Notch1信号通路调节成骨因子影响腰椎间盘钙化机制研究
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作者Author单位AddressE-Mail
方明 FANG Ming 宁波大学医学院附属医院, 浙江 宁波 315000 Affiliated Hospital of Medical College of Ningbo University, Ningbo 315000, Zhejiang, China fmdoc88473718@aliyun.com 
王兴武 WANG Xing-wu 宁波大学医学院附属医院, 浙江 宁波 315000 Affiliated Hospital of Medical College of Ningbo University, Ningbo 315000, Zhejiang, China  
期刊信息:《中国骨伤》2023年,第36卷,第5期,第473-479页
DOI:10.12200/j.issn.1003-0034.2023.05.015
基金项目:宁波市自然科学基金(编号:2019A610246)
中文摘要:

目的:探究Notch1信号通路调节成骨因子影响腰椎间盘钙化的机制。

方法:分离SD大鼠原代纤维环细胞并进行传代培养,分别加入诱导因子骨形态发生蛋白-2(bone morphogenetic protein-2,BMP-2)和碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)诱导钙化(分别为BMP-2组和b-FGF组),并设置对照组(正常培养基中培养)。随后进行细胞形态及细胞荧光鉴定、茜素红染色、ELISA检测、qRT-PCR实验,以确定诱导钙化的效果。再次进行细胞分组,分为对照组、钙化组(加入诱导因子BMP-2)、钙化+LPS组(加入诱导因子BMP-2和Notch1通路激活剂LPS)、钙化+DAPT组(加入诱导因子BMP-2和Notch1通路抑制剂DAPT),进行茜素红染色、流式细胞术检测细胞凋亡,ELISA检测成骨因子含量,Western blot检测BMP-2、b-FGF、Notch1蛋白表达情况。

结果:诱导因子筛选结果表明BMP-2组和b-FGF组大鼠纤维环细胞矿化结节数目均明显增加,且BMP-2组效果更显著;同时ELISA及Western blot结果表明诱导组细胞中BMP-2、b-FGF含量水平及BMP-2、b-FGF、Notch1 mRNA表达水平均显著升高(P<0.01)。探究Notch1信号通路影响腰椎间盘钙化机制结果表明:与钙化组相比,钙化+LPS组大鼠纤维环细胞矿化结节数目,细胞凋亡率,BMP-2、b-FGF含量水平,BMP-2、b-FGF、Notch1蛋白表达水平进一步显著增加;而钙化+DAPT组大鼠纤维环细胞矿化结节数目,细胞凋亡率,BMP-2、b-FGF含量水平,BMP-2、b-FGF、Notch1蛋白表达水平均降低(P<0.05或P<0.01)。

结论: Notch1信号通路通过正向调控成骨因子含量促进腰椎间盘钙化。
【关键词】Notch1信号通路  成骨因子  腰椎间盘钙化
 
Mechanism of the Notch1 signaling pathway regulating osteogenic factor influences lumbar disc calcification
ABSTRACT  

Objective To explore the mechanism of the Notch1 signaling pathway in regulating osteogenic factors and influencing lumbar disc calcification.

Methods Primary annulus fibroblasts from SD rats were isolated and subcultured in vitro. The calcification-inducing factors bone morphogenetic protein-2 (BMP-2) and basic fibroblast growth factor (b-FGF) were added to separate groups to induce calcification,which were referred to as the BMP-2 group and the b-FGF group,respectively. A control group was also set up,which was cultured in normal medium. Subsequently,cell morphology and fluorescence identification,alizarin red staining,ELISA,and quantitative real-time polymerase chain reaction (QRT-PCR) were performed to determine the effect of calcification induction. Cell grouping was performed again,including the control group,the calcification group (adding the inducer BMP-2),the calcification + LPS group(adding the inducer BMP-2 and the Notch1 pathway activator LPS),and the calcification + DAPT group (adding the inducer BMP-2 and the Notch1 pathway inhibitor DAPT). Alizarin red staining and flow cytometry were used to detect cell apoptosis,ELISA was used to detect the content of osteogenic factors,and Western blot was used to detect the expression of BMP-2,b-FGF,and Notch1 proteins.

Results The induction factor screening results showed that the number of mineralized nodules in fibroannulus cells in BMP-2 group and b-FGF group was significantly increased,and the increase was greater in the BMP-2 group Meanwhile,ELISA and Western blot results showed that BMP-2,b-FGF and mRNA expression levels of BMP-2,b-FGF and Notch1 in the induced group were significantly increased (P<0.01). The results of the mechanism of Notch1 signaling pathway affecting lumbar disc calcification showed that compared to calcified group,the number of fibroannulus cell mineralization nodules,apoptosis rate,BMP-2,b-FGF content,the expression levels of BMP-2,b-FGF,and Notch1 proteins were further increased significantly However,the number of mineralization nodules,apoptosis rate,BMP-2 and b-FGF levels,BMP-2,b-FGF and Notch1 protein expression levels were decreased in the calcified +DAPT group (P<0.05 or P<0.01).

Conclusion Notch1 signaling pathway promotes lumbar disc calcification through positive regulation of osteogenic factors.
KEY WORDS  Notch1 signaling pathway  Osteogenic factor  Calcification of the lumbar disc
 
引用本文,请按以下格式著录参考文献:
中文格式:方明,王兴武.Notch1信号通路调节成骨因子影响腰椎间盘钙化机制研究[J].中国骨伤,2023,36(5):473~479
英文格式:FANG Ming,WANG Xing-wu.Mechanism of the Notch1 signaling pathway regulating osteogenic factor influences lumbar disc calcification[J].zhongguo gu shang / China J Orthop Trauma ,2023,36(5):473~479
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