摘要
目的本研究旨在明确钙通道蛋白Orai2在调控成骨细胞增殖、凋亡以及分化方面发挥的作用。方法在成骨诱导条件下用Western blot和Real-Time PCR的方法检测Orai2的蛋白表达和mRNA转录水平。应用靶向Orai2的shRNA在MC3T3-E1成骨细胞中沉默Orai2的表达,转染无关干涉的shRNA作为阴性对照,将MC3T3-E1成骨细胞分为对照组和Orai2 shRNA组。在对照组和Orai2 shRNA组中,用MTT法检测细胞增殖变化、流式细胞仪检测细胞周期和细胞凋亡变化、Real-Time PCR检测Runt相关转录因子2(Runt-related transcription factor 2,Runx2)和锌指结构转录因子(Osterix)以及碱性磷酸酶(alkaline phosphatase,ALP)的转录水平,并用Western Blot检测Ras-ERK1/2信号通路活性。结果成骨诱导后Orai2的蛋白表达水平和mRNA转录水平逐渐增加(P<0.05)。与对照组相比较,转染靶向Orai2的shRNA后,MC3T3-E1细胞中Orai2的表达和转录均明显降低(P<0.05)。与对照组相比较,在MC3T3-E1细胞中沉默Orai2的表达后,MC3T3-E1细胞增殖减少、凋亡增加,并且Runx2和Osterix以及ALP的转录水平也显著下降(P<0.05);进一步研究发现,与对照组相比较,在MC3T3-E1细胞沉默Orai2的表达后,明显抑制了钙离子依赖的Ras-ERK1/2信号通路的活性(P<0.05)。结论Orai2的表达抑制可显著降低Ras-ERK1/2信号通路活性,并减少成骨细胞增殖、增加成骨细胞凋亡、抑制成骨分化,从而导致成骨细胞数量减少,参与骨质疏松症的发生与发展。
Objective To determine the role of Orai2 in regulating osteoblast proliferation,apoptosis and differentiation in osteoblasts.Methods Western blot and real-time PCR were used to detect the protein expression and mRNA transcription levels of Orai2 after osteoblastic induction.The expression of Orai2 was silenced in MC3T3-E1 osteoblasts by transfecting Orai2 shRNA,and unrelated interference shRNA was transfected as negative control.MC3T3-E1 osteoblasts were divided into control group and Orai2 shRNA group.In the control group and Orai2 shRNA group,cell proliferation and apoptosis,the transcription of Runx2,Osterix and ALP,and Ras-ERK1/2 signaling pathway activity were tested to dentify whether Orai2 is involved in the regulation of osteogenesis.Results After osteogenic induction,Orai2 protein expression level and mRNA transcription level were gradually increased(P<0.05).Compared with the control group,Orai2 was knockdown successfully after Orai2 shRNA transfected in MC3T3-E1 cells.Furthermore,compared with the control group,the proliferation were decreased and apoptosis were increased after Orai2 knockdown.Moreover,compared with the control group,the transcription of Runx2,Osterix and ALP,were decreased after Orai2 knockdown.We also found that Ras-ERK1/2 signaling pathway activity were inhibited compared with the control group.Conclusion The inhibition of Orai2 expression can significantly reduce the activity of Ras-Erk1/2 signaling pathway,reduce the proliferation of osteoblasts,increase the apoptosis of osteoblasts,and inhibit the differentiation of osteoblasts,resulting in the decrease in the number of osteoblasts and the involvement in the occurrence and development of osteoporosis.
作者
郭云山
郝定均
王晓东
胡慧敏
姜扩
黄研生
杨小卫
Guo Yunshan;Hao Dingjun;Wang Xiaodong(Department of Spinal Surgery,Honghui Hospital,Xi’an Jiaotong University,Xi’an 710054,China)
出处
《实用骨科杂志》
2019年第12期1092-1097,1109,共7页
Journal of Practical Orthopaedics
基金
国家自然科学基金青年项目(81502330)
中国博士后科学基金资助项目(2017T100763)
陕西省博士后科研项目资助(2017BSHQY XMZZ12)
关键词
Orai2
增殖
凋亡
分化
成骨细胞
Orai2
proliferation
apoptosis
differentiation
osteoblast
