[1]冯玮,谢正松,罗岑,等.过表达Notch配体Delta-like基因的MLO-Y4骨细胞株的建立及其对骨髓基质细胞成骨分化的作用[J].第三军医大学学报,2020,42(09):899-907.
 FENG Wei,XIE Zhengsong,LUO Cen,et al.Construction of MLO-Y4 cells with overexpression of Delta-like genes and their effects on osteogenic differentiation of bone marrow stromal cells[J].J Third Mil Med Univ,2020,42(09):899-907.
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过表达Notch配体Delta-like基因的MLO-Y4骨细胞株的建立及其对骨髓基质细胞成骨分化的作用(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
42卷
期数:
2020年第09期
页码:
899-907
栏目:
基础医学
出版日期:
2020-05-15

文章信息/Info

Title:
Construction of MLO-Y4 cells with overexpression of Delta-like genes and their effects on osteogenic differentiation of bone marrow stromal cells
作者:
冯玮谢正松罗岑涂小林
重庆医科大学生命科学研究院骨发育与再生平台
Author(s):
FENG Wei XIE Zhengsong LUO Cen TU Xiaolin

Bone Development and Regeneration Platform, Life Science Institute, Chongqing Medical University, Chongqing, 400016, China

关键词:
慢病毒Delta-like基因过表达小鼠骨样细胞骨髓基质细胞成骨分化DAPT
Keywords:
lentivirus Delta-like genes overexpression murine osteocyte-like cells bone marrow stromal cells osteogenic differentiation DAPT
分类号:
R322.71; R329.2; R394-33
文献标志码:
A
摘要:

目的建立稳定表达外源Dll1、Dll3、Dll4基因的MLO-Y4骨细胞株,研究骨细胞Notch信号Delta-like配体Dll1、Dll3、Dll4对骨髓基质细胞成骨分化的影响。方法用过表达Dll1、Dll3、Dll4基因的重组慢病毒液感染MLO-Y4样骨细胞,嘌呤霉素筛选分别稳定表达Dll1、Dll3及Dll4基因的MLO-Y4骨细胞株。采用实时荧光定量PCR(real time fluorescence quantitative,qPCR)对筛选出来的MLO-Y4骨细胞株中Dll1、Dll3及Dll4基因的表达进行鉴定。将3种细胞株分别与野生型小鼠骨髓基质细胞(bone marrow stromal cells,BMSCs)进行共培养,采用碱性磷酸酶(ALP)染色及ALP定量检测共培养3 d ALP活性变化;qPCR检测共培养3 d成骨相关标志基因及Notch信号靶基因的表达水平,在使用Notch信号抑制剂DAPT后检测上述指标。结果与阴性对照组相比,Dll1、Dll3及Dll4转染组中Dll1、Dll3及Dll4 mRNA的水平显著增加(P<0.05)。共培养3 d后,骨细胞中Dll4基因的过表达促进了BMSC的ALP活性,与阴性对照组相比有统计学差异(P<0.05)。qPCR结果显示,MLO-Y4-Dll4组的ALP、Runx2、OPN及CollagenⅠ成骨相关标志基因的表达均有所升高(P<0.05),Notch信号靶基因Hey1、HeyL、Hes1及Hes7的表达也有所升高(P<0.05)。而在加入DAPT后,MLO-Y4-Dll4组的ALP活性下降,成骨相关标志基因及Notch信号靶基因的表达有所下降(P<0.05)。结论建立了稳定表达外源Dll1、Dll3或Dll4基因的MLO-Y4骨细胞株,在骨细胞中过表达Dll4能促进骨髓基质细胞的早期成骨分化,经典Notch信号通路在其中可能发挥了重要作用。

Abstract:

ObjectiveTo construct the MLO-Y4 cell strains stably expressing exogenous genes of ligands for Notch receptors Delta like-1 (Dll1), Dll3, and Dll4 and determine the effects of these ligands on osteogenic differentiation of bone marrow stromal cells. MethodsMLO-Y4 cells were infected with recombinant lentiviral vectors carrying Dll1, Dll3 and Dll4 genes. Puromycin was used to choose the MLO-Y4 cells with stable expression of these genes. Then, real-time fluorescence quantitative (qPCR) was applied to identify the expression of above genes in the selected MLO-Y4 cells. The 3 cell strains were respectively co-cultured with wild-type mouse bone marrow stromal cells (BMSCs). In 3 d after co-culture, alkaline phosphatase (ALP) staining and ALP quantitative detection were used to detect changes in ALP activity, and qPCR was employed to detect the expression levels of osteogenesis-related markers and target genes of Notch signal. The above indicators were measured again after DAPT, a Notch signaling inhibitor was added in the culture medium. ResultsCompared with the blank group and the negative control, the mRNA levels of Dll1, Dll3, and Dll4 were significantly increased in the corresponding transfection cells (P<0.05). After 3 days of co-culture, the overexpression of Dll4 gene in MLO-Y4 cells promoted the ALP activity in the BMSCs, which was significantly stronger than that of the negative control group (P<0.05). qPCR results showed that the expression levels of osteogenesis-related marker genes, such as ALP, Runx2, OPN and CollagenⅠ in the MLO-Y4-Dll4 group were all risen (P<0.05), and so were those of Notch signaling target genes, Hey1, HeyL, Hes1 and Hes7 (P<0.05). However, the addition of DAPT inhibited the ALP activity in the MLO-Y4-Dll4 group, and decreased the expression levels of above osteogenesis-related markers and Notch signaling target genes (P<0.05).  ConclusionMLO-Y4 cell strains stably expressing the exogenous genes Dll1, Dll3 and Dll4 are established successfully. Overexpression of Dll4 in osteocytes can promote early osteogenic differentiation of BMSCs, and the classic Notch signaling pathway may play an important role in this process.

