[1]边佩鲜,杨冰,王靖雅,等.NICD表达下调对辐射损伤小鼠成骨细胞系MC3T3-E1增殖和功能基因表达的影响[J].国际放射医学核医学杂志,2018,(2):135-142.[doi:10.3760/cma.j.issn.1673-4114.2018.02.007]
　Bian Peixian,Yang Bing,Wang Jinya,et al.Effects of NICD expression downregulation on the proliferation and function-related gene expression of radiation-damaged MC3T3-E1 cells[J].International Journal of Radiation Medicine and Nuclear Medicine,2018,(2):135-142.[doi:10.3760/cma.j.issn.1673-4114.2018.02.007]
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NICD表达下调对辐射损伤小鼠成骨细胞系MC3T3-E1增殖和功能基因表达的影响(/HTML)
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《国际放射医学核医学杂志》[ISSN:1673-4114/CN:12-1381/R]

卷:
期数:
2018年第2期
页码:
135-142
栏目:
基础研究
出版日期:
2018-05-09

文章信息/Info

Title:
Effects of NICD expression downregulation on the proliferation and function-related gene expression of radiation-damaged MC3T3-E1 cells
作者:
边佩鲜1;  杨冰2;  王靖雅1;  孙元明1;  龙伟1
1. 300192 天津, 中国医学科学院北京协和医学院放射医学研究所, 天津市放射医学与分子核医学重点实验室;
2. 300070, 天津医科大学基础医学院细胞生物学系
Author(s):
Bian Peixian1;  Yang Bing2;  Wang Jinya1;  Sun Yuanming1;  Long Wei1
1. Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine, Institute of Radiation Medicine, Chinese Academy of Medical Sciences, Peking Union Medical College, Tianjin 300192, China;
2. Derpartment of Cell Biology, Institute of Medicine, Tianjin Medical University, Tianjin 300070, China
关键词:
Notch信号通路胞内结构域; RNA干扰; 辐射损伤; 实验性; MC3T3-E1细胞
Keywords:
Notch intracellular domin; RNA interference; Radiation injuries;  experimental; MC3T3-E1 cell
DOI:
10.3760/cma.j.issn.1673-4114.2018.02.007
摘要:
目的 利用RNA干扰抑制小鼠成骨细胞系MC3T3-E1表达Notch信号通路胞内结构域（NICD），探讨靶向抑制NICD表达对辐射损伤MC3T3-E1细胞的增殖和相关功能基因表达的影响。方法 建立抑制NICD表达的MC3T3-E1细胞株，利用实时定量PCR （qRT-PCR）和Western blot法检测其NICD基因的表达。MC3T3-E1细胞和NICD RNA干扰MC3T3-E1细胞经2 Gy γ射线照射后，用BrdU掺入法和qRT-PCR法检测上述细胞的增殖及相关功能基因的表达水平。使用Student-Newman-Keuls进行组间差异分析，两组间比较采用t检验。结果 用RNA干扰技术可靶向抑制MC3T3-E1细胞表达NICD。抑制NICD表达可干扰前体成骨细胞和成骨细胞的增殖。2 Gy照射后，前体成骨细胞和成骨细胞以及NICD RNA干扰的成骨细胞的增殖明显下降，各靶细胞的相关功能基因与照射前相比的变化如下：①2 Gy照射后的前体成骨细胞成骨特导性转录因子（Runx2）表达上调，差异有统计学意义（t=2.353，P<0.05），NICD RNA干扰的前体成骨细胞Runx2表达下调，差异有统计学意义（t=2.353，P<0.05）；②2 Gy照射后的前体成骨细胞和成骨细胞以及NICD RNA干扰的前体成骨细胞碱性磷酸酶（ALP）表达上调，差异有统计学意义（t=3.182、3.345、3.555，均P<0.05），NICD RNA干扰的成骨细胞ALP表达下调，差异有统计学意义（t=5.045，P<0.01）；③2 Gy照射后前体成骨细胞核因子κB受体活化因子配体（RANKL）表达下调，差异有统计学意义（t=2.541，P<0.05），成骨细胞和NICD干扰的前体成骨细胞RANKL表达上调，差异有统计学意义（t=3.299，P<0.05；t=10.212，P<0.01），而抑制NICD表达则发生相反变化，差异无统计学意义（t=0.765，P>0.05）；④2 Gy照射后的前体成骨细胞和成骨细胞骨保护素（OPG）表达下调，差异有统计学意义（t=2.994、2.782，均P<0.05），抑制NICD表达使前体成骨细胞OPG表达上调，差异有统计学意义（t=5.841，P<0.01），成骨细胞OPG表达下调，差异有统计学意义（t=2.544，P<0.05）；⑤2 Gy照射后各靶细胞巨噬细胞集落刺激因子（M-CSF）表达变化趋势与RANKL表达变化情况一致。结论 在不同阶段的成骨细胞中抑制NICD表达对辐射损伤表现出的作用是不同的：①可降低前体成骨细胞和成骨细胞的增殖，对辐射损伤后的前体成骨细胞的增殖有保护作用；②可通过调节Runx2从而明显抑制辐照后前体成骨细胞分化，减少骨质丢失；③辐照后各成骨细胞不会通过RANKL/OPG/RANK系统表现出对破骨细胞功能的调节作用；④成骨细胞经过调节M-CSF表现出对破骨细胞的功能抑制作用。
Abstract:
