RUNX1对牙髓干细胞的增殖及成骨、成脂分化的影响

The Roles of RUNX1 in the Proliferation and Osteogenic and Adipogenic Differentiation of Dental Pulp Stem Cells

目的研究Runt相关转录因子1(Runt-related transcription factor 1,RUNX1)对牙髓干细胞(dental pulp stem cell,DPSC)增殖、成骨向分化、成脂向分化的作用。方法转染建携带目的基因RUNX1和绿色荧光蛋白(GFP)的慢病毒载体至DPSC 48 h后,通过荧光标记GFP和Western blot确定转染效率。过表达RUNX1后,CCK-8法和克隆形成实验检测DPSC增殖能力和克隆形成能力,流式细胞术检测DPSC细胞周期。转染沉默RUNX1的siRNA至DPSC。矿化诱导后,通过碱性磷酸酶活性检测和茜素红染色,观察过表达/沉默RUNX1对DPSC成骨向分化的影响;成脂诱导后,通过油红O染色,观察过表达/沉默RUNX1对DPSC成脂向分化的影响。结果转染慢病毒后RUNX1蛋白在DPSC中过表达,荧光检测示慢病毒转染成功,稳定表达GFP蛋白的细胞均在70%以上。过表达RUNX1后DPSC细胞增殖速度增快、克隆形成能力增强、S期细胞比例增加(P<0.05)。RUNX1过表达后DPSC的碱性磷酸酶活性和矿化结节形成能力增强,脂滴减少(P<0.05);RUNX1敲低后DPSC的碱性磷酸酶活性和矿化结节形成能力减弱,脂滴增加(P<0.05)。结论RUNX1促进DPSC增殖和成骨向分化,抑制DPSC成脂向分化。

Objective To investigate the influence of Runt-related transcription factor 1(RUNX1)on the proliferation,osteogenic differentiation and adipogenic differentiation of dental pulp stem cells(DPSC)in vitro.Methods DPSCs were transfected through lentiviral vector carrying the target gene RUNX1 and green fluorescent protein(GFP).After 48 h,transfection efficiency was determined with the fluorescent marking of GFP and Western blot.The effect of the overexpression of RUNX1 on DPSC proliferation and colony formation was determined with CCK-8 and colony formation assay;cell cycle of DPSC was detected by flow cytometry.RUNX1 siRNA was transfected into the DPSCs.After mineralized induction,the effect of RUNX1 overexpression/silencing on the osteogenetic differentiation of DPSC was tested by alkaline phosphatase(ALP)staining and alizarin red staining.After adipogenic induction,oil red O staining was done in order to observe the effect of overexpression/silencing of RUNX1 on the adipogenic differentiation of DPSC.Results RUNX1 protein was overexpressed in DPSC after lentiviral transfection.Fluorescent test showed successful transfection of lentiviral transfection and over 70%of the cells showed stable expression of GFP protein.The proliferation and colony-formation efficiency of DPSC was enhanced significantly and the proportion of DPSCs in the S phase was significantly increased in the RUNX1-overexpessed group(P<0.05).ALP activity and mineralized nodule formation ability increased,while lipid droplets decreased in the RUNX1-overexpessed group(P<0.05).ALP activity and mineralized nodule formation ability decreased,while lipid droplets increased in the RUNX1 knockdown group(P<0.05).Conclusion RUNX1 promotes DPSC proliferation and osteogenic differentiation while it inhibits DPSC adipogenic differentiation.