HOXA10基因对牛骨骼肌卫星细胞增殖分化的影响
The Effect of HOXA10 Gene on Proliferation and Differentiation of Bovine Skeletal Muscle Satellite Cells
张萌 1张文天 1杨莉 1刘莉莉 1吕瑞雪 1付学鹏 1张伟伟1
作者信息
- 1. 齐齐哈尔大学生命科学与农林学院,齐齐哈尔 161006
- 折叠
摘要
该文探讨了HOXA10基因对牛骨骼肌卫星细胞(bovine skeletal muscle satellite cells,BSMSCs)增殖分化的作用.采用实时荧光定量PCR(qRT-PCR)检测增殖1、2、3天(P1、P2、P3)BSMSCs中HOXA10、PCNA基因的表达情况;将HOXA10基因过表达质粒、干扰质粒及对照质粒转染至BSMSCs中,利用qRT-PCR、蛋白质免疫印迹(Western blot)实验检测过表达及干扰效果;采用CCK-8、EdU、流式细胞术(Flow CytoMetry,FCM)、免疫荧光、qRT-PCR和Western blot检测细胞活力、细胞增殖、细胞周期、细胞分化以及细胞增殖和分化标志基因的mRNA及蛋白表达情况.结果显示,在BSMSCs增殖过程中HOXA10 mRNA水平显著升高(P＜0.01,P＜0.001);与对照组相比,转染24 h后过表达及干扰效果显著(P＜0.01,P＜0.001);过表达HOXA10基因后细胞活力增强,EdU阳性细胞比例显著增多(P＜0.001),G1期细胞百分比显著降低(P＜0.01),S期细胞百分比显著增加(P＜0.05),增殖标志基因PCNA、CCND1 mRNA和蛋白的表达水平显著升高(P＜0.05,P＜0.01);肌管形成水平显著降低,分化标志基因MyoG的mRNA和蛋白表达量显著减少(P＜0.01,P＜0.001).与对照组相比,干扰HOXA10基因后细胞活力降低,EdU阳性细胞比例显著减少(P＜0.05),G1期细胞百分比显著升高(P＜0.001),S期细胞百分比显著下降(P＜0.01),增殖标志基因PCNA、CCND1 mRNA和蛋白的表达水平显著降低(P＜0.05,P＜0.001);肌管形成水平显著上升,分化标志基因MyoG的mRNA和蛋白表达量显著减少(P＜0.001).综上,HOXA10基因促进BSMSCs增殖,抑制其分化.
Abstract
This study explored the effects of HOXA10 gene on the proliferation and differentiation of BSMSCs(bovine skeletal muscle satellite cells).qRT-PCR(real-time quantitative PCR)was used to detect the ex-pression of HOXA10 and PCNA genes in BSMSCs proliferating for 1,2 and 3 days(P1,P2 and P3).HOXA10 gene overexpression plasmids,interference plasmids,and control plasmids were transfected into BSMSCs.qRT-PCR and Western blot were used to detect overexpression and interference effects.CCK-8,EdU,FCM(Flow CytoM-etry),immunofluorescence,qRT-PCR and Western blot were used to detect cell viability,cell proliferation,cell cycle,cell differentiation,mRNA and protein expression of genes that mark cell proliferation and differentiation.The results showed that the mRNA of HOXA10 was significantly increased during the proliferation of BSMSCs(P＜0.01,P＜0.001).Compared with the control group,the overexpression and interference effects were signifi-cant after 24 hours of transfection(P＜0.01,P＜0.001);the cell viability and the proportion of EdU-positive cells in BSMSCs were increased(P＜0.001)after overexpression of HOXA10 gene,meanwhile the percentage of cells in G1 phase was significantly decreased(P＜0.01);the percentage of cells in S phase was significantly increased(P＜0.05);and the expression levels of proliferation marker genes PCNA,CCND1 mRNA and protein were sig-nificantly increased(P＜0.05,P＜0.01).The formation of myotubes was significantly reduced,and the mRNA and protein expressions of the differentiation marker gene MyoG were significantly reduced(P＜0.01,P＜0.001).Compared with the control group,after silencing of the HOXA10 gene,the cell viability decreased,the propor-tion of EdU-positive cells decreased(P＜0.05);the percentage of cells in G1 phase increased(P＜0.001);the per-centage of cells in S phase decreased significantly(P＜0.01).The levles of PCNA,CCND1 mRNA and protein were decreased significantly after silencing of the HOXA10 gene(P＜0.05,P＜0.001).The formation of myotubes was significantly increased,and the mRNA and protein expressions of the differentiation marker gene MyoG were significantly increased(P＜0.001).In conclusion,HOXA10 gene promotes the proliferation and inhibits dif-ferentiation of BSMSCs.
关键词
牛骨骼肌卫星细胞/HOXA10基因/增殖/分化Key words
bovine skeletal muscle satellite cells/HOXA10 gene/proliferation/differentiation引用本文复制引用
基金项目
黑龙江省自然科学基金(LH2021C099)
黑龙江省省属高等学校基本科研业务费科研项目(145209515)
齐齐哈尔大学研究生创新科研项目(QUZLTS_CX2023027)
出版年
2024
NETL
NSTL
万方数据