Objective RNA interference (RNAi) is used to inhibit Notch intracellular domain (NICD) expression in MC3T3-E1 cells. The aim of RNAi is to observe the effect of the inhibition of the NICD expression on cell proliferation and function-related gene expression in MC3T3-E1 cells exposed to 2 Gy radiation. Methods The MC3T3-E1 cells were established to inhibit the NICD. The NICD expression of cells was detected by using qRT-PCR and Western blot. MC3T3-E1 and inhibited NICD MC3T3-E1 cells were irradiated with 2 Gy. Then, the proliferation and function-related gene expression were detected through BrdU incorporation and qRT-PCR. Results NICD expression in MC3T3-E1 cells could be inhibited by the RNAi technology. The inhibition of NICD expression could interfere with the proliferation of precursor osteoblasts and osteoblasts. The proliferation of precursor osteoblasts, osteoblasts, and NICD RNAi osteoblasts significantly decreased after 2 Gy irradiation. The function-related gene expression of each target cell is as follows. ① The expression of Runt-related transcription factor 2 (Runx2) was upregulated in precursor osteoblasts (t=2.353, P<0.05) and downregulated in the NICD RNAi precursor osteoblasts (t=2.353, P<0.05) after 2 Gy irradiation. ② The expression of alkaline phosphatase (ALP) was upregulated in precursor osteoblasts and osteoblasts and the NICD RNAi precursor osteoblasts (t=3.182, 3.345, 3.555, all P<0.05) and was downregulated in the NICD RNAi osteoblasts (t=5.045, P<0.01) after 2 Gy irradiation. ③ The expression of receptor activator of nuclear factor κB ligand (RANKL) was downregulated in precursor osteoblasts (t=2.541, P<0.05) and was upregulated in osteoblasts and NICD RNAi precursors osteoblast (t=3.299, P<0.05; t=10.212, P<0.01) after 2 Gy irradiation. However, the inhibition of the NICD expression could cause an opposite change in other cells (t=0.765, P > 0.05). ④ The expression of osteoprotegerin (OPG) was downregulated in precursor osteoblasts and osteoblasts (t=2.994 and 2.782, P<0.05) after 2 Gy irradiation. However, the inhibition of the NICD expression could cause expression upregulation in precursor osteoblasts and expression downregulation in osteoblasts (t=5.841, P<0.01). ⑤ The expression of macrophage-colony stimulating factor (M-CSF) in the target cells exhibited the same trend as the expression of RANKL after 2 Gy irradiation. Conclusions The inhibition of the NICD expression exerts different effects on the differentiation of irradiated osteoblasts. The inhibition of the NICD expression could cause a series of changes including:①It may decrease the proliferation of precursor osteoblasts and osteoblasts and protect the proliferation of differentiating precursor osteoblasts after irradiation. ②It can significantly inhibit the differentiation of precursors osteoblasts after irradiation and reduce bone loss through regulating the expression of Runx2. ③The osteoblasts did not show the regulated function of the osteoclasts through the RANKL/OPG/RANK system. ④The osteoblasts can exhibit the inhibited function of the osteoclasts through the expression of M-CSF.

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备注/Memo

备注/Memo:
收稿日期:2018-01-25。
基金项目:国家自然科学基金（81673106）；天津市自然科学基金（14JCYBJC26700、16JCQNJC12100）；中国医学科学院医学与健康科技创新工程项目（2017-12M-1-012）
通讯作者:龙伟,Email:longwei@irm-cams.ac.cn
更新日期/Last Update: 2018-05-09