RhoU基因沉默对破骨细胞分化的影响

doi:10.11843/j.issn.0366-6964.2019.09.019

畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (9): 1904-1911.doi: 10.11843/j.issn.0366-6964.2019.09.019

RhoU基因沉默对破骨细胞分化的影响

郑嘉铭^1,2, 贺双江^1,2, 赵鸿雁^1,2, 宋瑞龙^1,2, 刘宗平^1,2*

1. 扬州大学兽医学院, 扬州 225009;
2. 江苏高校动物重要疫病与人兽共患病防控协同创新中心, 扬州 225009

收稿日期:2019-02-11 出版日期:2019-09-23 发布日期:2019-09-23
通讯作者: 刘宗平,主要从事动物营养代谢病与中毒病研究,E-mail:liuzongping@yzu.edu.cn
作者简介:郑嘉铭(1994-),男,广东湛江人,硕士生,主要从事动物营养代谢病与中毒病研究,E-mail:1743266610@qq.com
基金资助:
国家重点研发计划支持项目（2016YFD0501208）；国家自然科学基金（31672620；31702304；31872533）；江苏省自然科学基金青年基金项目（BK20150447）；江苏高校优势学科建设工程资助项目

Effect of RhoU Silencing on Osteoclast Differentiation

ZHENG Jiaming^1,2, HE Shuangjiang^1,2, ZHAO Hongyan^1,2, SONG Ruilong^1,2, LIU Zongping^1,2*

1. College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China;
2. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China

Received:2019-02-11 Online:2019-09-23 Published:2019-09-23

摘要/Abstract

摘要： 为了研究RhoU（Wrch1）在破骨细胞（osteoclast，OC）分化过程中的作用及其机理，试验以RAW 264.7细胞（单核巨噬细胞系）为基础材料，分别转染阴性慢病毒和siRhoU慢病毒并建立稳定的细胞系；在巨噬细胞集落刺激因子（M-CSF）和核因子κB受体活化因子配体（RANKL）存在的条件下，阴性对照组和RhoU沉默组细胞系分别诱导3或4 d。采用荧光定量聚合酶链式反应（qRT-PCR）技术检测RhoU和破骨细胞标志性mRNA的表达，蛋白免疫印迹（Western blot）技术检测RhoU和破骨细胞标志性蛋白的表达，抗酒石酸酸性磷酸酶（TRAP）染色观察OC的形成，激光共聚焦显微镜观察OC及其前体细胞的形态变化。结果显示，与阴性对照组相比，RhoU沉默组RhoU基因mRNA和蛋白表达量极显著降低（P<0.01）；OC形成的数量和面积极显著减少（P<0.01）；OC前体细胞丝状伪足形成受阻，破骨细胞缺乏完整的封闭带；OC特异性基因mRNA和蛋白表达量均显著或极显著下降（P<0.01或P<0.05），而TRAP基因mRNA变化不显著（P>0.05）。综上表明，沉默RhoU基因能抑制破骨细胞的分化，抑制前体细胞丝状伪足的形成进而影响其融合可能是其主要原因。

Abstract: In order to study the effect of RhoU (Wrch1) in osteoclast (OC) differentiation, we used RAW 264.7 cells (mononuclear macrophage cell line) as the basis for transfection of negative lentivirus and siRhoU lentivirus and established stable cell lines. In the presence of macrophage colony-stimulating factor (M-CSF) and nuclear factor-κB receptor activating factor ligand (RANKL), the negative control group and the RhoU-silencing group were induced for 3 or 4 days, respectively. The expression of RhoU and osteoclast-specific protein and its mRNA was detected by Western blot and quantitative polymerase chain reaction (qRT-PCR), respectively. The formation of osteoclasts was observed by tartrate acid phosphatase (TRAP) staining. The morphology of osteoclasts and their precursor cells was observed by confocal laser scanning microscope. The results showed that compared with the negative control group, the RhoU gene mRNA and protein expression in the RhoU-silencing group was significantly decreased (P<0.01).The number and area formation of osteoclasts were significantly decreased (P<0.01). The formation of filopodia in osteoclast precursor cells was blocked and it lacked complete sealing zone. The expression of osteoclast-specific gene mRNA and protein was significantly decreased (P<0.01 or P<0.05), while the TRAP gene mRNA was not changed (P>0.05). In conclusion, silencing RhoU can inhibit the differentiation of osteoclasts, inhibiting the formation of filamentous pseudopods in precursor cells and affecting their fusion may be the main mechanism.

中图分类号:

郑嘉铭, 贺双江, 赵鸿雁, 宋瑞龙, 刘宗平. RhoU基因沉默对破骨细胞分化的影响[J]. 畜牧兽医学报, 2019, 50(9): 1904-1911.

ZHENG Jiaming, HE Shuangjiang, ZHAO Hongyan, SONG Ruilong, LIU Zongping. Effect of RhoU Silencing on Osteoclast Differentiation[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(9): 1904-1911.

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RhoU基因沉默对破骨细胞分化的影响

Effect of RhoU Silencing on Osteoclast Differentiation

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