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

本研究采用人成骨样细胞系MG-63和小鼠成骨细胞原代培养的方法，在保持氧分压不变的密闭平板或烧瓶中向细胞内注入压缩的氦气，观察机械压缩对细胞增殖、DNA合成和蛋白质合成的影响。压力引起细胞数量和[~(3 H)]胸腺嘧啶核苷掺入呈时间和强度依赖性增加，以10min和80-15 OmmHg作用最明显。然而，当细胞被胰岛素样生长因子-I压缩时，这种效应也在更高的压力下出现，当细胞被胰岛素样生长因子-I压缩时，压力诱导的细胞增殖和DNA合成不被拉伸激活的双离子通道阻滞剂Gd^&lt；3+&gt；或蛋白激酶A、蛋白激酶C或钙调素依赖的蛋白激酶的抑制剂所抑制。然而，酪氨酸激酶抑制剂Genistein或Herbimycin A以浓度依赖的方式抑制这些压迫效应，上皮性Na~+通道阻滞剂苯扎米也是如此。加压60min后细胞获得的条件培养液可促进细胞增殖，而加压后30min或120min细胞的条件培养液则无此作用。RT-PCR分析显示，MG-63细胞在受压12小时后表达骨钙素。我们还检测了小鼠颅骨成骨细胞和骨细胞的原代培养形成的骨结节。按压(15 OmmHgx10min×1次/d)的骨结节形成率是不按压的1.6倍。这些结果表明，跨壁受压影响上皮细胞的Na+通道，然后触发自分泌-旁分泌因子的释放，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.