目的 观察不同浓度Periostin重组蛋白对体外培养的TWIST1+/-小鼠冠状缝颅缝细胞增殖及成骨分化的影响。方法 取TWIST1+/-小鼠冠状缝颅缝细胞,培养传代后随机分成4组,并分别加入不同浓度的重组Periostin蛋白(0μg/L、50μg/L、100μg/L、200μg/L)进行培养。采用CCK8法检测该蛋白对颅缝细胞增殖的影响;碱性磷酸酶定量试剂盒检测细胞内ALP的分泌;RT-PCR及Western Blot法检测细胞内成骨分化相关因子基因和蛋白的表达。结果CCK8法检测结果显示,与0μg/L组相比,Periostin 50μg/L、100μg/L、200μg/L浓度组分别培养1、3、5、7 d后,颅缝细胞活性均显著降低(P<0.05);碱性磷酸酶试剂盒检测结果显示,与0μg/L组相比,Periostin 50μg/L、100μg/L、200μg/L浓度组分别成骨诱导培养10 d后,ALP活性被抑制,表达量下降(P<0.05);RT-PCR和Western Blot检测结果显示,成骨诱导培养21 d后,与0μg/L组相比,Periostin 50μg/L、100μg/L、200μg/L浓度组颅缝细胞成骨分化相关因子OCN、OPN、BSP、COL-1表达量下降(P<0.05)。结论不同浓度(50μg/L、100μg/L、200μg/L)的重组Periostin蛋白对TWIST1+/-小鼠冠状缝颅缝细胞增殖、分化均有明显抑制作用。

Objective To observe the effects of recombinant Periostin on proliferation and osteogenic differentiation of cranial suture cells of coronal suture in TWIST1+/-mouse. Methods Cranial suture cells were separated and extracted from coronal suture of TWIST1+/-mouse. The cells were divided into 4 groups according to different concentration of recombinant Periostin: 0 μg/L, 50 μg/L, 100 μg/L, 200 μg/L group. CCK-8 method was used to detect the cell proliferation of cranial suture cells; Alkaline Phosphatase kit was used to detect the ALP activity; Real-Time PCR and Western Blot were used to detect the expression of osteogenic differentiation markers: OCN, OPN, BSP and COL-1. Results CCK-8 test showed that, compared with 0 μg/L group, other Periostin groups inhibited cranial suture cell proliferation in some degree (P<0.05). ALP activity test showed that, after osteoinduction for 10 days, compared with 0 μg/L group, ALP activity of 50 μg/L, 100 μg/L, 200 μg/L Periostin groups were significantly decreased (P<0.05). Real-Time PCR and Western Blot showed that, compared with 0 μg/L group, other groups significantly decreased the expression of OCN, OPN, BSP and COL-1 (P<0.05). Conclusion Different concentrations (50 μg/L, 100μg/L, 200μg/L) of recombinant Periostin can inhibit the proliferation and osteogenic differentiation of cranial suture cells of coronal suture in TWIST1+/-Mouse.