Lmo7对MC3T3-E1细胞增殖、迁移及成骨分化影响的实验研究

doi:10.12439/kqhm.1005-4979.2023.06.001

摘要/Abstract

摘要：

目的：研究Lmo7基因对小鼠胚胎成骨细胞前体细胞(mouse embryo osteoblast precursor cells，MC3T3-E1 cells)增殖、迁移及成骨能力的影响。方法：体外培养MC3T3-E1细胞并对其进行成骨诱导，在0、3、7、14 d时收样，通过实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）与蛋白质印迹法（Western blotting）验证Lmo7的表达变化。利用慢病毒转染pLV-shLmo7获得稳定干扰Lmo7 表达的细胞株，并通过CCK-8实验、EdU细胞增殖实验、细胞划痕实验、Transwell实验检测Lmo7对细胞增殖、迁移能力的影响。通过碱性磷酸酶（alkaline phosphatase，ALP）染色法检测ALP的表达活性，通过RT-qPCR检测成骨相关基因Runt相关转录因子2（Runt-related transcription factor 2，Runx2）、Osterix、ALP、骨钙素（osteocalcin，OCN）的表达。结果：Lmo7基因在成骨诱导的过程中表达上调。成功构建了Lmo7干扰的稳转细胞株，其增殖活性显著提高，而迁移能力受到抑制。对其进行成骨诱导后发现，与对照组相比，Lmo7干扰稳转株的ALP染色较浅，成骨相关基因Runx2、Osterix、ALP、OCN表达水平明显下调，且差异具有统计学意义（P<0.05）。结论：Lmo7低表达后可促进细胞增殖，抑制细胞迁移，下调Runx2、Osterix、ALP、OCN的表达能够抑制MC3T3-E1细胞体外成骨分化。

关键词: Lmo7, MC3T3-E1细胞, RNA干扰

Abstract:

Objective: To investigate the effects of Lmo7 gene in the process of proliferation, migration and osteogenic differentiation of MC3T3-E1 cells. Methods: The preosteoblasts MC3T3-E1 cells were cultured in vitro with osteogenic induction. The relative expression of Lmo7 was detected by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting at 0, 3, 7 and 14 d respectively. At the same time, MC3T3-E1 cells were transfected with pLV-shLmo7 and pLV-shControl to construct the stably interfering cell line. Cell proliferation and cell migration were detected by CCK-8 assay, EdU assay, wound healing assay and Transwell migration assay. While alkaline phosphatase (ALP) staining was used to detect the expression activity of ALP, and RT-qPCR was used to detect the expression of osteogenesis-related genes such as Runt-related transcription factor 2 (Runx2), Osterix, alkaline phosphatase (ALP) and osteocalcin (OCN). Results: Lmo7 gene expression level was up-regulated during the osteogenic differentiation of MC3T3-E1 cells in vitro. The stable MC3T3-E1 cell line with Lmo7 interference was successfully constructed, and its proliferation activity was significantly improved, while its migration ability was inhibited. After osteogenic induction, compared with the control group, the ALP staining of the Lmo7 interfering cells was lighter, and the expression levels of osteogenesis-related genes such as Runx2, Osterix, ALP, and OCN were significantly down-regulated (P<0.05). Conclusion: Lmo7 interference promoted the cell proliferation but inhibited the cell migration. Furthermore, the osteogenic differentiation ability of cells was suppressed.

Key words: Lmo7, MC3T3-E1 cells, RNA interference

中图分类号:

R782

毛佳奕, 王佐林. Lmo7对MC3T3-E1细胞增殖、迁移及成骨分化影响的实验研究[J]. 《口腔颌面外科杂志》, 2023, 33(6): 355-362.

MAO Jiayi, WANG Zuolin. Effect of Lmo7 on proliferation, migration and osteogenic differentiation of MC3T3-E1 cells in vitro[J]. 《Journal of Oral and Maxillofacial Surgery》, 2023, 33(6): 355-362.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2023/V33/I6/355

图/表 6

表1 RT-qPCR引物序列

Table 1 Primer sequences used for RT-qPCR

基因名称	正向引物序列(5'-3')	反向引物序列(5'-3')
β-actin	GTGACGTTGACATCCGTAAAGA	GCCGGACTCATCGTACTCC
Lmo7	TTGAGACTCTTTTAGGGCAAGC	CGATGTCACTTTCGCAACCTT
Runx2	AGAGTCAGATTACAGATCCCAGG	TGGCTCTTCTTACTGAGAGAGG
Osterix	GGAAAGGAGGCACAAAGAAGC	CCCCTTAGGCACTAGGAGC
ALP	GGCTGGAGATGGACAAATTCC	CCGAGTGGTAGTCACAATGCC
OCN	CTGACCTCACAGATCCCAAGC	TGGTCTGATAGCTCGTCACAAG

图1 RT-qPCR和Western blotting检测Lmo7在成骨诱导过程中的表达 A. RT-qPCR；B. Western blotting。*表示 P<0.05，**表示 P<0.01，与对照组比较。

Figure 1 RT-qPCR and Western blotting detection of Lmo7 expression during osteogenic induction

图2 慢病毒转染MC3T3-E1细胞后Lmo7的表达 A. RT-qPCR结果，**表示 P<0.01，与对照组比较；B. Western blotting结果；C. 荧光显微镜下转染慢病毒的MC3T3-E1细胞（×100），从左到右依次是红色荧光通道、明场、合并通道下的细胞图片。

Figure 2 Expression of Lmo7 after lentivirus transfection to MC3T3-E1 cells

图3 慢病毒转染后MC3T3-E1细胞的增殖情况 A. CCK-8实验检测细胞增殖能力；B. EdU标记细胞增殖情况（×40），从上到下依次是对照组和干扰组在荧光显微镜蓝色荧光通道、绿色荧光通道、合并通道下的细胞图片；C.2组细胞增殖率。*表示 P<0.05，**表示 P<0.01，与对照组比较。

Figure 3 Proliferation of MC3T3-E1 cells after lentivirus transfection

图4 慢病毒转染后MC3T3-E1细胞的迁移情况 A. 划痕实验检测细胞迁移能力（×40），从左到右依次是对照组和干扰组细胞在划痕后0、12、24 h时的生长情况；B. 2组划痕愈合率，**表示 P<0.01，与对照组比较；C. Transwell小室检测细胞迁移情况（×100）。

Figure 4 Migration of MC3T3-E1 cells after lentivirus transfection

图5 慢病毒转染后MC3T3-E1细胞的成骨分化情况 A—D. 成骨相关基因的表达水平，**表示 P<0.01，与对照组比较；E.2组ALP染色结果（×3）；F. 显微镜下观察2组ALP染色情况（×40）。

Figure 5 Osteogensis of MC3T3-E1 cells after lentivirus transfection

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