模拟微重力效应对骨细胞SOC通道功能的影响

Effects of Simulated Microgravity on Function of SOC Channels in Osteocytes MLO-Y4

目的探究模拟微重力效应下骨细胞钙池操纵Ca^2+通道(store-operated calcium channels,SOC)的活性变化以及其可能机制,阐明失重性骨丢失的发生机制。方法以小鼠骨细胞(MLO-Y4)为对象,分为回转模拟微重力效应组(simulated microgravity, SM)和正常重力组(control, CON)。分别旋转培养24、48 h后,激光共聚焦显微镜检测毒胡萝卜素引发细胞内质网钙库耗竭后胞内Ca^2+浓度水平,以反映SOC通道的活性;免疫荧光染色法观察膜骨架spectrin和内质网膜蛋白IP3R的分布情况,研究SOC通道功能变化的可能机制。结果在内质网钙库释放Ca^2+时期,24、48h SM组的胞内Ca^2+浓度水平与CON组相比均无显著差异,而在胞外Ca^2+经SOC通道内流时期,24h SM组只在前4min比CON组有显著性下降,48h SM组在整个时期均比CON组有显著性下降。与CON组相比,SM组膜骨架spectrin向细胞边缘聚集,而ER膜蛋白IP3R则向ER核被膜区域聚集,且48h组更为显著。结论模拟微重力效应可抑制骨细胞SOC通道活性。骨细胞膜骨架spectrin以及内质网膜上蛋白IP3R位置分布变化,可能影响SOC通道激活过程中蛋白间的构象耦合,进而降低骨细胞SOC通道的活性。

Objective To study the effect of simulated microgravity on activity of the store-operated calcium(SOC) channels in osteocytes and its possible mechanism, so as to elucidate the potential mechanism of weightlessness bone loss. Methods Osteocytes(MLO-Y4) as the experimental subjects were divided into simulated microgravity(SM) group and normal gravity group(CON). After rotating for 24 h and 48 h, confocal microscope was used to detect the intracellular calcium ion concentration level to reflect activity of the SOC channels after thapsigargin(TG)-induced endoplasmic reticulum(ER) depletion. Immunofluorescence staining was used to observe the distribution of ER membrane protein IP3R and spectrin membrane skeleton, in order to preliminarily explore the possible mechanism of functional changes of SOC channels. Results During the period of calcium release from ER,[Ca^2+]i had no significant difference between SM group and CON group for 24 h and 48 h;while during the period of extracellular calcium influx by SOC channels,[Ca^2+]i of SM group had significant differences in the first 4 minutes for 24 h, as well as in the whole time for 48 h. Compared with CON group, the spectrin membrane skeleton of SM group was gathered at the rim of membrane, while ER membrane protein IP3R of SM group was gathered at the nuclear envelope of ER. These two tendencies were more obvious for 48 h. Conclusions The stimulated microgravity could inhibit activity of SOC channels in osteocytes. Changes in the distribution of the spectrin membrane skeleton and ER membrane protein IP3R under the simulated microgravity might reduce the activity of SOC channels by affecting the conformation coupling process between the membrane and ER.