人胎盘间充质干细胞4种消化分离方法的效果比较

Comparison of four digestion methods in isolating mesenchymal stem cells from human placenta

背景:目前已从多种组织器官中分离出间充质干细胞,如何更高效地获取大批量纯度佳的干细胞仍是研究目标之一。目的:对人胎盘间充质干细胞采取4种不同的消化分离方法,比较其分离效果。设计、时间及地点:细胞学体外对比观察,于2007-07/2008-01在暨南大学医学院血液病研究所完成。材料:胎盘标本来源于足月正常剖宫产胎儿,由暨南大学附属第一医院提供。胶原酶Ⅱ、胶原酶Ⅳ为GIBCO产品,羟乙基淀粉为B.BRAUN产品,淋巴细胞分层液为上海试剂二厂产品。方法:胎盘组织剪碎后平均分为4组:胶原酶Ⅳ+羟乙基淀粉沉淀组、胶原酶Ⅱ+羟乙基淀粉沉淀组、胶原酶Ⅱ+淋巴细胞分层液分离组、胶原酶Ⅱ+氯化铵裂解红细胞组,5份标本/组。将第1组置于1g/L胶原酶Ⅳ中,另外3组置于1g/L胶原酶Ⅱ中,37℃消化45min。过筛后收集各组细胞悬液,按组别对应施以羟乙基淀粉沉淀法、淋巴细胞分层液分离法、氯化铵裂解红细胞法进行分离。主要观察指标:观察4种方法在获取细胞数、培养成功率、细胞出现伸展时间、原代培养时间方面的差异,并对培养得到的细胞进行表面标志检测。结果:与胶原酶Ⅱ+羟乙基淀粉沉淀组比较,其余3组获取的细胞数均明显减少(t=2.92～8.16,P<0.05)。在其他条件相同的情况下,胶原酶Ⅱ+羟乙基淀粉沉淀组培养成功率为100%,其余3组分别为80%,80%,20%。胶原酶Ⅱ+羟乙基淀粉沉淀组细胞出现伸展时间及原代培养时间均短于胶原酶Ⅱ+淋巴细胞分层液分离组(t=5.27～5.37,P<0.05),亦短于胶原酶Ⅳ+羟乙基淀粉沉淀组(2.46～2.50,P<0.05)。流式细胞仪检测示第3代人胎盘间充质干细胞强表达透明质酸受体CD44和整合素家族成员CD29,不表达造血干细胞标志CD34和CD45,也不表达内皮细胞标志CD106及HLA-DR。结论:使用胶原酶Ⅱ消化胎盘组织,结合羟乙基淀粉沉淀法能较好地分离人胎盘间充质干细胞。

BACKGROUND： Mesenchymal stem cells can be isolated from various tissue organs. How to effectively obtain high purity stem cells should be studied. OBJECTIVE： To investigate the various methods of separating mesenchymal stem cells from human placenta and to compare the isolation effectiveness. DESIGN, TIME AND SETTING： The in vitro controlled, cytological study was performed at the Institute of Hematology, Medical College, Jinan University from July 2007 to January 2008. MATERIALS： Placenta samples were collected from full-term fetus following normal uterine-incision delivery, which was supplied by First Affiliated Hospital, Jinan University. Collagenase Ⅱ and collagenase Ⅳ were purchased from Gibco. Hespan and lymphocyte layer solution were respectively bought from B.BRAUN and Shanghai Second Reagent Factory of China. METHODS： Human placenta mesenchymal stem cells were separated with four different methods： group A （collagenase Ⅳ ＋ Hespan method）, group B （collagenase Ⅱ ＋ Hespan method）, group C （collagenase Ⅱ ＋ Ficoll-hypaque method）, group D （collagenase Ⅱ ＋ NH4Cl solution）. Five samples were in each group. Samples in group A were incubated in 1 g/L collagenaseⅣ, whereas samples in groups B, C and D were incubated in 1 g/L collagenase Ⅱ at 37℃ for 45 minutes. Following cribration, cell suspension was collected from each group. Hespan method, Ficoll-hypaque method and NH4Cl solution were separately performed. MAIN OUTCOME MEASURES： Differences in cell count, successful culture rate, time of stretching and primary culture time using the culture methods were observed. The surface markers were determined. RESULTS： Compared with the group B, the cell number was significantly diminished in groups A, C and D （t=2.92-8.16, P〈 0.05）. Under the same conditions, the successful culture rate was 100% in group B, and 80%, 80% and 20% in group A, C and D, respectively. The time of stretching and the time of primary culture with Hespan were shorter in the group B than that in group C （t=5.27-5.37, P 〈 0.05） and in group A （2.46-2.50, P 〈 0.05）. Flow cytometry demonstrated that the third passage of human placenta mesenchymal stem cells were strongly positive for CD44, CD29, but negative for CD34, CD45, CD106 and HLA-DR. CONCLUSION： Collagenase Ⅱ digestion combined with Hespan method could effectively extract mesenchymal stem cells from human placenta.