Do bone cells have mechanosensor? Does Insulin-like growth factor-I play a role in signaltransduction of loading stress?

本研究采用体外培养的人成骨样细胞MG-63和小鼠成骨细胞，在保持一定氧分压的条件下，通过向培养皿或培养瓶中注入压缩氦气，观察机械压力对细胞增殖、DNA合成和蛋白质合成的影响。压缩导致细胞数量和[^3H]胸苷掺入的时间和强度依赖性增加，在10分钟和80 - 150 mmHg时最大效应明显。然而，当细胞被胰岛素样生长因子-I压迫时，在更高的压力下，即76 OmmHg，也可以观察到这种效应。钆（Gd^3+），一种牵张激活离子通道的阻断剂，或蛋白激酶A、蛋白激酶C或Ca^2+/钙调蛋白依赖性蛋白激酶的抑制剂，都不能抑制压迫诱导的细胞增殖和DNA合成。然而，酪氨酸激酶抑制剂染料木黄酮或除莠霉素A以浓度依赖的方式抑制了这些压迫效应，上皮Na^+通道阻断剂苯扎明也是如此。从压缩后60分钟的细胞获得的条件培养基诱导细胞增殖，但不从压缩后30或120分钟的细胞获得。RT-PCR分析显示MG-63细胞在加压后12小时表达骨钙素。我们还观察了小鼠颅骨成骨细胞和骨细胞混合培养形成的骨结节。骨结节形成伴透析扩张结果表明，透壁压迫可影响上皮细胞Na^+通道，从而引起自分泌-旁分泌因子的释放（或因子）通过酪氨酸激酶途径诱导细胞增殖和骨基质蛋白合成，最终增加骨细胞中的骨形成。

In the present study the effects of mechanical compression on cell proliferation, DNA synthesis and protein synthesis were examined in vitro with the human osteoblast-like cell line MG-63 and mouse osteoblast primary culture.Pressure was applied to cells by instilling compressed helium into sealed plates or flasks in which the partial pressure of oxygen were maintained constant. Compression resulted in time and intensity-dependent increases in cell number and [^3H]thymidine incorporation, with, maximum effects apparent at 10 min and 80 -l5OmmHg. However the effect was also seen at more intensive pressure, 76OmmHg, when cell were compressed with insulin-like growth factor-I.Compression-induced cell proliferation and DNA synthesis were not inhibited by gadolinium (Gd^<3+>), a blocker of stretch-activatedion channels, or by inhibitors of protein kinase A, protein kinase C, or Ca^<2+>/calmodulin-dependent protein kinases. However, the tyrosine kinase inhibitor genistein or herbimycin A inhibited these effects of compression in a concentration-dependent manner, and also epithelial Na^+ channel blocker benzamil did. Conditioned medium obtained from cells 60 minutes after compression induced cell proliferation, but not from cells 30 or 120 minutes after compression. RT-PCR analysis revealed that MG-63 cells expressed osteocalcin twelve hours after compression.We also examined bone nodule formed by the primary cultures of mixed osteoblast and osteocyte from mouse calvariae. Bone nodule formation with dialy compresion (l5OmmHg x 10 min x once / day) were increased 1.6 times as much as that without compression.These results suggest that transmural compression affects epithelial Na^+ channel and then triggers the release of a autocrine-paracrine factor (or factors) that induces cell proliferation and bone matrix protein synthesis through a tyrosine kinase pathway, then eventually increases bone formation in bone cells.