软骨细胞中生物钟基因Bmal1对细胞周期相关基因表达的影响

doi:10.16352/j.issn.1001-6325.2024.04.0496

基础医学与临床 ›› 2024, Vol. 44 ›› Issue (4): 496-502.doi: 10.16352/j.issn.1001-6325.2024.04.0496

软骨细胞中生物钟基因Bmal1对细胞周期相关基因表达的影响

杨春生¹, 王添兴², 张铁成¹, 武恒敏¹, 王宝兰^1*

新疆医科大学第一附属医院 1.康复医学科; 2.关节外科,新疆乌鲁木齐 830000

收稿日期:2023-11-06 修回日期:2024-01-24 出版日期:2024-04-05 发布日期:2024-03-25
通讯作者: ^*wbl0308@163.com
基金资助:
乌鲁木齐市青年科研起航专项基金(2022YFY-QKQN-32);康复医学四川省重点实验室开放课题(KFYXSZDSYS-10)

Effects of biological clock gene Bmal1 on the expression of cell cycle-associated genes in chondrocytes

YANG Chunsheng¹, WANG Tianxing², ZHANG Tiecheng¹, WU Hengmin¹, WANG Baolan^1*

1. Department of Rehabilitation Medicine; 2. Department of Joint Surgery, the First Affiliated Hospital of Xinjiang Medical University,Urumqi 830000, China

Received:2023-11-06 Revised:2024-01-24 Online:2024-04-05 Published:2024-03-25
Contact: ^*wbl0308@163.com

摘要/Abstract

摘要： 目的探讨生物钟和细胞周期在骨关节炎(OA)软骨细胞中内在的关系,主要是时钟基因Bmal1对细胞周期相关基因的调控。方法将胰岛素-转铁蛋白-硒(ITS)诱导后的软骨样ATDC5细胞分为normal组、OA组、LV-Bmal1组。采用CCK8法检测各组细胞活力;RT-qPCR法检测Bmal1、Per1、Wee1、Cdk1、Ccnb1和Mmp13 mRNA在各组中的表达;Western blot检测BMAL1、PER1、WEE1、CDK1、CCNB1和MMP13蛋白在各组中的表达水平;流式细胞测量术分析Bmal1对细胞周期中不同分期及细胞凋亡的影响;分析Bmal1对Per1、Wee1、Cdk1、Ccnb1和Mmp13的调控和它们在OA中发挥的作用。结果与normal组相比,OA组细胞活力提高,Bmal1、Wee1的mRNA相对表达量下降,Per1、Cdk1、Ccnb1、Mmp13的mRNA相对表达量明显增加;LV-Bmal1组细胞活力下降,Bmal1、Wee1的mRNA相对表达量上升,Per1、Cdk1、Ccnb1、Mmp13的mRNA相对表达量下降(P＜0.05);相关性分析显示,Bmal1与Wee1呈正相关,二者与Per1、Cdk1、Ccnb1或Mmp13存在负相关;Western blot结果显示,不同组别中蛋白表达水平的结果与PCR趋势一致;细胞周期和细胞凋亡检测结果显示,和normal组相比,OA组S期、G2/M期缩短,细胞比例明显下降,细胞早期和晚期凋亡比例增加,LV-Bmal1组S期、G2/M期延长,细胞比例增加, 早期和晚期凋亡比例下降。结论炎性软骨细胞中生物钟基因Bmal1可能参与调节细胞周期相关基因的表达。

关键词: 生物钟, 细胞周期, 骨关节炎, 软骨细胞, 凋亡

Abstract: Objective To explore the intrinsic relationship between circadian clock and cell cycle in osteoarthritis(OA) chondrocytes, especially the regulation of cell cycle-related genes by the clock gene Bmal1. Methods The chondroid ATDC5 cells induced by insulin-transfering-selenium(ITS) were divided into control group, OA group and LV-Bmal1 group. The cell viability of each group was detected by CCK8 method. The expression of Bmal1, Per1, Wee1, Cdk1, Ccnb1 and Mmp13 mRNA in each group was detected by RT-qPCR. The expression of BMAL1, PER1, WEE1, CDK1, CCNB1 and MMP13 protein in each group was detected by Western blot. The effects of Bmal1 on different stages of cell cycle and apoptosis was analyzed by flow cytometry. The regulation of Bmal1 on Per1, Wee1, Cdk1, Ccnb1 and Mmp13 and their roles in OA were analyzed. Results Compared with the normal group, the cell viability of the OA group was increased, the relative mRNA expression of Bmal1 and Wee1 in the OA group decreased, and the relative mRNA expression of Per1, Cdk1, Ccnb1 and Mmp13 increased significantly. The cell viability of LV-Bmal1 group decreased, the relative expression of Bmal1 and Wee1 mRNA increased, and the relative expression of Per1, Cdk1, Ccnb1 and Mmp13 mRNA decreased(P＜0.05). Correlation analysis showed that Bmal1 was positively correlated with Wee1 and they were negatively correlated with Per1, Cdk1, Ccnb1 or Mmp13. The results of Western blot showed that protein expression in different groups were consistent with the trend of PCR. The results of cell cycle and apoptosis showed that compared with the normal group, the S phase and G2/M phase of the OA group were shortened, the proportion of cells decreased significantly, and the proportion of early and late apoptosis increased. The S phase and G2/M phase of the LV-Bmal1 group were prolonged, the proportion of cells was increased, and the proportion of early and late apoptosis was decreased. Conclusions Circadian clock gene Bmal1 in inflammatory chondrocytes might regulate the expression of cell cycle-related genes.

Key words: biological clock, cell cycle, osteoarthritis, chondrocytes, apoptosis

中图分类号:

杨春生, 王添兴, 张铁成, 武恒敏, 王宝兰. 软骨细胞中生物钟基因Bmal1对细胞周期相关基因表达的影响[J]. 基础医学与临床, 2024, 44(4): 496-502.

YANG Chunsheng, WANG Tianxing, ZHANG Tiecheng, WU Hengmin, WANG Baolan. Effects of biological clock gene Bmal1 on the expression of cell cycle-associated genes in chondrocytes[J]. Basic & Clinical Medicine, 2024, 44(4): 496-502.

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软骨细胞中生物钟基因Bmal1对细胞周期相关基因表达的影响

Effects of biological clock gene Bmal1 on the expression of cell cycle-associated genes in chondrocytes

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