Department of Prosthodontics, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai, China; Department of Prosthodontics, the 2nd Affiliated Hospital, Harbin Medical University, Harbin 150001, China.
Bone. 2009 Dec;45(6):1146-52. doi: 10.1016/j.bone.2009.08.009. Epub 2009 Aug 13.
To assess the influence of high extracellular glucose levels on the osteogenic differentiation of bone marrow stromal cells (BMSCs) and to determine if Sonic hedgehog (Shh) protein can alleviate those effects. BMSCs were incubated with NG (normal glucose), NG+Shh (200 ng/ml Shh in normal glucose), NG+Shh+Gan (200 ng/ml Shh and 5 micromol/L GANT61 in normal glucose), HG (high glucose), HG+Shh (200 ng/ml Shh in high glucose), and HG+Shh+Gan (200 ng/ml Shh and 5 micromol/L GANT61 in high glucose). The expression levels of Shh signaling pathway genes Patched 1 (PTCH1) and osteogenesis-related genes were tested, which included bone morphogenetic protein 4 (BMP4), runt-related transcription factor 2 (Runx2), and osteopontin (OPN). Alkaline phosphatase (ALPase) activity and mineralized matrix formation were also investigated. Immunofluorescent staining of Gli1 was tested for Shh signaling activation. We found that recombinant Shh in normal-glucose medium could promote osteogenic differentiation of BMSCs, while inhibiting Shh signaling by GANT61 could antagonize this differentiation. Besides that high glucose impaired the Shh signaling as well as osteogenic differentiation of BMSCs, reactivation of Shh signal pathway by addition of Shh protein could mitigate the inhibition while further deactivation by Shh inhibitor GANT61 could retain their osteogenic inhibitions. The above data suggest that Shh pathway activity is involved in the HG condition mediated osteoblastic differentiation deficiency for BMSCs and that recombinant Shh could alleviate this inhibitory effect.
为了评估高细胞外葡萄糖水平对骨髓基质细胞（BMSCs）成骨分化的影响，并确定 Sonic hedgehog（Shh）蛋白是否可以减轻这些影响。将 BMSCs 与 NG（正常葡萄糖）、NG+Shh（正常葡萄糖中 200ng/ml Shh）、NG+Shh+Gan（正常葡萄糖中 200ng/ml Shh 和 5μmol/L GANT61）、HG（高葡萄糖）、HG+Shh（高葡萄糖中 200ng/ml Shh）和 HG+Shh+Gan（高葡萄糖中 200ng/ml Shh 和 5μmol/L GANT61）孵育。测试 Shh 信号通路基因 Patched 1（PTCH1）和骨生成相关基因的表达水平，包括骨形态发生蛋白 4（BMP4）、 runt 相关转录因子 2（Runx2）和骨桥蛋白（OPN）。还研究了碱性磷酸酶（ALPase）活性和矿化基质形成。测试 Gli1 的免疫荧光染色以检测 Shh 信号激活。我们发现正常葡萄糖培养基中的重组 Shh 可以促进 BMSCs 的成骨分化，而 GANT61 通过抑制 Shh 信号可以拮抗这种分化。此外，高葡萄糖还会损害 BMSCs 的 Shh 信号和成骨分化，添加 Shh 蛋白重新激活 Shh 信号通路可以减轻这种抑制作用，而 Shh 抑制剂 GANT61 的进一步失活则可以保留其成骨抑制作用。上述数据表明，Shh 通路活性参与了高葡萄糖条件下 BMSCs 成骨分化不足的过程，而重组 Shh 可以减轻这种抑制作用。
