Effect of ubiquitin-specific protease 4 on osteogenic differentiation of adipose -derived stem cells

doi:10.3969/j.issn.1005-4979.2019.02.002

Abstract

Abstract: Objective: This study investigated the effect of USP4 on the osteogenic differentiation of ASCs by using the ubiquitin-specific protease 4 (USP4) inhibitor Vialinin A to interfere the growth and differentiation of rat adipose stem cells (ASCs). Methods: The ASCs cells isolated and cultured to the third passage were divided into 6 groups and cultured for 10 days with 0, 0.1, 0.5, 1, 5, and 10 μmol / L Vialinin A. Cell proliferation of each group was detected by CCK-8. The third generation cells were divided into experimental group, positive and negative control groups, and cultured with osteogenic induction solution containing 0.5 μmol / L Vialinin A, osteogenic induction solution without Vialinin A, and ordinary α-MEM. The expressions of USP4, β-catenin, alkaline phosphatase (ALP) and osteopontin (OPN) genes in the 7th day of each group were detected. Cells from the three groups were cultured to 4, 7, 10, and 14 days for ALP staining to detect ALP activity; to 21 days, alizarin red staining was used to detect the formation of mineralized nodules. The t-test was used to compare the data difference between the groups. The difference was statistically significant at P <0.05. Results: There was no statistically significant difference in cell proliferation trend between the experimental group treated with 0.5 μmol / LVialinin A and the control group, indicating that the concentration of Vialinin A had no inhibitory effect on cell proliferation and was used as the concentration of subsequent experimental groups. The results of ALP and Alizarin Red staining showed that the staining effect of the experimental group was more significant than the other groups. RT-qPCR results showed that compared with the negative control group, the expression levels of USP4 and β-catenin in the cells of the positive control group and the experimental group were lower, and the difference was statistically significant. There was no significant difference between the experimental group and the positive control group. Compared with the positive control group, the expression levels of ALP and OPN in the experimental group were significantly increased, and the difference was statistically significant. CONCLUSION: 0.5 μmol / L Vialinin A can promote the expression of ALP and OPN genes in rat ASCs, and it can promote its osteogenic differentiation to a certain extent. The low expression of USP4 and β-catenin may promote the osteogenic differentiation of rat ASCs.

Key words: adipose-derived stem cells；USP4；Vialinin A； &beta, -catenin； osteogenic differentiation

摘要： 目的：本研究通过使用泛素特异性蛋白酶4（USP4）抑制剂Vialinin A干预大鼠脂肪干细胞（ASCs）的生长分化，探究USP4对ASCs的成骨分化的影响。方法：将分离培养至第3代的ASCs细胞分为6组分别用含0、0.1、0.5、1、5、10μmol/L Vialinin A的培养基培养10d，CCK-8检测各组细胞增殖情况。将第3代细胞分为实验组、阳性以及阴性对照组，分别用含0.5μmol/L Vialinin A的成骨诱导液、不含Vialinin A的成骨诱导液及普通的α-MEM培养。检测各组第7d细胞内USP4、β-catenin、碱性磷酸酶（ALP）及骨桥蛋白（OPN）基因的表达。将3组细胞培养至第4、7、10、14d进行ALP染色，检测ALP的活性；培养至21d时进行茜素红染色，检测细胞矿化结节的形成。采用t检验比较组间数据差异，P<0.05时差异具有统计学意义。结果： 0.5 μmol/LVialinin A处理的实验组细胞增殖趋势与对照组的差异无统计学意义，表明该浓度的Vialinin A对细胞增殖无抑制作用，作为后续实验组浓度。ALP与茜素红染色结果显示实验组染色效果均较其他组显著。RT-qPCR结果显示与阴性对照组相比，阳性对照组与实验组细胞内USP4与β-catenin表达量较低，差异具有统计学意义。实验组与阳性对照组相比无显著性差异。与阳性对照组相比，实验组的ALP与OPN表达量显著升高，差异具有统计学意义。结论： 0.5μmol/L 的Vialinin A可以促进大鼠ASCs内的ALP与OPN基因的表达，对其前期成骨分化有一定的促进作用。USP4与β-catenin的低表达对大鼠ASCs前期的成骨分化可能有一定促进作用。

关键词: 脂肪干细胞；USP4；Vialinin A；&beta, -catenin；成骨分化

CLC Number:

LU Yue-feng, FAN Zhen. Effect of ubiquitin-specific protease 4 on osteogenic differentiation of adipose -derived stem cells[J]. 《Journal of Oral and Maxillofacial Surgery》, doi: 10.3969/j.issn.1005-4979.2019.02.002.

卢跃峰, 范震. 泛素特异性蛋白酶4对脂肪干细胞成骨分化的影响[J]. 《口腔颌面外科杂志》, doi: 10.3969/j.issn.1005-4979.2019.02.002.

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URL: https://journal06.magtech.org.cn/Jweb_joms/EN/10.3969/j.issn.1005-4979.2019.02.002

https://journal06.magtech.org.cn/Jweb_joms/EN/Y2019/V29/I2/69

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