[1]李松涛,孙靖,陈武桂,等.缺氧通过HIF-1α介导NRP-1上调抑制破骨细胞分化 [J].第三军医大学学报,2019,41(11):1052-1058.
 LI Songtao,SUN Jing,CHEN Wugui,et al.Hypoxia inhibits osteoclast differentiation by HIF-1α-mediated upregulation of NRP-1[J].J Third Mil Med Univ,2019,41(11):1052-1058.
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缺氧通过HIF-1α介导NRP-1上调抑制破骨细胞分化
 
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
41卷
期数:
2019年第11期
页码:
1052-1058
栏目:
基础医学
出版日期:
2019-06-15

文章信息/Info

Title:
Hypoxia inhibits osteoclast differentiation by HIF-1α-mediated upregulation of NRP-1
作者:
李松涛孙靖陈武桂马敏张莹牛晓健周驰雨初同伟
陆军军医大学(第三军医大学)第二附属医院骨科
Author(s):
LI Songtao SUN Jing CHEN Wugui MA Min ZHANG Ying NIU Xiaojian ZHOU Chiyu CHU Tongwei  

Department of Orthopaedics, Second Affiliated Hospital, Army Medical University (Third Military Medical University), Chongqing, 400037, China

关键词:
HIF-1&alphaNRP-1缺氧破骨细胞分化RAW 264.7细胞
Keywords:
HIF-1&alpha NRP-1 hypoxia osteoclast differentiation RAW 264.7 cells
分类号:
R322.71; R329.21; R329.28
文献标志码:
A
摘要:

目的 研究低氧/HIF-1α通路对RAW264.7细胞向成熟破骨细胞分化的调控作用,并初步探讨其可能的分子机制。方法 采用短发夹RNA(short hairpin RNA,shRNA)慢病毒感染法,分别建立HIF-1α和NRP-1稳定低表达的RAW264.7细胞系。将RAW264.7细胞分组培养:对照组,分化组(RANKL 100 ng/mL+50%成骨细胞CM),缺氧组(RANKL 100 ng/mL+50%成骨细胞CM+100 μmol/L CoCl2氯化钴),缺氧+HIF-1α-NC组,缺氧+HIF-1α-shRNA干扰组,缺氧+NRP-1-NC组及缺氧+NRP-1-shRNA干扰组。TRAP染色观察破骨细胞的分化情况;RT-PCR检测Cath K、MMP-9、TRAP、HIF-1α和NRP-1 mRNA的表达;Western blot检测HIF-1α和NRP-1蛋白的表达。结果 ①缺氧组破骨细胞分化相关基因Cath K、MMP-9、TRAP mRNA的表达量及TRAP染色阳性细胞数均明显低于分化组(P<0.05)。②缺氧组细胞中HIF-1α和NRP-1的表达均明显高于分化组(P<0.05)。③与缺氧组相比,缺氧+HIF-1α-shRNA干扰组TRAP染色阳性细胞数及破骨细胞分化相关基因的表达均明显增加(P<0.05);且HIF-1α和NRP-1表达均明显减少(P<0.05)。④与缺氧组相比,缺氧+NRP-1-shRNA干扰组TRAP染色阳性细胞数及破骨细胞分化相关基因的表达均明显增加(P<0.05);且NRP-1表达明显减少(P<0.05)。结论 在RAW264.7细胞中,CoCl2诱导的缺氧能够通过上调HIF-1α增强NRP-1的表达,从而抑制其向成熟破骨细胞分化。

Abstract:

Objective To investigate the regulation effect of hypoxic/HIF-1α pathway on the differentiation of RAW264.7 cells into mature osteoclasts, and to explore the possible molecular mechanism. Methods RAW264.7 cell lines with stable low expression of HIF-1α and NRP-1 were respectively established by short hairpin RNA (shRNA) lentivirus infection. Then the RAW264.7 cells were cultured in groups, that is, control group, differentiation group [100 ng/mL RANKL+50% osteoblast conditioned medium (CM)], hypoxia group (100 ng/mL RANKL+50% osteoblast CM+100 μmol/L CoCl2), hypoxia+HIF-1α-NC group, hypoxia+HIF-1α shRNA group, hypoxia+NRP-1-NC group, and hypoxia+NRP-1-shRNA group. TRAP staining was used to observe osteoclast differentiation. The mRNA expression of Cath K, MMP-9, TRAP, HIF-1α and NRP-1 were detected by RT-PCR. Western blotting was used to detect the expression of HIF-1α and NRP-1 proteins. Results ① The mRNA expression levels of Cath K, MMP-9 and TRAP and the number of TRAP positive cells in the hypoxia groups were significantly lower than those in the differentiation groups (P<0.05). ② The expression levels of HIF-1α and NRP-1 were significantly higher in the hypoxic groups than the differentiation groups (P<0.05). ③ Compared with the hypoxia groups, the number of TRAP positive cells and the expression levels of osteoclast differentiation related genes were significantly increased, while those of HIF-1α and NRP-1 were significantly reduced in the hypoxic+HIF-1α shRNA group (P<0.05). ④ Compared with the hypoxia groups, the number of TRAP positive cells and the expression levels of osteoclast differentiation related genes were significantly increased, while the level of NRP-1 was significantly decreased in the hypoxia+NRP-1-shRNA group (P<0.05). Conclusion The hypoxia-mediated HIF-1α-dependent up-regulation of NRP-1 inhibits the differentiation of RAW264.7 cells into mature osteoclasts.

 

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更新日期/Last Update: 2019-06-06