人脐带间充质干细胞的新分离方法及其作为骨组织工程种子细胞的相关研究

New isolation method of human umbilical cord mesenchymal stem cells and relative research of them as seed cells in tissue engineering bone

目的探讨人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,h UCMSCs)的新分离方法及其作为骨组织工程(tissue engineering bone,TEB)种子细胞的特点。方法处理脐带组织,将含有杂质较多的组织去除,使剩余组织尽可能为Wharton’s jelly。采用组织块培养皿(实验组)及普通培养皿(对照组)分别分离脐带间充质干细胞,统计组织块培养成功率及获取原代脐带间充质干细胞的数量。流式细胞仪检测3代细胞的表面分子,并进行成骨、成软骨、成脂诱导以鉴定其多向分化潜能。将获取的3代脐带间充质干细胞与DBM骨体外构建组织工程骨,并检测细胞与组织工程骨的黏附率,激光共聚焦及总蛋白检测来确定细胞在支架材料上的生长情况。结果实验组与对照组培养12 d时组织块漂浮数为0 vs 13;培养成功率为(86.0±2.7)%vs(44±4)%,细胞数量为(24.7±3.6)×104vs(5.1±1.0)×104,2组相比差异均有统计学意义(P<0.01)。P3细胞阳性表达CD44+、CD73+、CD90+、CD105+,阴性表达CD34+、CD45+,具有向成骨、脂肪、软骨分化潜能。细胞在DBM上的黏附率为(81.3±4.7)%,相容性好,构建TEB上的种子细胞随时间增殖。结论新分离方法可以简化、规范操作过程,提高组织块的利用率,获取大量细胞(提高15~20倍)。细胞与DBM相容性好,可成功构建TEB。

Objective To investigate a new method of isolating human umbilical cord mesenchymal stem cells( h UC-MSCs) by explants culture and explore their features as seed cells in tissue engineering bone( TEB). Methods Fresh sterile umbilical cord tissues were obtained after approval of ethics committee of our hospital and signature of informed consent. The tissues containing more impurities cells were removed to make sure that the remained tissue as much as possible for Wharton’s jelly. Tissue culture dish( experiment group)and common culture dish( control group) were used to isolate UC-MSCs from the umbilical cord tissues. The successful rate of tissue culture and the number of primary cells were noted. Flow cytometry was used to detect the expression of cell surface molecules,and the potentials of multi-directional differentiation in osteogenicity,chondrogenicity,and adipogenicty in P3 cells. The obtained P3 cells were used to construct the TEB with demineralized bone matrix( DBM). The cell adhesion was detected. The growth of the cells was observed by laser confocal microscopy,and the amount of total proteins were also determined. Results In 12 d after culture,significant differences were seen between the experiment group and control in the number of floating explants( 0 vs 13,P < 0. 01),successful culture rate [( 86. 0 ± 2. 7) % vs( 44 ± 4) %,P < 0. 01 ],and number of cells [( 24. 7 ± 3. 6) × 104vs( 5. 1 ± 1. 0) × 104,P < 0. 01]. The obtained P3 cells were positive to CD44,CD73,CD90 and CD105+and negative to CD34 and CD45,with osteogenic,chondrogenic,and adipogenic differentiation capabilities. The cells had an adhesive rate of( 81. 3 ± 4. 7) %,exhibited good compatibility,and grew well and proliferated with time on TEB. Conclusion Tissue culture dish is a simple and standardized procedure to isolate h UC-MSCs,with high utilization rate,and harvests abundant cells( increased by about 15 to 20 times). Those cells have very good compatibility with DBM,and TEB are successfully constructed in vitro.