法的比较

Comparison between two isolation methods of human mesenchymal stem cells in vitro

目的:目前尚缺乏标准的骨髓间充质干细胞分离方法,虽然Percoll密度梯度离心法被认为是较经典的方法,但该方法操作较复杂。比较全骨髓培养法与常用的Percoll密度梯度离心法分离成人骨髓间充质干细胞的差异,以寻找一种简便、经济、实用的人骨髓间充质干细胞体外分离方法。方法:实验于2006-09/2007-06在青岛大学医学院附属医院中心实验室完成。实验材料:行自体干细胞移植治疗的糖尿病患者骨髓由青岛大学医学院附属医院内分泌科提供,患者对实验知情同意,并经医院伦理委员会批准。实验方法:采用全骨髓培养法与Percoll密度梯度离心法从成人骨髓中分离骨髓间充质干细胞,比较两种方法所获得的贴壁细胞克隆数、细胞形态、细胞表面标志及向脂肪细胞的分化情况。结果:①全骨髓培养法获得的贴壁细胞克隆数明显多于Percoll密度梯度离心法(P<0.05)。两种方法获得的人骨髓间充质干细胞形态无明显差异,均为长梭形,克隆样生长。②免疫荧光显示,全骨髓培养法分离培养的第3代骨髓间充质干细胞CD44阳性表达率和CD34阴性表达率均略低于Percoll密度梯度离心法,但两者之间的差异无统计学意义(t=2.639,P<0.01)。③两种方法获得的第3代骨髓间充质干细胞经地塞米松、胰岛素诱导后均可分化脂肪细胞。结论:与传统的Percoll密度梯度离心法比较,全骨髓培养法获得的骨髓间充质干细胞贴壁较快,传代时间略早,细胞数量多。

AIM： There is no standard isolation method of mesenchymal stem cells. Percoll density gradient centrifugation is treated as the traditional method among numerous methods but it is so complicated. This study compares whole bone marrow culture to traditional Percoll density gradient centrifugation in order to establish a better method in vitro. METHODS： Experiments were conducted at Central Laboratory of Affiliated Hospital of Qingdao University Medical College from September 2006 to June 2007. Bone marrow was supported by patients who had autologous stem cell transplantation by endocrinology department for diabetes treatment. The experiments had known by the patients and authorized by the hospital ethic committee. The two different methods, whole bone marrow culture and Percoll density gradient centrifugation, were compared in the number of cell clone, cell morphology, cell superficial mark, the differentiation into fat cell. RESULTS： （1） The number of clone cell by whole bone marrow culture was more than by Percoll density gradient centrifugation （P 〈 0.05 ） . There was no remarkable differentiation of morphological expression about mesenchymal stem cells using the two methods. They were both like long shuttle, clone growth. （2）Immunofluorescence showed that positive CD44 expression and negative CD34 expression in the third generation of mesenchymal stem cells cultured by the whole bone marrow method was less than by Percoll density gradient centrifugation （t =2.639,P 〈 0.01）. （3）The third generation of mesenchymal stem cells gotten by the two methods could differentiate into fat cell by insulin and dexamethasone. CONCLUSION： Compared with the Percoll density gradient centrifugation method, whole bone marrow method with advantages of rapid adherence, operated simply and high gain ratio is better isolation method of mesenchymal stem cells.