冻存脐带间充质干细胞向成骨细胞的分化

Differentiation of cryopreserved umbilical cord mesenchymal stem cells into osteoblasts

背景:在骨组织工程中,脐带间充质干细胞是的一种新兴的种子细胞。目前认为低温冻存是长期保存细胞的有效方法。目的:探究冻存的脐带间充质干细胞能否被诱导分化成成骨细胞。方法:采用组织块贴壁法从脐带的华尔通氏胶组织中分离出间充质干细胞。然后,用倒置显微镜观察原代细胞的细胞形态。脐带间充质干细胞的免疫表型和细胞周期用流式细胞仪检测。在冻存6个月后,复苏第2代脐带间充质干细胞进行冻存复苏,并传代培养至12代。对第12代的脐带间充质干细胞进行成骨诱导,它的成骨能力分别通过碱性磷酸酶活性检测,骨钙素和骨涎蛋白的免疫荧光检测以及茜素红染色检测来确定。结果与结论:原代脐带间充质干细胞呈现典型的成纤维细胞样形态。流式细胞仪显示培养的细胞高表达间充质干细胞的表面标志CD73、CD105和CD90,但是不表达造血细胞的表面标志CD34和CD45。复苏后细胞的存活率是90%。细胞周期显示P8的细胞有75%处于G0/G1期,25%处于S+G2M期。经成骨诱导液处理的第12代细胞显示出比对照组更高的碱性磷酸酶活性(P<0.01)。此外,在成骨诱导液中诱导的细胞对骨钙素和骨涎蛋白的染色呈阳性,并形成矿化了结节。冻存后的脐带间充质干细胞仍保持了它们的生物学特性,并且在成骨诱导液中能被诱导分化成成骨细胞。

BACKGROUND： Human umbilical cord mesenchymal stem cells are considered as novel seed cells in bone tissue engineering. Cryopreservation is an effective method for storing cells for a long time. OBJECTIVE： To explore whether umbilical cord mesenchymal stem calls of cryopreservation could be induced to differentiated into osteoblasts. METHODS： Mesenchymal stem cells were isolated from the Wharton＇s jelly of human umbilical cord tissue by the tissue explant adherent method. Morphology of primitive cells was observed by inverted microscopy. Immunophenotypes and cell cycle of umbilical cord mesenchymal stem cells were measured using flow cytometry. After frozen s^orage for 6 months, the second passage of umbilical cord mesenchymal stem cellswas thawed and subcultured to passage 12. Upon induction with osteogenic inductive medium, the osteogenic ability of passage 12 of umbilical cord mesenchymal stem cell was evaluated by alkaline phosphatase activity, the immunofluorescent analysis of osteocalcin and bone sialoprotein and the assay of alizarin red staining separately. RESULTS AND CONCLUSION： Primary umbilical cord mesenchymal stem cells displayed a typical fibroblast-like morphology. Flow cytometry showed that the cultured cells expressed high levels of the mesenchymal stem cells surface markers CD73, CD105 and CD90, but did not express hematopoietic cells surface markers CD34 and CD45. The survival rate of umbilical cord mesenchymal stem cells after resuscitation was 90%. The cell cycle analysis indicated that 75% of the cells of passage 8 were in G0/G1 phase and 25% in S＋G2M phase. Passage 12 cells treated with osteogenic inductive medium displayed a higher alkaline phosphatase activity compared with control cells （P 〈 0.01）. Moreover, the cells, induced in osteogenic inductive medium, were positive for osteocalcin and bone sialoprotein staining and formed the mineralized nodules. Umbilical cord mesenchymal stem cells still maintain their biological characteristics after cryopreservation, and can be induced into osteoblasts with osteogenic inductive medium.