HOXC10基因对体外人牙髓干细胞增殖和成脂分化的调控作用机制研究

The regulatory mechanism of HOXC10 gene on the proliferation and adipogenic differentiation of human dental pulp stem cells in vitro

目的:探讨HOXC10基因对体外人牙髓干细胞增殖和成脂分化的调控作用及其分子机制。方法:原代分离培养人牙髓干细胞,分别将si-HOXC10、si-con转染至人牙髓干细胞,分别记作si-HOXC10组、si-con组,未经转染的人牙髓干细胞记作NC组,同时将人牙髓干细胞进行成脂诱导。q PCR与Western blotting分别检测HOXC10 mRNA及蛋白表达量;采用Western blotting检测成脂诱导后细胞中过氧化物酶体增殖因子活化受体γ(PPARγ)、重组人CCAAT增强子结合蛋白-α(C/EBP-α)蛋白水平。甲基噻唑基四唑(MTT)检测细胞增殖;Transwell小室实验检测细胞迁移能力;采用酶标仪检测细胞的黏附能力;Western blotting检测沉默HOXC10对PPARγ、C/EBP-α、WNT通路相关蛋白糖原合成酶激酶-3(GSK3β)、β-连环蛋白(β-catenin)表达的影响。结果:与培养前相比,成脂分化诱导后人牙髓干细胞中HOXC10 mRNA及蛋白表达水平显著降低(P<0.05),PPARγ、C/EBP-α的表达水平显著降低(P<0.05);与si-con组相比,si-HOXC10组人牙髓干细胞活性显著降低(P<0.05);相较于si-con组,si-HOXC10组人牙髓干细胞迁移细胞数显著增多(P<0.05),细胞黏附能力显著升高(P<0.05);与si-con组相比,siHOXC10组人牙髓干细胞中PPARγ、C/EBP-α的表达水平显著降低(P<0.05);与si-con组相比,si-HOXC10组人牙髓干细胞WNT通路中GSK3β蛋白水平显著降低(P<0.05),β-catenin蛋白水平显著升高(P<0.05)。结论:沉默HOXC10基因可抑制人牙髓干细胞增殖及成脂分化,其作用机制可能是通过激活WNT通路而发挥作用。

Objective: To investigate the regulatory effect and mechanism of HOXC10 gene on the proliferation and adipogenic differentiation of human dental pulp stem cells in vitro. Methods: Primary human dental pulp stem cells were isolated and cultured. Si-HOXC10 and si-con were transfected into human dental pulp stem cells,and recorded as si-HOXC10 group and si-con group, respectively. Human dental pulp stem cells without transfection were recorded as NC group. Then the human dental pulp stem cells were induced into adipogenesis. qPCR and Western blotting were used to detect HOXC10 mRNA and protein expression, respectively. Western blotting was used to detect the protein levels of peroxisome proliferator-activated receptor γ(PPARγ) and recombinant human CCAAT/enhancer binding protein-α(C/EBP-α) in cells after adipogenic induction. MTT assay and Transwellchamber test were used to detect cell proliferation and migration, respectively. The adhesion ability was tested. The effects of silencing HOXC10 on the expression of PPARγ, C/EBP-α, and WNT pathway related proteins glycogen synthase kinase 3β(GSK3β) and β-catenin were investigated by Western blotting. Results: The HOXC10 m RNA and protein expression levels and the protein levels of PPARγ and C/EBP-α were significantly reduced in human dental pulp stem cells after adipogenic differentiation induction(P<0.05). Compared with si-con group, the cell viability and the expressions of PPARγ, C/EBP-α and GSK3β in siHOXC10 group were significantly reduced, while the number of migrated cells, cell adhesion ability, and the protein expression level of β-catenin were increased(P<0.05). Conclusion: Silencing HOXC10 gene can inhibit the proliferation and adipogenic differentiation of human dental pulp stem cells, and its mechanism may be related to activating the WNT pathway.