Su Xinlin, Yu Mei, Qiu Guixing, Zheng Yongwei, Chen Yuhong, Wen Renren, Fu Guoping, Zhu Wen, Chen Jun, Wu Nan, Ma Pei, Chen Weisheng, Wu Zhihong, Wang Demin
Department of Orthopaedics Surgery, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical SciencesBeijing 100730, China; Blood Research Institute, Blood Center of WisconsinMilwaukee, WI53226, USA.
Blood Research Institute, Blood Center of Wisconsin Milwaukee, WI53226, USA.
Am J Transl Res. 2016 Mar 15;8(3):1447-59. eCollection 2016.
To compare Osterix and Nestin-Cre/Loxp system in studying the biological functions of murine osteoblastic cells including primary osteoblasts (OBs) and osteolineage mesenchymal progenitor cells (MPCs).
We isolated primary osteoblasts (OBs) from neonatal Nestin-cre-R26-loxP-YFP (Nes-OBs) and Osterix-cre-R26-loxP-YFP (Osx-OBs) mice and bone marrow mesenchymal stromal cells (BMMSCs) from the adults (termed as Nes-BMMSCs and Osx-BMMSCs). Then we detected the percentage of YFP(+) subpopulation in Nes/Osx-OBs and the percentage of CD45(-)YFP(+) progenitor population in Nes/Osx-BMMSCs and sorted them out (termed as Nes/Osx-YFP(+) OBs and Nes/Osx-CD45(-)YFP(+) MPCs) by using the sorting machine. We also analyzed the expression of surface antigens on Nes/Osx-YFP(+) OBs and Nes/Osx-CD45(-)YFP(+) MPCs by Flow cytometry. PDGF-BB induced proliferation of Nes/Osx-YFP(+) OBs and Nes/Osx-CD45(-)YFP(+) MPCs was measured by H3-Thymidine incorporation assay. We then did OB maturation and mineralization assays of Nes/Osx-YFP(+) OBs and CFU and multi-lineage differentiation assays of Nes/Osx-CD45(-)YFP(+) MPCs.
YFP(+)% in Nes-OBs and Osx-OBs and CD45(-)YFP(+)% in Nes-BMMSCs and Osx-BMMSCs was respectively 5.56%±3.56% (n=5), 10.12%±2.7% (n=4), 1.29%±0.98% (n=13) and 16.38%±6.98% (n=17). Both Nes-YFP(+) OBs and Osx-YFP(+) OBs were positive for CD51. Nes/Osx-CD45(-)YFP(+) MPCs were positive for CD51, CD105 and Sca1, and negative for CD31 and CD45. PDGFR expression in Osx-YFP(+) OBs was a bit higher than that in Nes-YFP(+) OBs, and slightly higher in Osx-CD45(-)YFP(+) MPCs than in Nes-CD45(-)YFP(+) MPCs. Proliferation ability of Nes/Osx-YFP(+) OBs increased dramatically after stimulated with PDGF-BB for 48 h, while it was not statistically significant that PDGF-BB induced the increase of proliferation ability in either Nes-CD45(-)YFP(+) MPCs or Osx-CD45(-)YFP(+) MPCs. We observed that no significant difference of OB maturation and mineralization ability existed between Nes-YFP(+) OBs and Osx-YFP(+) OBs, and there was little difference of self-renewal and multi-lineage differentiation potential between Nes-CD45(-)YFP(+) MPCs and Osx-CD45(-)YFP(+) MPCs, either.
Both Nestin and Osterix could be selected as useful markers for the osteoblastic cells, while Osterix was a prior choice due to larger number of Osterix-expressing cells than Nestin-expressing cells in distinct subpopulations of bone-forming cells.
比较Osterix和Nestin-Cre/Loxp系统在研究小鼠成骨细胞（包括原代成骨细胞（OBs）和成骨谱系间充质祖细胞（MPCs））生物学功能中的作用。
我们从新生的Nestin-cre-R26-loxP-YFP（Nes-OBs）和Osterix-cre-R26-loxP-YFP（Osx-OBs）小鼠中分离出原代成骨细胞（OBs），并从成年小鼠中分离出骨髓间充质基质细胞（BMMSCs）（分别称为Nes-BMMSCs和Osx-BMMSCs）。然后我们检测了Nes/Osx-OBs中YFP(+)亚群的百分比以及Nes/Osx-BMMSCs中CD45(-)YFP(+)祖细胞群的百分比，并使用分选仪将它们分选出来（分别称为Nes/Osx-YFP(+) OBs和Nes/Osx-CD45(-)YFP(+) MPCs）。我们还通过流式细胞术分析了Nes/Osx-YFP(+) OBs和Nes/Osx-CD45(-)YFP(+) MPCs表面抗原的表达。通过H3-胸腺嘧啶掺入法检测血小板衍生生长因子-BB（PDGF-BB）诱导的Nes/Osx-YFP(+) OBs和Nes/Osx-CD45(-)YFP(+) MPCs的增殖。然后我们对Nes/Osx-YFP(+) OBs进行了成骨细胞成熟和矿化检测，对Nes/Osx-CD45(-)YFP(+) MPCs进行了集落形成单位（CFU）和多谱系分化检测。
Nes-OBs和Osx-OBs中的YFP(+)%以及Nes-BMMSCs和Osx-BMMSCs中的CD45(-)YFP(+)%分别为5.56%±3.56%（n = 5）、10.12%±2.7%（n = 4）、1.29%±0.98%（n = 13）和16.38%±6.98%（n = 17）。Nes-YFP(+) OBs和Osx-YFP(+) OBs的CD51均呈阳性。Nes/Osx-CD45(-)YFP(+) MPCs的CD51、CD105和Sca1呈阳性，而CD
