+15 Gz重复暴露状态小鼠骨髓间充质干细胞转录组分析

Transcriptome analysis of mouse bone marrow mesenchymal stem cells under repeated+15 Gz exposure

目的分析高正加速度(+Gz)重复暴露状态小鼠骨髓间充质干细胞(BMSCs)转录组的变化,并初步探讨+Gz重复暴露对小鼠BMSCs成骨相关基因表达的影响。方法利用动物离心机分别建立1周与3周+15 Gz重复暴露小鼠模型。将20只C57BL/6小鼠随机分为4组(+15 Gz暴露1周组、+15 Gz暴露3周组、1周对照组和3周对照组),+15 Gz暴露组小鼠分别用动物离心机连续1、3周以+15 G、15 min/d进行重复暴露,对照组以0 G、15 min/d分别处理1、3周,暴露完成24 h后进行转录组测序,分析各组小鼠BMSCs转录组的变化,并筛选出相关差异表达基因;通过GO、KEGG富集分析探讨差异表达基因涉及的生物学过程、信号通路,并采用RT-qRCR对成骨相关差异基因的表达进行验证。结果GO富集分析显示,与对照组相比,+15 Gz重复暴露1周小鼠BMSCs上调的基因主要富集在蛋白质翻译和磷酸化水平负性调节的生物学过程中,而+15 Gz重复暴露3周小鼠BMSCs上调的基因主要富集在细胞黏附和成骨分化的生物学过程中;KEGG通路富集分析结果显示,与对照组相比,+15 Gz暴露1周组上调的基因主要富集在细胞黏附分子、ErbB和Ras信号通路,重复暴露3周小鼠BMSCs上调的基因主要富集在基质与胞外受体相互作用、黏着斑、PI3K/Akt、cGMP/PKG和Wnt信号通路;RT-qPCR结果表明,+15 Gz暴露1周小鼠BMSCs成骨相关差异基因Alpl和Bmp 2转录水平有增加趋势,而+15 Gz暴露3周小鼠BMSCs成骨相关差异基因Alpl转录水平显著上升(P<0.01)。结论+15 Gz重复暴露影响小鼠BMSCs转录组状态,暴露3周可显著增强小鼠BMSCs成骨分化相关基因的表达水平。

Objective To analyze the transcriptome changes of mouse bone marrow mesenchymal stem cells(BMSCs)under repeated exposure of high positive acceleration(+Gz),and to preliminarily investigate the effects of repeated+Gz on the expression of osteogenesis-related genes of mouse BMSCs.Methods Animal centrifuge was used to establish mouse model of 1-and 3-week repeated+15 Gz exposure.Twenty C57BL/6 mice were randomly divided into four groups(1-week+15 Gz exposure group,3-week+15 Gz exposure group,1-week control group,and 3-week control group).Mice in+15 Gz exposure groups were repeatedly exposed to+15 Gz for one week and three weeks(15 min/d),respectively,and mice in control groups were treated with 0 Gz for one week and three weeks(15 min/d),respectively.After 24 h of exposure,transcriptome sequencing was performed to analyze the transcriptome changes of mouse BMSCs in each group and screen out related differentially expressed genes.GO and KEGG enrichment analysis were used to explore the biological processes and signaling pathways involved in differentially expressed genes,and RT-qRCR was used to verify the expression of osteogenesis-related differentially expressed genes.Results GO enrichment analysis showed that compared with the control groups,the upregulated geneses of BMSCs in mice exposed to repeated+15 Gz for one week were mainly enriched in the biological processes of protein translation and negative regulation of the level of phosphorylation,while the upregulated genes of BMSCs in mice exposed to repeated+15 Gz for three weeks were mainly enriched in the biological processes of cell adhesion and osteogenic differentiation.KEGG pathway enrichment analysis showed that compared with the control groups,the upregulated genes in 1-week+15 Gz exposure group were mainly enriched in cell adhesion molecules,ErbB and Ras signaling pathways,while the upregulated genes of BMSCs in mice exposed to repeated+15 Gz for three weeks were mainly enriched in extracellular matrix-receptor interaction,focal adhesion,PI3K-Akt,cGMP-PKG and Wnt signaling pathways.The results of RT-qPCR showed that the transcription levels of osteogenesis-related differentially expressed genes Alpl and Bmp 2 in BMSCs were increased after 1 week of+15 Gz exposure,while the transcription level of osteogenesis-related differentially expressed gene Alpl in BMSCs was significantly increased after 3 weeks of+15 Gz exposure(P<0.01).Conclusion Repeated+15 Gz exposure influences the transcriptome state of mouse BMSCs,and repeated+15 Gz exposure for three weeks can significantly enhance the expression of genes related to osteogenic differentiation of mouse BMSCs.