模拟微重力诱导小鼠成骨细胞微丝变化对碱性磷酸酶及骨钙蛋白的影响

目的 研究模拟微重力诱导小鼠成骨细胞微丝结构改变对碱性磷酸酶（alkaline phosphatase,ALP）及骨钙蛋白（osteocalcin,OCN）的影响,探讨失重导致骨质疏松的机制.方法本研究以已建株的小鼠成骨样细胞株（MC3T3-E1）作为对象,以回转器模拟微重力效应.实验将细胞分为4组：模拟微重力组、0.5 μg/ml细胞松弛素B组、模拟微重力＋0.5 μg/ml细胞松弛素B组及正常重力组（对照组）.培养48 h后,利用异硫氰酸荧光素鬼笔环肽（fluoresceine isothiocyanate phalloidin,FITC-phalloidin）进行免疫荧光染色,在激光共聚焦显微镜下观察各组细胞微丝的变化;同时用ALP试剂检测各组细胞培养液内ALP的酶活性,采用ELISA法检测各组细胞裂解液中OCN的表达量.结果 除正常重力组外,其余各组细胞微丝结构都出现了不同程度的解聚,张力纤维减少,失去方向性,以模拟微重力＋0.5 μg/ml细胞松弛素B组改变最为明显.和正常重力组比较,其余各组ALP的酶活性和OCN的表达量均成下降趋势（F=17.23、121.91,P〈0.01）,且模拟微重力＋0.5 μg/ml细胞松弛素B组与模拟微重力组和0.5 μg/ml细胞松弛素B组这两组之间的差别都有统计学意义（P〈0.05）.结论 微重力影响成骨细胞ALP的酶活性及OCN的表达可能与微重力诱导细胞骨架微丝解聚相关.

Disruption of mouse's microfilament cytoskeleton induced by simulated microgravity on alkaline phosphatase and osteocalcin

Objective To research the effects of the mouse's microfilament cytoskeleton change induced by simulated microgravity on alkaline phosphatase (ALP) and osteocalcin (OCN), and then to explore the mechanism of weightlessness leading osteoporosis. Methods Research object was the osteoblast-like cell line of mouse (MC3T3-E1). Microgravity was simulated by rotary cell culture system. Cells were divided into four groups: simulated microgravity group, 0.5 μg/ml cytochalasin B group, simulated microgravity+0.5 μg/ml cytochalasin B group and normal gravity group. Cells had been cultivated for 48 h when immunofluorescence staining was made by fluoresceine isothiocyanate phalloidin (FITC-phallacidin). Then the changes of cell microfilament were observed by laser confocal microscope. At same time, the activity of ALP in the cell culture medium was tested by ALP detecting reagent, and the expression of OCN in cell lysate was measured with ELISA method. Results The microfilament cytoskeleton depolymerization, reduction of tension fibers and disorder were observed in all but normal gravity group, especially in the simulated microgravity+0.5 μg/ml cytochalasin B group. Compared with normal gravity group, the activity of ALP and the expression of OCN in other groups were in decreased tendency (F=17.23, 121.91, P<0.01), of which significant difference was found between simulated microgravity+0.5 μg/ml cytochalasin B group and 0.5 μg/ml cytochalasin B group (P<0.05). Conclusion Microgravity affects the activity of ALP and the expression of OCN and these may be associated with the disruption of microfilament cytoskeleton that induced by simulated microgravity.