Journal of Prevention and Treatment for Stomatological Diseases ›› 2022, Vol. 30 ›› Issue (2): 89-96.DOI: 10.12016/j.issn.2096-1456.2022.02.003

• Basic Study • Previous Articles     Next Articles

Effect of silencing HDAC9 on the proliferation and differentiation of periodontal ligament stem cells

GE Wenbin1(), ZHANG Kun1, LUO Shitong1, ZHOU Zhi2(), LIU Yali1()   

  1. 1. Department of Orthodontics, Stomatological Hospital of Kunming Medical University, Kunming 650106, China
    2. Department of Orthodontics, the Affiliated Hospital of Yunnan University, Kunming 650021, China
  • Received:2021-04-15 Revised:2021-08-28 Online:2022-02-20 Published:2021-11-25
  • Contact: ZHOU Zhi, LIU Yali
  • Supported by:
    National Natural Science Foundation of China(31860326);the Joint Fund of Yunnan Provincial Science and Technology Office and Kunming Medical University(2017FE468-227);the Joint Fund of Yunnan Provincial Science and Technology Office and Kunming Medical University(2018FE001-260);the Graduate Innovation Fund of Kunming Medical University(2020S021);Scientific Research Fund of Yunnan Provincial Department of Education(2020Y0134)

沉默HDAC9对牙周膜干细胞增殖与成骨分化的影响

戈文斌1(), 张琨1, 罗世通1, 周治2(), 刘亚丽1()   

  1. 1.昆明医科大学附属口腔医院正畸科,云南 昆明(650106)
    2.云南大学附属医院正畸科,云南 昆明(650021)
  • 通讯作者: 周治,刘亚丽
  • 作者简介:戈文斌,医师,硕士,Email: 469039529@qq.com
  • 基金资助:
    国家自然科学基金项目(31860326);云南省科技厅联合专项(2017FE468-227);云南省科技厅联合专项(2018FE001-260);昆明医科大学研究生创新基金(2020S021);云南省教育厅科学研究基金(2020Y0134)

Abstract:

Objective To investigate the effect of silencing histone deacetylase 9 (HDAC9) expression on the proliferation and osteogenic differentiation of periodontal ligament stem cells (PDLSCs). Methods PDLSCs were isolated, cultured and identified in vitro. An siRNA construct specific for HDAC9 was transfected into PDLSCs (siHDAC9 group), and a nontargeting siRNA was used as a control (siNC group). The interference effect was determined by qRT-PCR. The cell cycle progression of PDLSCs was detected using flow cytometry. The proliferation activity of PDLSCs was detected via CCK-8 assay. Western blotting was used to detect the protein expression of proliferating cell nuclear antigen (PCNA). The mRNA expression of runt-related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP) was investigated by qRT-PCR. The protein expression of RUNX2 was detected by western blotting. In addition, the formation of mineralized nodules was assessed by alizarin red staining. Results Compared with that in the siNC group, the mRNA expression of HDAC9 in the siHDAC9 group was lower (P < 0.01). Moreover, compared with those in the siNC group, the proliferation index (P<0.01), proliferation activity (P<0.05) and protein expression of PCNA (P<0.01) in the siHDAC9 group were all increased. Compared with the siNC group, the siHDAC9 group exhibited higher mRNA expression of RUNX2 and ALP (P < 0.05), and the protein expression of RUNX2 showed the same results (P < 0.01). The results of alizarin red staining showed that compared to the siNC group, the siHDAC9 group formed more mineralized nodules. Conclusion Silencing HDAC9 expression can promote the proliferation and osteogenic differentiation of PDLSCs.

Key words: histone deacetylase 9, periodontal ligament stem cells, proliferation, osteogenic differentiation, proliferating cell nuclear antigen, mineralized nodules, runt-related transcription factor 2, alkaline phosphatase

摘要:

目的 探讨沉默组蛋白去乙酰化酶9(histone deacetylase 9,HDAC9)对牙周膜干细胞(periodontal ligament stem cells,PDLSCs)增殖与成骨分化的影响。方法 体外分离培养及鉴定PDLSCs,构建HDAC9的siRNA转染至PDLSCs中,实验分为阴性对照组(siNC组)和实验组(siHDAC9组),qRT-PCR检测干扰效果,流式细胞术检测细胞周期,CCK-8检测细胞增殖活性,Western blot检测增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的蛋白表达;qRT-PCR检测Runt相关转录因子2(runt-related transcription factor 2,RUNX2)、碱性磷酸酶(alkaline phosphatase,ALP)的mRNA表达,Western blot检测RUNX2的蛋白表达,茜素红染色检测矿化结节形成。结果 相较于siNC组,siHDAC9组HDAC9 mRNA表达降低(P<0.01)。siHDAC9组PDLSCs增殖指数大于siNC组(P<0.01),增殖活性强于siNC组(P<0.05),PCNA的蛋白表达高于siNC组(P<0.01)。siHDAC9组RUNX2、ALP的mRNA表达高于siNC组(P<0.05),RUNX2蛋白表达高于siNC组(P<0.01),矿化结节数多于siNC组。结论 沉默HDAC9可促进PDLSCs的增殖与成骨分化。

关键词: 组蛋白去乙酰化酶9, 牙周膜干细胞, 增殖, 成骨分化, 增殖细胞核抗原, 矿化结节, Runt相关转录因子2, 碱性磷酸酶

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