Zfp260对MC3T3-E1细胞体外成骨分化及增殖、迁移影响的实验研究

doi:10.12439/kqhm.1005-4979.2024.06.002

摘要/Abstract

摘要：

目的：探究锌指蛋白260（zinc‐finger protein 260，Zfp260）对小鼠胚胎成骨细胞前体细胞(mouse embryo osteoblast precursor cells，MC3T3-E1 cells)体外成骨分化及增殖、迁移的影响。方法：体外培养并诱导MC3T3-E1细胞成骨分化，实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）检测诱导7、14 d后Zfp260的表达量变化。用小干扰RNA（small interfering RNA，siRNA）转染MC3T3-E1细胞，并通过RT-qPCR测定Zfp260的敲降效率及敲降组与对照组碱性磷酸酶（alkaline phosphatase，ALP）、人骨形态发生蛋白2（bone morphogenetic protein 2，BMP2）等成骨生物标志物表达量的变化。通过Transwell、细胞划痕实验检测敲降Zfp260后MC3T3-E1细胞迁移能力的变化。通过CCK8实验检测敲降Zfp260后MC3T3-E1细胞增殖能力的变化。结果：体外诱导MC3T3-E1细胞成骨分化后，Zfp260的表达量明显上调（P<0.05）。使用siRNA敲降Zfp260后，ALP及BMP2的表达量明显下调（P<0.05）。Transwell及细胞划痕实验证明敲降Zfp260后，MC3T3-E1细胞的迁移能力受到抑制。CCK8实验证明敲降Zfp260后，MC3T3-E1细胞的增殖能力增加。结论：Zfp260可促进MC3T3-E1细胞的成骨分化。

关键词: 锌指蛋白260, 小鼠胚胎成骨细胞前体细胞, 成骨分化

Abstract:

Objective: To explore the effect of zinc‐finger protein 260 (Zfp260) on the osteogenic differentiation, proliferation and migration of mouse embryo osteoblast precursor cells (MC3T3-E1 cells) in vitro. Methods: MC3T3-E1 cells were cultured in vitro and induced to differentiate into osteoblasts. The expression of Zfp260 was detected by real-time quantitative polymerase chain reaction (RT-qPCR) when it was induced for 7 days and 14 days. MC3T3-E1 cells were transfected with siRNA, and the knockdown efficiency of Zfp260 and the expression of alkaline phosphatase (ALP), human bone morphogenetic protein 2 (BMP2) and other osteogenic biomarkers in knockdown group and control group were measured by RT-qPCR. The change of migration ability of MC3T3-E1 cells after knockdown of Zfp260 was detected by Transwell, cell scratch assay. The change of proliferation ability of MC3T3-E1 cells after knockdown of Zfp260 was detected by CCK8 experiment. Results: The expression of Zfp260 was significantly up-regulated after inducing osteogenic differentiation of MC3T3-E1 cells in vitro (P<0.05). After using siRNA to knock down Zfp260, the expression of ALP and BMP2 were significantly decreased (P<0.05). Transwell and cell scratch assays showed that the migration ability of MC3T3-E1 cells was inhibited after knocking down Zfp260. CCK8 experiment showed that the proliferation ability of MC3T3-E1 cells increased after knocking down Zfp260. Conclusion: Zfp260 can promote the osteogenic differentiation of MC3T3-E1 cells.

Key words: zinc-finger protein 260, mouse embryo osteoblast precursor cells, osteogenic differentiation

中图分类号:

R782

吴军, 王佐林. Zfp260对MC3T3-E1细胞体外成骨分化及增殖、迁移影响的实验研究[J]. 《口腔颌面外科杂志》, 2024, 34(6): 421-426.

WU Jun, WANG Zuolin. Experimental study on the effect of Zfp260 on osteogenic differentiation, proliferation and migration of MC3T3-E1 cells in vitro[J]. 《Journal of Oral and Maxillofacial Surgery》, 2024, 34(6): 421-426.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2024/V34/I6/421

图/表 8

表1 引物序列

Table 1 Primer sequences

基因名	引物序列5'-3'	引物序列3'-5'
GAPDH	AGGTCGGTGTGAACGGATTTG	TGTAGACCATGTAGTTGAGGTCA
Zfp260	ATCTTCTGCATGACGAACCTG	GGCTTGTGAGGACTCTTCGC
ALP	CCAACTCTTTTGTGCCAGAGA	GGCTACATTGGTGTTGAGCTTTT
BMP2	GGGACCCGCTGTCTTCTAGT	TCAACTCAAATTCGCTGAGGAC

图1 成骨诱导后Zfp260表达变化 ***表示P<0.001，与对照组比较。

Figure 1 Expression change of Zfp260 after osteogenesis induction

图2 siRNA沉默效率 **表示P<0.01，与对照组比较。

Figure 2 siRNA silence efficiency

图3 成骨诱导7 d和14 d时Zfp260、ALP、BMP2基因表达量比较 A—C.成骨诱导7 d时Zfp260、ALP、BMP2基因相对表达量；D—F.成骨诱导14 d时Zfp260、ALP、BMP2基因相对表达量。*表示P<0.05，**表示P<0.01，与对照组比较。

Figure 3 Comparison of BMP2, Zfp260, ALP gene expression at 7 d and 14 d after osteogenic induction

图4 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 4 Changes in cell proliferation after siRNA knockdown

图5 荧光显微镜观察敲降组和对照组迁移细胞情况 A. Transwell实验检测敲降组细胞迁移情况（×200）；B. Transwell实验检测对照组细胞迁移情况（×200）；C.敲降组和对照组细胞迁移数比较，**表示P<0.01，与对照组比较。

Figure 5 Fluoroscopic microscopy migration of cells in knockdown group and control group

图6 敲降组和对照组细胞划痕后0~24 h的生长情况 A—C.依次为对照组细胞划痕实验后0、12、24 h的生长情况（×40）；D—F.依次为敲降组细胞划痕实验后0、12、24 h的生长情况（×40）；G.敲降组与对照组细胞划痕实验后12 h划痕愈合率比较；H.敲降组与对照组细胞划痕实验后24 h划痕愈合率比较。**表示P<0.01，***表示P<0.001，与对照组比较。

Figure 6 The growth of cells in knockdown group and control group at 0-24 h after scratch

图7 成骨诱导7 d后的ALP染色 A.对照组；B.敲降组。

Figure 7 ALP staining 7 days after osteogenesis induction

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