不同强度低频脉冲电磁场对小鼠MC3T3-E1成骨样细胞增殖、分化及矿化影响

Effect of low-frequency pulsed electromagnetic field on the mice MC3T3-E1 cells proliferation,differentiation and mineralization

目的探究不同强度低频脉冲电磁场(LFPEMFs)对小鼠成骨细胞株MC3T3-E1细胞增殖、分化和矿化的影响。方法将MC3T3-E1成骨样细胞分成4组,实验取材分为2个时间点。常规培养环境下分别接受频率一定(0.15 Hz),强度不同(1.10 m T,3.10 m T,4.10 m T)脉冲磁场刺激;不接受任何磁场刺激组作为对照组。细胞处理24 h后行细胞毒性检测(CCK8);细胞处理14 d后行碱性磷酸酶(ALP)染色、骨钙素(OCN)酶联免疫吸附测定(ELISA),Runt相关转录因子2(RUNX 2)的蛋白免疫印迹(WB)实验;对处理21 d后的4组细胞行茜素红(Alizarin red)染色、骨钙素ELISA检测和Ⅰ型胶原(Cola Ⅰ)WB实验。结果 MC3T3-E1成骨样细胞接受不同强度LFPEMFs处理24 h后没有发现毒性反应,各组CCK 8的OD值差异无统计学意义(P>0.05)。MC3T3-E1细胞接受不同强度LFPEMFs处理14d后,ALP、OCN、RUNX 2表达与对照组相比明显增强(P<0.05),随着强度的递增蛋白表达也相应增加(P<0.05);ALP表达随着磁场强度增大而增加(P<0.05),数量最多出现在4.10 m T组中(P<0.05);LFPEMFs促进OCN的分泌(P<0.05)并呈现强度依赖性(P<0.05);随着磁场强度的增加,RUNX 2蛋白表达增加(P<0.05)。处理21 d后,LFPEMFs提升MC3T3-E1成骨样细胞的矿化能力,随着磁场强度的增加,矿化结节数量相应增多(P<0.05),最大数量出现在4.10 m T组中(P<0.05);同时OCN的表达随着磁场强度的增加而增加(P<0.05),并出现浓度依赖性(P<0.05);Cola Ⅰ的的表达随着磁场强度的增强而增加(P<0.05),表达峰值出现在4.10 m T中(P<0.05)。结论固定频率脉冲电磁场在1.10~4.10 m T的强度下对小鼠MC3T3-E1细胞的增殖能力无明显影响,但能促进细胞成骨分化及矿化能力,并呈现磁场强度依赖性,这为临床上干预骨折愈合、抗骨质疏松等治疗提供实验依据。

ObjectiveTo investigate the effect of low frequency pulsed electromagnetic fields(LFPEMFs)on the mice MC3T3-E1cells proliferation,differentiation and mineralization in vitro.Methods MC3T3-E1pre-osteoblasts were divided into four groups,the time for sam-pling was divided into two time points.Cells were received frequency(0.15HZ),different intensities(1.10mT,3.10mT and4.10mT)pulse magnetic field in conventional cultivation environmen.Those without any magnetic stimulation group were selected as control group.Cell counting kit(CCK8)assay was examined after24hours-cultured.When14days cultured,cells were performed alkaline phosphatase(ALP)staining,enzyme-linked immuno sorbent assay(ELISA)of osteocalcin(OC),western blotting(WB)of runt-related transcription factor2(RUNX2);In addition,cells received alizarin red staining,ELISA of OC and WB of Collagen I(Cola I).ResultsThere was no toxic effects of LFPEMFs on MC3T3-E1cells after24hour culture(P>0.05).The expressions of ALP,OC and RUNX2were up-regulated after14days culture(P<0.05)and in a intensity-dependence manner(P<0.05).The expression of ALP was increased with magnetic stimulation(P<0.05)and the peak of ALP was at4.10mT group(P<0.05).LFPEMFs up-regulated the expression of OC(P<0.05)with a intensity-dependence manner(P<0.05).With the increasing of magnetic field intensity,RUNX2was up-regulated(P<0.05).With the increase of magnetic field intensity,mineralized nodule number of MC3T3-E2was increased accordingly(P<0.05)and LFPEMFs increased the mineralization ability of MC3T3-E1cells after21days culture.The expressions of OC and Cola I were up-regulated after21days culture(P<0.05)and in a intensity-dependence manner(P<0.05).ConclusionLFPEMFs has no effect of MC3T3-E1cell proliferation at the indensity of(1.10mT^4.10mT).However,LFPEMFs promoted osteoblasts differentiation and mineralization ability in a intensity-dependence manner.This provides experimental data of LFPEMFs for clinical intervention such as fracture healing,anti osteoporosis and so on.