参考文献/References:

[1]BLACK D M, ROSEN C J. Postmenopausal osteoporosis[J]. N Engl J Med, 2016, 374(3): 254-262. DOI:10.1056/NEJMcp1513724.
[2]BONEWALD L F. The role of the osteocyte in bone and nonbone disease[J]. Endocrinol Metab Clin North Am, 2017, 46(1): 1-18.DOI:10.1016/j.ecl.2016.09.003.
[3]DEMPSTER D W, LAMBING C L, KOSTENUIK P J, et al. Role of RANK ligand and denosumab, a targeted RANK ligand inhibitor, in bone health and osteoporosis: a review of preclinical and clinical data[J]. Clin Ther, 2012, 34(3): 521-536.DOI:10.1016/j.clinthera.2012.02.002.
[4]DRAKE M T, KHOSLA S. Hormonal and systemic regulation of sclerostin[J]. Bone, 2017, 96: 8-17. DOI:10.1016/j.bone.2016.12.004.
[5]OMINSKY M S, BOYCE R W, LI X D, et al. Effects of sclerostin antibodies in animal models of osteoporosis[J]. Bone, 2017, 96: 63-75. DOI:10.1016/j.bone.2016.10.019.
[6]TU X L, DELGADO-CALLE J, CONDON K W, et al. Osteocytes mediate the anabolic actions of canonical Wnt/β-catenin signaling in bone[J]. Proc Natl Acad Sci USA, 2015, 112(5): E478-E486. DOI:10.1073/pnas.1409857112.
[7]MUGURUMA Y, HOZUMI K, WARITA H, et al. Maintenance of bone homeostasis by DLL1-mediated notch signaling[J]. J Cell Physiol, 2017, 232(9): 2569-2580. DOI:10.1002/jcp.25647.
[8]TU X L, JOENG K S, NAKAYAMA K I, et al. Noncanonical Wnt signaling through G protein-linked PKCdelta activation promotes bone formation[J]. Dev Cell, 2007,12(1):113-127.DOI:10.1016/j.devcel.2006.11.003.
[9]BONEWALD L F. The amazing osteocyte[J]. J Bone Mineral Res,2011,26(2):229-238. DOI:10.1002/jbmr.320.
[10]VAN DER PLAS A, NIJWEIDE P J. Isolation and purification of osteocytes[J]. J Bone Miner Res, 2009,7(4):389-396.DOI:10.1002/jbmr.5650070406.
[11]NIJWEIDE P J, VAN DER PLAS A, ALBLAS M J, et al. Osteocyte isolation and culture[J]. Methods Mol Med, 2003, 80: 41-50.DOI:10.1385/1-59259-366-6:41.
[12]STERN A R, STERN M M, VAN DYKE M E, et al. Isolation and culture of primary osteocytes from the long bones of skeletally mature and aged mice[J]. Bio Techniques, 2012, 52(6): 361-373. DOI:10.2144/0000113876.
[13]KALAJZIC I, MATTHEWS B G, TORREGGIANI E, et al. In vitro and in vivo approaches to study osteocyte biology[J]. Bone, 2013, 54(2): 296-306. DOI:10.1016/j.bone.2012.09.040.
[14]BONEWALD L F. Establishment and characterization of an osteocyte-like cell line, MLO-Y4[J]. J Bone Miner Metab, 1999, 17(1): 61-65. DOI:10.1007/s007740050066.
[15]DUNWOODIE S L, CLEMENTS M, SPARROW D B, et al. Axial skeletal defects caused by mutation in the spondylocostal dysplasia/pudgy gene Dll3 are associated with disruption of the segmentation clock within the presomitic mesoderm[J]. Development, 2002, 129(7): 1795-1806.
[16]WONG P C, ZHENG H, CHEN H, et al. Presenilin 1 is required for Notch1 and DII1 expression in the paraxial mesoderm[J]. Nature, 1997, 387(6630): 288-292. DOI:10.1038/387288a0.
[17]NOBTA M, TSUKAZAKI T, SHIBATA Y, et al. Critical regulation of bone morphogenetic protein-induced osteoblastic differentiation by Delta1/Jagged1-activated Notch1 signaling[J]. J Biol Chem, 2005, 280(16): 15842-15848. DOI:10.1074/jbc.M412891200.
[18]SEKINE C, KOYANAGI A, KOYAMA N, et al. Differential regulation of osteoclastogenesis by Notch2/delta-like 1 and Notch1/Jagged1 axes[J]. Arthritis Res Ther,2012,14(2):R45. DOI:10.1186/ar3758.
[19]ATSUMI T, IKAWA Y, MIWA Y, et al. A chondrogenic cell line derived from a differentiating culture of AT805 teratocarcinoma cells[J]. Cell Differ Dev, 1990,30(2):109-116.DOI:10.1016/0922-3371(90)90079-c.
[20]BENEDITO R, ROCA C, SRENSEN I, et al. The notch ligands Dll4 and Jagged1 have opposing effects on angiogenesis[J]. Cell, 2009, 137(6): 1124-1135. DOI:10.1016/j.cell.2009.03.025.
[21]RAMASAMY S K, KUSUMBE A P, WANG L, et al. Endothelial Notch activity promotes angiogenesis and osteogenesis in bone[J]. Nature, 2014, 507(7492): 376-380. DOI:10.1038/nature13146.

更新日期/Last Update: 2020-05-06