一种从骨髓血凝块中分离间充质干细胞的方法

An Approach of Isolating Mesenchymal Stem Cells from Human Bone Marrow Clots

目的探讨一种从骨髓血凝块中分离培养间充质干细胞的简易方法。方法采集肝素化骨髓标本7份,部分加入凝血酶以模拟血液凝固过程,并于0、8和16 h分别使用尿激酶或机械处理,分为尿激酶处理组、机械处理组、凝固未处理组及未凝固对照组。各组标本经氯化铵溶解红细胞后,分别进行成纤维细胞集落形成单位(CFU-F)和MSC传代培养。计每组CFU-F及第0、1和2代MSC数量。流式细胞仪测定细胞表型,组织化学法检测细胞体外成骨和成脂肪能力。结果尿激酶处理组样本CFU-F数平均为(33.71±23.54),接近于未凝固对照组样本(40.43±21.29)(n=7,P>0.05),显著高于机械法处理组(13±11.91)(n=7,P<0.01)和凝固未处理组(3.71±3.89)(n=7,P<0.01)。储存8或16 h后的标本,各组CFU-F形成能力下降,而未凝固对照组和尿激酶处理组数量仍显著高于另外两组(P<0.05)。标本储存不同时间进行MSC传代培养,未凝固对照组及尿激酶处理组细胞数量无明显差别,但均显著高于凝固未处理组及机械处理组。各组MSC均表达CD73和CD90,不表达CD31和CD45。经特异诱导后,各组MSC均呈现碱性磷酸酶活性,细胞内出现亲油红O染料的脂肪滴。结论对于已经凝固的骨髓标本而言,尿激酶预处理是分离培养MSC的良好途径。

Objective To establish an easily-handled method to isolate and culture-expand MSC from marrow clots.Methods Seven heparinized bone marrow samples were harvested from healthy subjects.Thrombin were added to part of the samples to mimic the coagulation process.After being stored for 0,8 and 16 hours,all the samples were divided into 4 groups： urokinase-treated group,mechanically-cut group,untreated group and control group.The clots were mechanically cut into pieces or treated with urokinase,followed by red blood cell lysis with ammonium chloride.Colony-forming units of fibroblast（CFU-F） and MSC culture were further developed,and the numbers of CFU-F and MSC at passage 0-2 were counted.The phenotypic features were analyzed with flow cytometry.Histological staining was used to observe the in vitro osteogenesis and adipogenesis.Results The average CFU-F number of urokinase-treated group was 33.71 ±23.54,which was comparable to that of control group（40.43±21.29）（n=7,P＆gt;0.05）.Both of them were significantly higher than those of mechanically-cut group（13±11.91）（n=7,P＆lt;0.01） and untreated group（3.71±3.89）（n=7,P＆lt;0.01）.The CFU-F numbers decreased after samples were stored for 8 or 16 hours,but those of control and urokinase-treated group remained higher than the other two groups.MSC culture from samples stored at different times was developed.The cell numbers at different passages were similar between control and urokinase-treated group,while they were greatly higher than those from untreated and mechanically-cut group（P ＆lt;0.05 for all time points and passages respectively）.MSC from different groups expressed CD73 and CD90 and they were negative for CD31 and CD45.After specific induction of differentiation,the cells became positive for alkaline phosphatase activity and intracellular Oil-red O-binding lipid droplets appeared.Conclusion The results here suggest that urokinase pretreatment is an optimal strategy to culture mesenchymal stem cells from coagulated marrow samples.