《Journal of Oral and Maxillofacial Surgery》 ›› 2023, Vol. 33 ›› Issue (2): 77-82. doi: 10.3969/j.issn.1005-4979.2023.02.003

• Basic Scientific Study • Previous Articles     Next Articles

Effects of Hmcn1 on osteogenic differentiation, migration and proliferation of MC3T3-E1 cells in vitro

ZHOU Tao(), WANG Zuolin()   

  1. Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Department of Oral Implantology, School and Hospital of Stomatology, Tongji University, Shanghai 200072, China
  • Revised:2023-03-06 Accepted:2022-12-08 Online:2023-04-28 Published:2023-05-05

Hmcn1对MC3T3-E1细胞体外成骨分化、迁移及增殖影响的实验研究

周涛(), 王佐林()   

  1. 上海牙组织修复与再生工程技术研究中心,同济大学口腔医学院,同济大学附属口腔医院口腔种植科,上海 200072
  • 通讯作者: 王佐林,教授. E-mail:zuolin@tongji.edu.cn
  • 作者简介:

    周涛,硕士研究生. E-mail:

  • 基金资助:
    国家自然科学基金(81670962); 国家重点研发计划(2018YFE0202200); 中央高校基本业务费专项资金(22120180196)

Abstract:

Objective: To investigate whether Hemicentin 1 (Hmcn1) had an effect on the process of osteogenic differen- tiation, proliferation and migration of mouse embryo osteoblast precusor cells (MC3T3-E1 cells). Methods: MC3T3-E1 cells were cultured in vitro and osteogenic induction was conducted to observe the expression of Hmcn1. The relative expression of Hmcn1 was detected by real-time quantitative polymerase chain reaction (RT-qPCR) at 0, 3, 7 and 14 d respectively. MC3T3-E1 cells were transfected with small interfering RNA (siRNA) to construct Hmcn1 knockdown MC3T3-E1 cells, and the knockdown efficiency was detected RT-qPCR. RT-qPCR was used to detect the expression changes of bone morphogenetic protein-2 (BMP-2), Osterix (OSX), osteocalcin (OCN), Runt related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) before and after knockdown. The effect of Hmcn1 knockdown on osteogenic differentiation of MC3T3-E1 cells was observed by ALP and alizarin red S (ARS) staining. The effect of Hmcn1 knockdown on the migration of MC3T3-E1 cells was observed by cell scratch assay and Transwell assay. The effect of Hmcn1 knockdown on proliferation of MC3T3-E1 cells was observed by cell counting kit-8 (CCK-8) experiment. Results: The expression of Hmcn1 gene was up-regulated after osteogenic induction of MC3T3-E1 cells. After Hmcn1 gene was knocked down by siRNA, the BMP-2 expression was down-regulated, the migration ability of MC3T3-E1 cells decreased and proliferation ability increased, at the same time, the staining of ALP knockdown group was slightly shallow. Conclusion: This study demonstrated that Hmcn1 can promote the migration of MC3T3-E1 cells, inhibit the proliferation of MC3T3-E1 cells, and promote the osteogenic differentiation of MC3T3-E1 cells in vitro by up-regulating the expression of BMP-2.

Key words: Hmcn1, MC3T3-E1 cells, osteogenic differentiation, bone morphogenetic protein-2

摘要:

目的:研究Hemicentin1(Hmcn1)基因对小鼠胚胎成骨细胞前体细胞(mouse embryo osteoblast precursor cells,MC3T3-E1 cells)成骨、迁移及增殖能力的影响。方法:体外培养MC3T3-E1细胞,对其进行成骨诱导,诱导后第0、3、7、14 天时收样,通过实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)观察Hmcn1的表达量变化。使用小干扰RNA(small interfering RNA,siRNA)转染MC3T3-E1细胞,构建Hmcn1敲降的MC3T3-E1细胞,采用RT-qPCR检测敲降效率。采用RT-qPCR检测敲降前后骨形态发生蛋白-2(bone morphogenetic protein-2,BMP-2)、成骨细胞特异性转录因子(Osterix,OSX)、骨钙素(osteocalcin,OCN)、Runt相关转录因子2(Runt related transcription factor 2,Runx2)、碱性磷酸酶(alkaline phosphatase,ALP)表达水平的变化。通过ALP和茜素红S(alizarin red S,ARS)染色观察Hmcn1敲降对MC3T3-E1细胞成骨分化的影响。通过细胞划痕实验和Transwell实验观察Hmcn1敲降对MC3T3-E1细胞迁移能力的影响。通过细胞计数试剂盒-8(cell counting kit-8,CCK8)实验观察Hmcn1敲降对MC3T3-E1细胞增殖能力的影响。结果:对MC3T3-E1细胞进行体外成骨诱导后,Hmcn1基因的表达出现上调。使用siRNA敲降Hmcn1基因后,BMP-2表达下调。敲降Hmcn1后,MC3T3-E1细胞的迁移能力下降,增殖能力提高,ALP染色敲降组细胞着色较少。结论:Hmcn1基因可通过上调BMP-2的表达,促进MC3T3-E1细胞的迁移,抑制MC3T3-E1细胞的增殖,促进MC3T3-E1细胞体外成骨分化。

关键词: Hmcn1, MC3T3-E1细胞, 成骨分化, 骨形态发生蛋白-2

CLC Number: