摘要
背景:研究发现miR-25的下调与活化T细胞核因子c1(nuclear factor of activated T cells c1,NFATc1)过度激活相关,而NFATc1是破骨细胞分化成熟中的核心转录因子,提示miR-25的表达水平能调控破骨细胞分化。目的:探讨过表达miR-25对Ti颗粒诱导的破骨细胞分化的影响,并探索其中的信号通路。方法:体外培养RAW264.7细胞系,CCK-8测定Ti颗粒对细胞增殖的影响;转染miR-25模拟物(mimics)至RAW264.7细胞,并与Ti颗粒培养诱导破骨细胞的分化;免疫荧光检测NFATc1的激活状态,TRAP染色及计数验证各组破骨细胞分化情况,实时荧光定量PCR检测Ca2+/NFATc1通路分子NFATc1、CaMKⅡ、CaMKⅣmRNA表达。结果与结论:(1)低浓度的Ti颗粒(0.1 g/L)对细胞增殖无明显影响;(2)与阴性对照组相比,miR-25过表达组破骨细胞(TRAP阳性细胞)生成明显减少(P <0.05);(3)细胞内转染miR-25模拟物后,免疫荧光检测见NFATc1的激活减少,荧光强度降低;(4)miR-25过表达后信号通路分子NFATc1、CaMKⅡ、CaMKⅣmRNA的表达显著下降(P <0.05)。(5)结果表明,miR-25过表达能抑制Ti颗粒诱导的破骨细胞分化,这一过程可能是通过Ca2+/NFATc1通路实现的。
BACKGROUND: Studies have found that the down-regulation of miR-25 is related to the excessive activation of nuclear factor of activated T cells c1(NFATc1), and NFATc1 is the core transcription factor in osteoclasts differentiation and maturation, suggesting that the expression level of miR-25 can regulate osteoclasts differentiation.OBJECTIVE: To explore the effect of mi R-25 overexpression on titanium(Ti) particles-induced osteoclasts differentiation and investigate the signaling pathways.METHODS: RAW264.7 cell line was cultured in vitro. Cell counting kit-8 was used to determine the effect of Ti particles on cell proliferation. mi R-25 mimics were transfected into RAW264.7 cells, and cultured with Ti particles to induce osteoclasts differentiation. Immunofluorescence was used to detect the activation status of NFATc1, TRAP staining and counting were adopted to verify the differentiation of osteoclasts, and real-time PCR was employed to detect and analyze the differential expression of NFATc1, Ca MKII, and Ca MKIV m RNAs.RESULTS AND CONCLUSION: The low concentration of Ti particles(0.1 g/L) had no significant effect on cell proliferation. Compared with the negative control group, there was less production of osteoclasts(TRAP positive) in the mi R-25 overexpression group(P < 0.05). After intracellular transfection with mi R-25 mimic, immunofluorescence detection showed that the activation of NFATc1 decreased and the fluorescence intensity decreased. The expression of signaling pathway molecules NFATc1, Ca MKII, and Ca MKIV m RNAs decreased significantly after mi R-25 overexpression(P < 0.05). These findings indicate that mi R-25 overexpression can inhibit the differentiation of Ti particles-induced osteoclasts. This process may be achieved through the Ca2+/NFATc1 pathway.
作者
胡维帆
郑力
李大地
孙阳
赵凤朝
Hu Weifan;Zheng Li;Li Dadi;Sun Yang;Zhao Fengchao(Graduate School of Xuzhou Medical University,Xuzhou 221000,Jiangsu Province,China;Department of Orthopedics,Affiliated Hospital of Xuzhou Medical University,Xuzhou 221000,Jiangsu Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2022年第5期682-687,共6页
Chinese Journal of Tissue Engineering Research
