Ano5基因敲除小鼠成骨增强机制的初步研究

Preliminary study on the mechanism of osteogenic enhancement in Ano5 knockout mice

目的初步探讨导致Ano5基因敲除小鼠(Ano5^(-/-))成骨增强的潜在机制。方法选取出生2~3天小鼠颅骨成骨细胞(mCOB)进行原代培养,CCK-8实验观察细胞增殖能力,荧光探针染色检测胞内Zn^(2+)离子浓度。利用Agilent Mouse lncRNA V3芯片检测12周雄鼠胫骨组织差异表达基因,实时定量PCR(qRT-PCR)检测胫骨组织及m COB成骨分化过程中锌离子转运体和作用相关基因的表达水平。结果Ano5^(-/-)小鼠的mCOB增殖能力明显增强;细胞内Zn^(2+)浓度在成骨分化的第21天升高。锌离子调控基因Pde4d在骨组织和m COB成骨分化晚期的表达均下降,介导Zn^(2+)胞内转运的基因Zip-8、Zip-14表达增强,而介导Zn^(2+)胞外转运的基因Znt-5、Znt-7表达均降低。结论Ano5基因敲除后通过调控Zn^(2+)转运体相关基因表达改变成骨细胞内Zn^(2+)浓度,可能造成Pde4d-cAMP-CREB轴功能障碍,导致Ano5^(-/-)小鼠成骨功能增强。

Objective To investigate the potential mechanism of enhanced osteogenesis in Ano5 knockout mice(Ano5^(-/-) ).Methods Mouse calvarial osteoblasts(mCOB)were isolated from postnatal 2-3 days littermates and cultured.The cell proliferation was evaluated by CCK-8 assay.The concentrations of zinc in mCOB were determined by fluorescent probe staining.The Agilent Mouse lncRNA V3 chip was used to detect the genes expressed differentially in the tibia tissue of 12-week-old male mice.The real-time quantitative reverse transcriptase-polymerase chain reaction(qRT-PCR)was performed to examine the expression levels of zinc ion transporters or related genes in tibia tissue and mCOB respectively during the osteogenic differentiation.Results The proliferation of Ano5^(-/-) mCOB was significantly enhanced and intracellular zinc ion concentration was increased during osteogenic differentiation.The expression of Pde4d was decreased in tibias and the late stage of mCOB osteogenic differentiation.The expression of Zip-8 and Zip-14 as Zn^(2+) importer genes was increased.Meanwhile,the expression of Znt-5 and Znt-7 as Zn^(2+) exporter genes was reduced.Conclusions The Ano5 gene knockout leads to the abnormal Zn^(2+) concentration in osteoblasts via altering the expression of Zn^(2+) transport-related genes,which may affect Pde4d-cAMP-CREB axis and cause osteogenic enhancement in Ano5^(-/-) mice.