甲状旁腺激素对成骨细胞中ClC-3氯通道表达及成骨分化影响的研究

Effect of PTH on the ClC-3 Expression and Osteodifferentiation in MC3T3-E1 Cells

目的:观察甲状旁腺激素(PTH)对成骨细胞中ClC-3氯通道表达及成骨分化影响,初步探索ClC-3介导PTH在细胞成骨分化中的作用。方法:采用10-8M、10-9M、10-10M PTH持续刺激和间断刺激MC3T3-E1细胞72 h后,通过CCK-8试剂盒法检测MC3T3-E1细胞的增殖情况,Real-Time PCR法检测MC3T3-E1细胞中Clcn3及成骨相关基因Alp、Runx2的表达情况,免疫荧光法检测10-9M PTH不同给药方式下对ClC-3蛋白表达的影响。结果 :经不同浓度PTH连续和间断处理72 h后,结果显示10-9 M PTH间断刺激的MC3T3-E1细胞的增殖能力最强,且其Alp、Runx2 m RNA表达均高于10-8 M组和10-10 M组(P<0.05),而相同浓度间断刺激的MC3T3-E1细胞成骨相关基因的表达均高于持续刺激组,以10-9M间断刺激组差异最显著(P<0.05),而10-8 M和10-10M均无统计学差异(P>0.05),10-9 M PTH刺激的MC3T3-E1细胞中ClC-3蛋白表达也显著增加(P<0.05)。结论 :成骨细胞的ClC-3氯通道能够响应PTH的刺激发生变化,并伴随着成骨相关基因Alp、Runx2表达的增强。

Objective： To observe the effects of parathyroid hormone（PTH） on the expression of ClC-3 and osteogenic genes expression in MC3T3-E1 cells, and investigate the role of CIC-3 in osteoblast differentiation through PTH. Methods： 10-8M, 10-9M, 10-10 M PTH were used to continuously and intermittently stimulate the MC3T3-E1 cells for 72 h, then Cell Counting Kit-8 was used performed to detect the proliferation of MC3T3-E1 cells, Real-Time PCR was applied to measure the genes expression levels of Alp,Runx2 and Clcn3, immunofluorescence analysis was used to detect the expression of ClC-3 protein. Results： After the continuous and intermittent stimulation of different concentrations of PTH for 72 h, the proliferation of MC3T3-E1 cells treated by intermittent stimulation of 10-9M PTH was the most strongest, in which the Alp, Runx2 m RNA expressions were both significantly higher than those of 10-8M and 10-10 M PTH（P0.05）. The osteogenic genes expressions of intermittent stimulation with the same concentration of PTH were all significantly higher than those of the continuous stimulation, which were the highest in intermittent stimulation with 10-9M PTH（P0.05）, while no significant difference was found between 10-8M and 10-10 M group（P0.05）. The ClC-3 protein expression in MC3T3-E1 cells stimulated by 10-9M PTH was also obviously increased（P0.05）. Conclusions： PTH stimulation could promote the ClC-3 expression in MC3T3-E1 cells, along with the enhancement of osteogenic genes（Alp, Runx2） expression.