解剖学报

解剖学报 ›› 2016, Vol. 47 ›› Issue (5): 628-632.doi: 10.16098/j.issn.0529-1356.2016.05.008

• 细胞和分子生物学 • 上一篇    下一篇

间充质干细胞的成骨细胞分化和免疫组织化学鉴定

蔡敏1 林建立2* 侯建明2 江亚涛3 晋龙4   

  1. 1. 福建省立医院 干部特诊一科; 2.内分泌科;  3.妇产科;  4.病理科， 福州 350001
  • 收稿日期:2016-06-20 修回日期:2016-07-09 出版日期:2016-10-06 发布日期:2016-10-06
  • 通讯作者: 林建立 E-mail:linjianli@hotmail.com
  • 基金资助:

    福建省自然科学基金

Differentiation of mesenchymal stem cells into osteoblasts and immunohistochemical identification

CAI Min1 LIN Jian-li 2* HOU Jian-ming2 JIANG Ya-tao3 JIN Long4   

  1. 1.Department of the First Cadres Special Medicine; 2.Endocrinology; 3.Gynaecology and Obstetrics; 4.Pathology, Fujian Provincial Hospital, Fuzhou 350001, China

  • Received:2016-06-20 Revised:2016-07-09 Online:2016-10-06 Published:2016-10-06
  • Contact: LIN Jian-li E-mail:linjianli@hotmail.com

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摘要：

目的 探讨脐带的间充质干细胞分化为成骨细胞的影响因素和免疫组织化学鉴定。 方法 收集306例冻存的健康胎儿脐带，采用华尔通胶组织块贴壁法从脐带组织中分离间充质干细胞，利用荧光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采用免疫组织化学检测。复苏冻存的脐带间充质干细胞，并传代培养至10代。对第10代的脐带间充质干细胞进行成骨诱导，其成骨能力分别通过钙结节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 免疫组织化学结果显示，培养的细胞高表达间充质干细胞的表面标志物CD73、CD90和CD105，但是不表达造血细胞的表面标志物CD34和CD45。复苏后细胞的存活率是90%。细胞周期显示，第10代的细胞有80%处于G0/G1期，20%处于S+G2/M期。成骨细胞刺激因子诱导干细胞的骨涎蛋白染色呈阳性，并形成矿化的钙结节。 结论 冻存的脐带间充质干细胞在成骨细胞刺激因子下能被诱导分化为成骨细胞，具有高度自我增殖和多向分化能力。

关键词: 脐带间充质干间充质干细胞, 成骨细胞, 骨涎蛋白, 细胞三维培养, 免疫组织化学, 人

Abstract:

Objective To study if the umbilical cord mesenchymal stem cells of cryopreservation could be induced to differentiated into osteoblasts and immunohistochemical evaluation. Methods Totally 306 cases of healthy fetal umbilical cord were collected and frozen. 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 immunohistochemical analysis. Umbilical cord mesenchymal stem cells were thawed and subcultured to passage 10. Upon induction with osteogenic inductive medium， the osteogenic ability of passage 10 of umbilical cord mesenchymal stem cell was evaluated by calcium nodules， the immunofluorescent analysis of bone sialoprotein and the assay of alizarin red staining separately. Results Immunohistochemical analysis showed that the cultured cells expressed high levels of the mesenchymal stem cells surface markers CD73, CD90 and CD105, 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 80% of the cells of passage 10 were in G0/G1 phase and 20% in S+G2/M phase. Passage 10 cells treated with osteogenic inductive medium displayed a higher calcium nodule expression compared with control cells. Moreover, the cells, induced in osteogenic inductive medium, were positive for osteocalcin and bone sialoprotein staining and formed the mineralized nodules. Conclusion Umbilical cord mesenchymal stem cells can be induced into osteoblasts with osteogenic inductive medium and. still maintain their highly self-proliferation and multi-directional differentiation.

Key words: Umbilical cord Mesenchymal stem cell, Osteoblast, Bone sialoprotein, Three-dimensional cell culture, Immunohistochemistry, Human