Hmcn1对MC3T3-E1细胞体外成骨分化、迁移及增殖影响的实验研究

doi:10.3969/j.issn.1005-4979.2023.02.003

摘要/Abstract

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

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

中图分类号:

R782

周涛, 王佐林. Hmcn1对MC3T3-E1细胞体外成骨分化、迁移及增殖影响的实验研究[J]. 《口腔颌面外科杂志》, 2023, 33(2): 77-82.

ZHOU Tao, WANG Zuolin. Effects of Hmcn1 on osteogenic differentiation, migration and proliferation of MC3T3-E1 cells in vitro[J]. 《Journal of Oral and Maxillofacial Surgery》, 2023, 33(2): 77-82.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2023/V33/I2/77

图/表 5

图1 RT-qPCR检测成骨诱导后Hmcn1表达的变化 *表示P<0.05，与对照组比较。

Figure 1 RT-qPCR analysis of Hmcn1 expression after osteogenic stimulation

图2 siRNA敲降效率及敲降后成骨标志物表达量的变化 A.siRNA的敲降效率；B.成骨诱导3 d时，成骨标志物表达量的变化；C.成骨诱导7 d时，成骨标志物表达量的变化；D.成骨诱导14 d时，成骨标志物表达量的变化。*表示P<0.05，**表示P<0.01，与对照组比较。

Figure 2 siRNA knockdowm efficiency and the expression of osteogenic markers changes after knockdown

图3 siRNA敲降后细胞增殖能力的变化 *表示P<0.05，与对照组比较。

Figure 3 Changes of cell proliferation capacity after siRNA knockdown

图4 siRNA敲降对MC3T3-E1细胞迁移能力的影响 A.划痕实验(×40)；B.划痕实验显示细胞迁移数；C. Transwell实验(×100)，2组显微镜下的随机视野；D. Transwell实验显示穿过小室的细胞计数；E.结晶紫洗脱后测得的OD值。*表示P<0.05，**表示P<0.01，与对照组比较。

Figure 4 Effect of siRNA knockdown on migration of MC3T3-E1 cells

图5 siRNA敲降对MC3T3-E1细胞成骨分化的影响 A.2组ALP染色结果（×2）；B.2组ARS染色结果（×2）。

Figure 5 Effect of siRNA knockdown on osteogenesis of MC3T3-E1 cells

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