BMP-2、iRoot BP Plus联合对牙髓细胞成牙本质分化的实验研究

Experimental study on the combination of BMP-2 and iRoot BP Plus for the odontoblast differentiation of dental pulp cells

目的研究BMP-2、iRoot BP Plus、BMP-2与iRoot BP Plus联合对牙髓细胞增殖、分化方面的影响,以期为活髓保存治疗提供实验参考。方法采用改良组织块酶消化法进行人牙髓细胞(hDPCs)的原代及传代培养。经流式细胞仪对所培养的牙髓细胞进行鉴定。取生长状态良好的3-5代牙髓细胞分为四组:A组为对照组,B、C、D组分别加入0.2 mg/mL iRoot BP Plus、0.2 mg/mL iRoot BP Plus+100 ng/mL BMP-2及100 ng/mL BMP-2。培养1、3、5、7 d后绘制各组细胞生长曲线;培养5、7 d通过qPCR检测成牙本质相关基因ALP、DSPP、BSP的mRNA相对表达量;培养7、14d行ALP活性检测。结果成功分离培养出人牙髓细胞,经流式细胞仪检测证明所培养的牙髓细胞具有间充质干细胞特性。随着培养时间的延长,各组牙髓细胞OD值逐渐增长,3~5 d时为对数增长期,7 d以后缓慢增长;培养第1 d各组OD值之间无明显差异(P>0.05),第3、5、7 d时C组OD值均高于其它各组,差异具有统计学意义(P<0.05),其中第3、5 d时B组与D组之间无明显差异(P>0.05),第7 d时D组OD值明显高于B组OD值,差异具有统计学意义(P<0.05)。qPCR结果显示:培养5 d时,除BSP基因外,ALP、DSPP基因mRNA相对表达量均提高,其中C组显著高于B、D组,D组高于B组,差异具有统计学意义(P<0.05);培养7 d时,各组基因表达量均上调,DSPP、ALP、BSP基因mRNA相对表达量均高于A组,其中C组高于B、D组,D组高于B组,差异具有统计学意义(P<0.05)。ALP活性检测显示:培养7、14 d时,B、C、D组ALP活性均高于A组,其中C组最高,D组次之,最后是B组,差异具有统计学意义(P<0.05)。结论BMP-2较iRoot BP Plus在诱导hDPCs增殖、分化方面更具优势;两者联合可发挥协同作用,较单独使用对人牙髓细胞增殖及成牙本质分化有明显促进作用。

Objective To study the effects of BMP-2,iRoot BP Plus,BMP-2 combined with iRoot BP Plus on the proliferation and differentiation of dental pulp cells,so as to provideexperimental reference for improving vital pulp therapy.Methods The primary culture and subculture of human dental pulp cells were carried out by improved tissue block enzyme digestion.The cultured dental pulp cells were identified by flow cytometry.The 3-5generations of dental pulp cells with good growth were divided into four groups:group A was the control group,and groups B,C and D were added with 0.2 mg/mL iRoot BP Plus,0.2 mg/mL iRoot BP Plus+100 ng/mL BMP-2and 100 ng/mL BMP-2 respectively.After 1,3,5 and 7 days of culture,the growth curves of cells in each group were drawn.The mRNA relative expression of dentin-related genes ALP,DSPP and BSP was detected by qPCR on5th and 7th day of culture.The ALP activity was detected after 7 and 14 days of culture.Results Human dental pulp cells were successfully isolated and cultured.Flow cytometry showed that the cultured dental pulp cells had the characteristics of mesenchymal stem cells.With the prolongation of culture time,the OD value of dental pulp cells in each group gradually increased,with a logarithmic growth period at 3~5 days and a slow growth after 7days.There was no significant difference in the OD values of each group on the 1st day of culture(P>0.05),and the OD values of group C were higher than those of other groups on the 3rd,5th and 7th days(P<0.05).There was no significant difference between group B and group D on the 3rd and 5th days(P>0.05),and the OD values of group D were significantly higher than those of group B on the 7th day(P<0.05).The qPCR results showed that after 5 days of culture,except BSP gene,the relative mRNA expressions of ALP and DSPP genes were all increased,among which group C was significantly higher than that of group B and D,and group D was higher than that of group B,with statistical significance(P<0.05).At the 7th day of culture,the gene expression levels of each group were all up-regulated,and the relative mRNA expression levels of DSPP,ALP and BSP genes were higher than those of group A,among which group C was higher than those of groups B and D,and group D was higher than those of group B,with statistical significance(P<0.05).The ALP activity test showed that the ALP activity of groups B,C and D was higher than that of group A after 7 and 14 days of culture,with the highest activity in group C,the second in group D and the last in group B(P<0.05).Conclusion BMP-2 is superior to iRoot BP Plus in inducing hDPCs proliferation and differentiation.The combination of the two can play a synergistic role,which can obviously promote the proliferation and odontoblast differentiation of human dental pulp cells compared with the single use.