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α-MSH对MC3T3-E1增殖、分化、矿化及其对OPG、RANKL、M-CSF表达的影响
作者:黄利华  桂华伟  雷德利 
单位:武汉市第四医院 病理科, 湖北 武汉 430030
关键词:α-促黑色素 成骨细胞 增殖 分化 矿化 
分类号:R589.5
出版年·卷·期(页码):2023·51·第一期(9-16)
摘要:

目的: 探讨α-促黑色素(α-MSH)对小鼠成骨细胞MC3T3-E1增殖、分化、矿化及其对骨保护素(OPG)、核因子-κβ受体活化因子配体(RANKL)、巨噬细胞集落刺激因子(M-CSF)表达的影响。方法: 设立空白组,α-MSH(10-9 mol·L-1、10-8 mol·L-1和10-7 mol·L-1)作为处理组,逆转录聚合酶链反应检测黑皮质素受体(MCR)表达情况;采用CCK-8法检测细胞增殖,PNPP法检测ALP活性,实时定量聚合酶链反应测定Runx-2、BMP-2、OCN、ALP、OPG、RANKL、M-CSF的基因表达, 蛋白质印迹法测定上述指标的蛋白表达, 细胞茜红素法测定矿化情况。 结果: MC3T3-E1细胞表达MC1R、MC2R、MC4R、MC5R 4种受体,与空白组相比,不同浓度组α-MSH均促进细胞增殖(P<0.01),且明显促进Runx-2、BMP-2、OCN蛋白及基因表达(P<0.01), 提高ALP活性及其基因表达(P<0.01),同时促进OPG基因和蛋白表达(P<0.01), 显著降低RANKL和M-CSF基因及蛋白表达(P<0.01), 且呈剂量依赖性。 结论: α-MSH通过MCR作用于MC3T3-E1细胞,可能通过调控Runx-2的表达促进成骨分化相关基因及蛋白BMP-2、ALP、OCN的表达,同时促进OPG表达,减少RANKL和M-CSF表达,从而调控骨代谢。

Objective: To explore the effects of α-MSH on the proliferation, differentiation and mineralization of mouse osteoblast MC3T3 E1 and its effects on the expression of OPG and RANKL and M-CSF. Methods: Blank group was set up, and treatment groups taking α-MSH(10-9 mol·L-1,10-8 mol·L-1 and 10-7 mol·L-1) were set up as well. The expression of MCR was detected by RT-PCR; Cell proliferation and ALP activity were detected with CCK-8 method and PNPP respectively; the gene expression of Runx-2, BMP-2, OCN, ALP, OPG, RANKL, M-CSF was determined using real-time quantitative polymerase chain reaction; the protein expression of the above indicators was determined using Western blot method; and the mineralization was analyzed with cytochrome method.Results: MC3T3 E1 cells expressed four kinds of receptors of MC1R, MC2R, MC4R and MC5R. Compared with the blank group, α-MSH of different concentration groups could promote cell proliferation(P<0.01); the expression levels of Runx-2, BMP-2, OCN protein and gene were significantly increased(P<0.01); the activity of ALP and its gene expression were increased(P<0.01). Moreover, the expression levels of OPG protein and gene were increased(P<0.01), and the expression levels of RANKL and M-CSF gene and protein were markedly decreased(P<0.01) in a dose-dependent manner.Conclusion: α-MSH may act on MC3T3 E1 cells through MCRs, which may regulate the expression of Runx-2, promote the expression of osteogenic differentiation related genes and proteins BMP-2, ALP and OCN, promote the expression of OPG, reduce the expression of RANKL and M-CSF, and thus regulate bone metabolism.

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