Liu Feng, Akiyama Yasuto, Tai Sachiko, Maruyama Kouji, Kawaguchi Yoshihiro, Muramatsu Kouji, Yamaguchi Ken
Immunotherapy Division, Shizuoka Cancer Center Research Institute, 1007 Shimonagakubo, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8777, Japan.
J Bone Miner Metab. 2008;26(4):312-20. doi: 10.1007/s00774-007-0842-0. Epub 2008 Jul 4.
Mesenchymal stem cells (MSCs) are well known to possess multipotential differentiation and are becoming a good tool for clinical research. However, specific markers for their purification and the mechanism of their osteogenic differentiation remain to be elucidated. In the present study, we compared the expression of CD106, and osteogenic differentiation-related proteins and genes in human bone marrow (BM)-derived MSCs, before and after differentiation by FACS, histochemical staining, immunohistochemical staining, RT-PCR, and real-time PCR. It was found that MSCs were positive for CD13, CD29, CD44, CD73, CD90, CD105, and CD166, but negative for CD14, CD31, CD34, CD62E, CD45, and GlyA. Notably, CD106 was detected before osteogenic induction, but its expression was downregulated 10 fold after 2 weeks of osteogenic differentiation as determined by flow cytometry. The results of RT-PCR and real-time PCR revealed that the expression of CD106 mRNA in MSCs significantly decreased by 7.1-, 4.2-, and 5.1-fold, respectively after osteogenic, chondrogenic, and adipogenic differentiation. In contrast, other MSC-positive markers described above did not change significantly even after differentiation. Compared to levels in control cells, after 2 weeks of osteogenic differentiation, mRNA levels of alkaline phosphatase, bone sialoprotein, osteocalcin, and transcript factors RUNX2 and Osterix showed more than 2-fold, 5-fold, 1.5-fold, 2-fold, and 5-fold increase, respectively. Thus, we speculate that CD106 might be a useful surface marker for BMMSCs. Moreover, alkaline phosphatase, type I collagen, osteonectin, osteopontin, and biglycin were expressed in the early stages of osteogenic differentiation before bone sialoprotein and osteocalcin. The present study should help to provide a novel marker for isolating purified MSCs and characterizing osteogenic differentiation.
间充质干细胞（MSCs）具有多向分化潜能，已成为临床研究的良好工具。然而，其纯化的特异性标志物及其成骨分化机制仍有待阐明。在本研究中，我们通过荧光激活细胞分选（FACS）、组织化学染色、免疫组织化学染色、逆转录聚合酶链反应（RT-PCR）和实时定量聚合酶链反应（real-time PCR），比较了人骨髓（BM）来源的MSCs在分化前后CD106以及成骨分化相关蛋白和基因的表达。结果发现，MSCs对CD13、CD29、CD44、CD73、CD90、CD105和CD166呈阳性反应，但对CD14、CD31、CD34、CD62E、CD45和GlyA呈阴性反应。值得注意的是，在成骨诱导前可检测到CD106，但通过流式细胞术测定，在成骨分化2周后其表达下调了10倍。RT-PCR和real-time PCR结果显示，在成骨、软骨和成脂分化后，MSCs中CD106 mRNA的表达分别显著降低了7.1倍、4.2倍和5.1倍。相比之下，上述其他MSCs阳性标志物即使在分化后也没有明显变化。与对照细胞水平相比，成骨分化2周后，碱性磷酸酶、骨唾液蛋白、骨钙素以及转录因子RUNX2和Osterix的mRNA水平分别增加了2倍以上、5倍、1.5倍、2倍和5倍。因此，我们推测CD106可能是BMMSCs的一个有用表面标志物。此外，在骨唾液蛋白和骨钙素之前，碱性磷酸酶、I型胶原、骨连接蛋白、骨桥蛋白和双糖链蛋白聚糖在成骨分化的早期阶段就有表达。本研究有助于为分离纯化的MSCs和鉴定成骨分化提供一种新的标志物。
