MYSM1对大鼠颌骨骨髓间充质干细胞增殖和成骨分化能力的影响

杨琳; 杨燕美; 李朋礼; 陈琦; 胡雅雯; 顾斌

doi:10.12435/j.issn.2095-5227.2024.086

MYSM1对大鼠颌骨骨髓间充质干细胞增殖和成骨分化能力的影响

Effect of MYSM1 on proliferation and osteogenic differentiation of rat jaw bone marrow mesenchymal stem cells

摘要

摘要:
背景去泛素化酶能够调控骨骼生长发育，MYSM1基因缺失会造成全身骨骼疏松，当前并无MYSM1对颌骨发育影响的研究。
目的探讨MYSM1对颌骨来源的骨髓间充质干细胞成骨分化能力的影响。
方法分离10只8周Wistar大鼠下颌骨，利用组织消化法分离培养颌骨骨髓间充质干细胞(jaw bone marrow mesenchymal stem cells，JBMMSCs)。取生长良好的第3 ~ 5代JBMMSCs进行成骨诱导，利用qPCR、Western blot技术检测成骨标志物OCN、Runx2、ALP及去泛素化酶MYSM1的变化。分别利用空转染慢病毒和MYSM1敲低的慢病毒转染JBMMSCs，设置对照组和敲低组细胞，qPCR检测敲低效率。CCK-8、细胞克隆形成实验检测其敲低MYSM1对细胞的增殖能力影响，细胞划痕实验检测细胞迁移能力。空转染慢病毒和敲低MYSM1的JBMMSCs成骨诱导7 d后利用qPCR技术、ALP、茜素红染色检测成骨的表达变化。
结果 JBMMSCs成骨诱导成功后，MYSM1的mRNA和蛋白表达水平升高(P＜0.05)。CCK-8显示对照组和敲低组的细胞增殖能力无统计学差异(P＞0.05)，两组间细胞集落形成实验和细胞迁移能力有统计学差异(P＜0.05)。敲低组JBMMSCs在成骨诱导7 d后OCN、Runx2、ALP的mRNA表达水平降低(P＜0.05)，ALP、茜素红染色浅于对照组。
结论敲低MYSM1并未对JBMMSs的增殖和迁移能力造成显著影响，而敲低MYSM1的JBMMSCs成骨分化能力降低。

Abstract:
Background Deubiquitination enzyme can regulate bone growth and development, and the deletion of Myb-like, SWIRM, and MPN domains 1 (MYSM1) gene may cause osteoporosis in the whole body. Currently, no studies have been found on the effect of MYSM1 on jaw development.
Objective To investigate the effect of MYSM1 on osteogenic differentiation of bone marrow mesenchymal stem cells derived from jaws.
Methods Jaw bone marrow mesenchymal stem cells (JBMMSCs) of 10 8-week-old Wistar rats were isolated and cultured by tissue digestion. The 3-5 generations of well-grown JBMMSCs were selected for osteogenic induction, and the changes of osteogenic markers OCN, Runx2, ALP and deubiquitination enzyme MYSM1 were detected by qPCR and Western Blot. JBMMSCs were transfected with lentivirus with empty transfection and lentivirus with MYSM1 knockdown, respectively, cells were divided into the control group and knockdown group, and qPCR was used to detect the knockdown efficiency. The effect of MYSM1 knockdown on cell proliferation was detected by CCK8 and cell clonal formation assay, and the cell migration ability was detected by cell scratch assay. Osteogenic expression of JBMMSCs transfected with lentivirus and MYSM1-knockdown JBMMSCS was detected by qPCR, ALP and alizarin red staining at 7 days after osteogenic induction.
Results After successful osteogenic induction of JBMMSCs, mRNA and protein expression levels of MYSM1 were increased (P＜0.05). CCK8 showed no difference in cell proliferation between control group and knockdown group (P＞ 0.05), the cell colony formation test and cell migration ability were significantly different between the two groups (P＞0.05). The mRNA expression levels of OCN, Runx2 and ALP of JBMMSCs in the knockdown group decreased at 7 days after osteogenic induction (P＜ 0.05), ALP and alizarin red staining were lighter than control group.
Conclusion MYSM1 knockdown does not significantly affect the proliferation and migration ability of JBMMSs, but the osteogenic differentiation ability of JBMMSCs with MYSM1 knockdown is decreased.

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