人脐带沃顿胶间充质干细胞体外分离条件的优化及传代效应的流式细胞学检测

Optimization the methodology of isolating human ubilical cord mesenchymal stromal cells from Wharton's jelly and examination of their passage effect on immune phenotype using flow cytometry

目的观察不同胶原酶消化方法对人脐带沃顿胶间充质干细胞（mesenchymal stem cell,MSC）分离和培养结果的影响,并鉴定其分化潜能;探讨传代效应对其免疫表型的影响。方法将制备好的脐带标本分别加入Ⅰ型、Ⅱ型及Ⅳ型胶原酶,持续消化4-18 h,筛网过滤,离心收集细胞,用DMEM/F12培养基重悬细胞,调整细胞密度4.8×10^3-1×10^4/cm^2,接种培养,比较不同消化法分离人脐带沃顿胶MSC的效果。Von kossa钙结节染色、四环素荧光标记鉴定人脐带沃顿胶MSC向成骨方向分化的能力,RT-PCR鉴定其向心肌细胞分化的能力。应用流式细胞仪检测连续传代后MSC的免疫表型变化。结果Ⅰ型胶原酶消化法能够从人脐带沃顿胶获取了数量较多、活力较高的MSC,而且细胞出现伸展的时间及原代培养时间均短于Ⅱ型胶原酶及Ⅳ型胶原酶消化法。表面标记分析显示：随着传代次数的增加,阳性标记CD29、CD44、CD73、CD90、CD105的表达百分率没有变化,而阴性标记CD31、CD34和HLA-DR的表达率增加明显。体外诱导实验表明：来源于人脐带沃顿胶的MSC具有体外成骨和成心肌样细胞分化的能力。结论Ⅰ型胶原酶消化法简单易行,对细胞损伤小,能稳定、高效地从人脐带沃顿胶中分离出MSC。

Objective To observe the effects of different collagenase digestions on isolating human umbilical cordmesenchymal stromal cells（MSC） from Wharton＇s jelly, to exam their differentiation ability and to investigate their passageeffect on the immune phenotype. Methods Human umbilical cord samples were digested by collagenaseⅠ or Ⅱor Ⅳ for4-18 hours then were passed through sieves. Cells were collected by centrifugation then inoculated in DMEM/F12 mediumat concentration within range of 4.8×10^3-1×10^4/cm^2 to compare the effect of different digestions on MSC. Von kossa stainingand tetracycline fluorescence was used to label the osteogenic differentiation capacity of MSC. Also RT-PCR was employedto identify the differentiate capacity of MSC into myocardial-like cells. The immunophenotype of MSCs were detected byflow cytometry after subculture. Results Using collagenaseⅠ digestion, the number of MSCs isolated from human umbilicalcord in Wharton＇s jelly and their vitality were much higher while the period to show cell extension and primary culture timewere shorter than those using collagenaseⅡ or Ⅳ digestions. The analysis of surface marker revealed that the expression ofpositive markers include CD29, CD44, CD73, CD90 and CD105 did not change with passages while the negative markerssuch as CD31, CD34 and HLA-DR increased significantly with passages; Differential experiments induced in vitro show thathuman umbilical cord MSC in wharton＇s jelly had the ability to differentiate into osteoblasts and myocardial-like cells. Conclusion The human umbilical cord MSC in Wharton＇s jelly was successfully isolated by collagenaseⅠ digestion. This meth-od was simple with a high success rate while cell loss and damage were minimum. This makes large-scale cultivation possible. Negative markers increased with cell passages. This phenomenon revealed that MSC showed directional differentiation.