转化生长因子-β3 对牙髓干细胞成骨早期蛋白的影响

Effect of transforming growth factor-β3 on early osteogenic protein of dental pulp stem cells

目的 研究转化生长因子-β3(TGF-β3)对体外培养的兔牙髓干细胞(DPSCs)成骨向分化的影响。方法 采用酶消化法分别将兔牙髓与颅骨分离培养获得DPSCs与成骨细胞(OB),通过光镜观察细胞形态,免疫组化染色和茜素红染色对OB进行鉴定。取第3代DPSCs与OB,分为3组:DPSCs组(空白对照组)、OB组(阳性对照组)及DPSCs+TGF-β3组(实验组)。培养第5天采用免疫组化染色检测各组细胞内Runt相关转录因子2(Runx-2)的表达;培养第7天采用碱性磷酸酶(ALP)活性测定试剂盒检测各组细胞内ALP的活性;培养第1、3、5、7天采用蛋白质印迹法(Western Blot)检测各组成骨特异性标志物Runx-2及TGF-β3蛋白的表达。结果 兔DPSCs多呈长梭形,突触多,OB多呈短梭形或成纤维样细胞形态,突触少而丰满;DPSCs与OB鉴定阳性。培养第5天,OB组与DPSCs+TGF-β3组细胞中的Runx-2蛋白表达均呈强阳性;培养第7天,两组细胞内的ALP活性差异无统计学意义(P>0.05)。Western Blot结果显示,培养各时间点DPSCs组与另外两组比较Runx-2和TGF-β3蛋白相对表达量差异均具有统计学意义(均P<0.05);培养第7天,DPSCs+TGF-β3组Runx-2和TGF-β3蛋白相对表达量均高于另外两组,差异均具有统计学意义(均P<0.01)。结论 TGF-β3可促进DPSCs成骨早期特异性蛋白的表达。

Objective To investigate the osteogenic differentiation of rabbit dental pulp stem cells (DPSCs) induced by transforming growth factor-β3 (TGF-β3) in vitro. Methods DPSCs and osteoblasts (OBs) were respectively obtained from rabbit dental pulp and skull by enzymetic digestion method. The morphology of the cells was observed by a light microscopy. Immunohistochemical staining and alizarin red staining were carried out to identify OBs. The third generation of DPSCs and OBs were divided into three groups, including DPSCs group (blank control), OBs group (positive control) and DPSCs+TGF-β3 group (experimental group). The expression of Runt-related transcription factor 2 (Runx-2) in each group was detected by immunohistochemical staining on the 5th day of culture. The activity of alkaline phosphatase (ALP) in each group was detected by assay kit on the 7th day of culture. Western Blot was used to detect the expression of the bone-specific markers Runx-2 and TGF-β3 proteins on the 1st, 3rd, 5th and 7th days of culture. Results The rabbit DPSCs were mostly long spindle-shaped with many synapses. The OBs were mostly short spindle-shaped or fibroblast-like, and plump with few synapses. The identification result showed that the DPSCs and OBs were positive. On the 5th day of culture, the expression of Runx-2 protein in the OBs group and DPSCs+TGF-β3 group showed strong positive. On the 7th day of culture, there was no significant difference in ALP activity between the above two groups (P>0.05). The results of Western Blot showed that the relative expressions of Runx-2 and TGF-β3 protein in the DPSCs group were significantly different from those in the other two groups, and the differences were statistically significant (all P<0.05). On the 7th day of culture, the relative expression of Runx-2 and TGF-β3 protein in the DPSCs+TGF-β3 group was higher than that in the other two groups, and the differences were statistically significant (all P<0.01). Conclusions TGF-β3 can promote the expression of early osteogenic specific proteins in DPSCs.