人脐带间充质干细胞获得和纯化及向软骨细胞分化能力的研究

Research on the acquirement and purification of human umbilical cord mesenchymal stem cells and their differentiation abilities to chondrocytes

目的研究脐带间充质干细胞获得纯化的高效方法及向软骨细胞的分化能力。方法取人的正常分娩或剖腹产胎儿的脐带，用胶原酶和胰酶初步消化后，将流式细胞仪筛选得到的CD45（-）、CD90（＋）细胞作为原代细胞进行培养，并用复合胶原酶差速消化法逐级传代、纯化细胞，每次传代后取部分细胞用作流式细胞仪分析细胞的CD45、CD90阳性比率；对P3代细胞向软骨细胞进行诱导。结果流式细胞仪检测提示，采用流式细胞仪进行初次筛选并用复合胶原酶差速消化法逐级传代获得的P1、P2、P3代细胞中CD90的阳性率为（80．86±7．85）％、（95．86±3．28）％、（97．15±1．43）％，而CD45的阳性率为（2．534-0．28）％、（0．97±0．48）％、（0．05±0．01）％；向软骨细胞诱导结果提示P3代细胞有向终末软骨细胞分化能力，具有干细胞的特性。结论采用流式细胞仪进行初次筛选并用复合胶原酶差速消化法能快速获得高纯度的具有向终末软骨细胞分化能力的人脐带间充质干细胞。

Objective To explore the efficient way to purify and to enrich umbilical cord mesenchymal stem cells which have abilities to differentiate into chondrocytes. Methods Umbilical cord was acquired from new born infants and chopped into 1 mm^2 cubics, then they were pretreated with colleganase and trypsin so as to get the mixed cell suspension. CD45（-）,CD90（±） cells were acquired from the mixed cell suspension via FCM sorting and were cultured in the medium as umbilical cord mesenchymal stem PO cells. The PO cells were detached and passaged by multiplex collagenase NB4 and the positive rate of CD90 and CD45 of the P1-P3 cells were observeed via FCM; the P3 cells were induced to differentiated into chondrocytes. Results FCM results showed that the positive rate of CD90 on P1, P2, P3 was （80.86 ± 7.85）%, （95.86 ± 3.28）%, （97.15 ± 1.43）%, while the positive rate of CD45 on P1, P2, P3 cells was （2.53 ± 0.28）% and （0.97 ± 0.48）% and （0.05 ± 0.01）% respectively; P3 cells had differenciated into chondrocytes. Conclusion FCM presorting and Multiplex collagenase detachment procedures can swiftly acquire highly purified human umbilical cord mesenchymal stem cells which have the ability to differentiate into cbondrocytes.