Hey1基因参与调控BMP9诱导的C3H10T1/2细胞成骨分化

Hey1 Gene Is Involved in Regulating BMP9-induced Osteogenic Differentiation of C3H10T1/2 Cells

目的:观察发状分裂相关增强子Hey1在骨形态发生蛋白9(BMP9)诱导的小鼠间充质干细胞(MSCs)C3H10T1/2成骨分化中的作用。方法:包装Hey1、BMP9以及GFP的过表达慢病毒,并分别作用于C3H10T1/2细胞,RT-PCR和Western blot检测Hey1、BMP9以及GFP的慢病毒是否包装成功,碱性磷酸酶(ALP)检测早期成骨指标ALP的变化,茜素红S染色检测晚期成骨指标钙盐沉积,MTT检测Hey1对BMP9调控的C3H10T1/2细胞增值的影响,流式细胞术检测Hey1对BMP9调控的C3H10T1/2细胞周期的影响。结果:Hey1、BMP9以及GFP的慢病毒包装成功;在成骨分化早期,过表达Hey1基因可促进BMP9调控的C3H10T1/2细胞的成骨分化与增殖;在成骨分化晚期,过表达Hey1基因可促进BMP9诱导的C3H10T1/2细胞的成骨分化,并将BMP9调控的C3H10T1/2细胞周期阻滞在G1期。结论:Hey1基因可参与调控BMP9诱导的小鼠C3H10T1/2细胞的早晚期成骨分化,并对细胞的增殖与周期有一定的影响。

Objective： To analysis the effect of Hey1 gene on BMP9-induced osteogenic differentiation of mesenchymal stem cells C3H10T1 /2. Methods： Assemble lentivirus LV5-GFP,LV5-BMP9 and LV5-Hey1,the expression of GFP,BMP9 and Hey1 were detected by RT-PCR and Western blot in C3H10T1 /2 cells. Then C3H10T1 /2 cells was infected with LV5-GFP,LV5-Hey1 and / or LV5-BMP9 respectively,the early osteogenic marker ALP activity was detected by quantitative assay,the later osteogenic marker calcium deposition was detected by Alizarin Red S staining,the cell proliferation and cycles regulated by BMP9 were detected by MTT and flow cytometry assay. Results： LV5-GFP,LV5-BMP9 and LV5-Hey1 promoted the expression of GFP,BMP9 and Hey1. LV5-Hey1 can increase the early and later osteogenic differentiation of C3H10T1 /2 cells induced by BMP9,and promote the cell proliferation in the early stage,but inhibit cell cycles in the later stage regulated by BMP9. Conclusion： Hey1 gene can influence BMP9-induced osteogenic differentiation and BMP9-regulated cell proliferation and cell cycles of C3H10T1 /2 cells.