Lmo7对ATDC5细胞体外成软骨分化及增殖、迁移影响的实验研究

doi:10.12439/kqhm.1005-4979.2025.01.002

摘要/Abstract

摘要：

目的： 探究Lmo7（LIM domain 7）基因对ATDC5细胞体外成软骨分化及增殖、迁移的影响。方法： 体外培养ATDC5细胞，通过免疫荧光染色观察Lmo7在细胞中的分布。对细胞进行成软骨诱导，通过实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）观察分化过程中Lmo7表达水平的变化。转染小干扰RNA（small interfering RNA，siRNA）以干扰Lmo7基因在ATDC5细胞的表达水平，通过RT-qPCR检测成软骨分化标志物的基因表达变化，CCK8法和划痕实验分别检测Lmo7敲低对ATDC5细胞增殖、迁移能力的影响。结果：Lmo7分布于细胞核及细胞质内。在体外诱导ATDC5细胞成软骨分化后，成软骨分化标志物表达上调，Lmo7的表达也随之上调。使用siRNA敲降Lmo7后，成软骨分化标志物SRY相关高迁移率族盒蛋白9（SRY-related high mobility group-box gene 9，Sox9）、Ⅱ型胶原蛋白（typeⅡcollagen，Col2）表达显著上升。CCK8实验显示敲降组的增殖水平高于对照组。在划痕实验中，敲降组的划痕愈合率低于对照组。结论：Lmo7基因在ATDC5细胞成软骨分化期间上调，干扰Lmo7基因表达可以促进ATDC5细胞的成软骨分化和增殖能力，并可抑制ATDC5细胞的迁移能力。

关键词: Lmo7, ATDC5细胞, 分化, 增殖, 迁移

Abstract:

Objective: To investigate the effects of LIM domain 7 (Lmo7) on chondrogenic differentiation, proliferation and migration of ATDC5 cells in vitro.Methods: Incubate ATDC5 cells in vitro, cell immunofluorescence was used to determine the location of Lmo7 in ATDC5 cells. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the genetic expression of Lmo7 during chondrogenic differentiation. ATDC5 cells was transfected with small interfering RNA (siRNA) to inhibit the genetic expression level of Lmo7, markers of chondrogenic differentiation were detected by RT-qPCR after chondrogenic induction. The effect of Lmo7 knockdown on proliferation was observed by CCK8 assay. Wound-healing assay was used to determine the effect of Lmo7 knockdown on migration.Results: Lmo7 is located in the nucleus and cytoplasm of ATDC5 cells. After chondrogenic induction in vitro, Lmo7 upregulated significantly together with chondrogenic marker. After knockdown of Lmo7 by siRNA, chondrogenic marker SRY-related high mobility group-box gene 9 (Sox9), type Ⅱ collagen (Col2) upregulated significantly. CCK8 assay showed that the proliferation of knockdown group was higher than that of control group. In wound-healing assay, the healing efficiency of knockdown group was lower than that of control group.Conclusion: Lmo7 is upregulated during chondrogenic differentiation in ATDC5 cells. Knockdown of Lmo7 leads to promotion of chondrogenic differentiation of ATDC5 cells. Inhibiting the expression level of Lmo7 promoted proliferation but inhibited migration of ATDC5 cells.

Key words: Lmo7, ATDC5 cells, differentiation, proliferation, migration

中图分类号:

R782

陶然, 王佐林. Lmo7对ATDC5细胞体外成软骨分化及增殖、迁移影响的实验研究[J]. 《口腔颌面外科杂志》, 2025, 35(1): 7-13.

TAO Ran, WANG Zuolin. Effects of Lmo7 on chondrogenic differentiation, proliferation and migration of ATDC5 cells in vitro[J]. 《Journal of Oral and Maxillofacial Surgery》, 2025, 35(1): 7-13.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2025/V35/I1/7

图/表 8

表1 基因引物序列

Table 1 Primer sequences

基因名称	正向序列	反向序列
GAPDH	AGGTCGGTGTGAACGGATTTG	TGTAGACCATGTAGTTGAGGTCA
Lmo7	TTGAGACTCTTTTAGGGCAAGC	CGATGTCACTTTCGCAACCTT
Sox9	TGAAGAACGGACAAGCGGAG	CTTGCACGTCGGTTTTGGG
Col2	GTCAATAATGGGAAGGCGGGAGG	CGAGGGCAACAGCAGGTTCACATAC

图1 ATDC5细胞Lmo7免疫荧光染色图 A. DAPI染色；B. Lmo7染色；C.合并。上排图片为低倍视野（×200），下排图片为上排虚线矩形框的高倍视野（×400）。

Figure 1 Immunofluorescence staining of Lmo7 in ATDC5 cells

图2 ATDC5软骨分化过程中Lmo7及软骨相关基因表达水平 A. Lmo7 mRNA相对表达量；B. Sox9 mRNA相对表达量；C. Col2 mRNA相对表达量；D. Col10 mRNA相对表达量。*表示P<0.05，**表示P<0.01，***表示P<0.001，与0 d时比较。

Figure 2 Expression level of Lmo7 and chondrogenic related genes during ATDC5 chondrogenic differentiation

图3 Lmo7敲降效率 A. Lmo7 mRNA相对表达量，**表示P<0.01，与阴性对照组比较（n=3）；B. Lmo7蛋白表达条带。

Figure 3 Knockdown efficiency of Lmo7

图4 siRNA转染后软骨分化标志物表达量的变化 A. Sox9 mRNA相对表达量；B. Col2 mRNA相对表达量。*表示P<0.05，**表示P<0.01，与对照组比较（n=3）。

Figure 4 The expression of chondrogenic differentiation markers changed after siRNA transfection

图5 抑制Lmo7表达对ATDC5细胞增殖的影响 *表示P<0.05，***表示P<0.001，与对照组比较（n=3）。

Figure 5 Effect of inhibiting Lmo7 expression on ATDC5 proliferation

图6 抑制Lmo7表达水平对ATDC5细胞迁移的影响 A.敲降组和对照组细胞0、12、24 h划痕实验结果（×40）；B.敲降组和对照组12 h和24 h划痕愈合率统计结果。**表示P<0.01，与对照组比较（n=3）。

Figure 6 Effect of inhibiting Lmo7 expression level on ATDC5 cells migration

图7 软骨分化诱导7 d时对照组和敲降组的阿利新蓝染色 A.对照组；B.敲降组。

Figure 7 Alcian blue staining of Si-Control and Si-Lmo7 groups on the 7th day of cartilage differentiation induction

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