人重组转化生长因子β1促进牙髓干细胞的增殖和矿化

Recombinant human transforming growth factor β_1 promotes dental pulp stem cells proliferation and mineralization

目的:研究人重组转化生长因子β1(recombinant human transforming growth factorβ1,rh TGF-β1)对牙髓干细胞生物学性能的影响,包括确定促进牙髓干细胞增殖的最佳作用浓度和该浓度下对牙髓干细胞分化的作用。方法:分离培养人健康第三磨牙牙髓干细胞,分别加入1μg/L、6μg/L、10μg/L的rh TGF-β1,CCK-8(cell counting kit-8)法检测对牙髓干细胞增殖的影响,选择出最佳浓度在成骨/成牙本质诱导条件下连续培养,酶标仪检测碱性磷酸酶(alkaline phosphatase,ALP)光密度值,二喹啉甲酸(bicinchoninic acid,BCA)蛋白质定量试剂盒计算总蛋白含量,两者比值作为ALP相对活性的指标。茜素红染色观察矿化结节形成能力,染色液洗脱后检测光密度值,比较rh TGF-β1对牙髓干细胞增殖和分化的作用。结果:牙髓干细胞具有体外形成矿化结节的能力,6μg/L rh TGF-β1可促进牙髓干细胞增殖;连续培养7 d后,6μg/L rh TGF-β1组细胞ALP的光密度值为0.31±0.03,显著高于对照组0.02±0.01(P<0.05);6μg/L rh TGF-β1组总蛋白含量为(2 775.46±83.54)mg/L,对照组为(1 432.20±110.83)mg/L(P<0.05);ALP相对光密度值,6μg/L rh TGF-β1组较对照组提高了6倍。茜素红染色下显示矿化结节形成增加,洗脱液光密度值6μg/L rh TGF-β1组和对照组分别为0.83±0.02和0.55±0.05,P<0.05。结论:6μg/L rh TGF-β1具有促进牙髓干细胞增殖和促进体外成牙本质分化的作用。

Objective： To explore suitable concentration of recombinant human transforming growth factor β1（rh TGF-β1） usage and study the effect of rh TGF-β1on differentiation of dental pulp stem cells（DPSCs）. Methods： DPSCs were isolated from the undiseased third molars of people aged 18-25 years and cultured according to instructions in vitro. Different concentrations（1,6,10 μg/L） of rh TGF-β1were added to the culture medium to examine DPSCs proliferation by CCK-8（cell counting kit-8） assay.The suitable concentration was then selected. For differentiation,the DPSCs were incubated for 7 or14 days with rh TGF-β1supplemented with osteo/odontoblastic induction medium containing 10 nmol/L dexamethasone, 10 mmol/L b-glycerophosphate, 50 g/L ascorbate phosphate, 10 nmol/L 1,25-dihydroxyvitamin D3 and 10% fetal bovine serum. The cells were then washed 3 times with phosphatebuffered saline and sonicated with 1% Triton X-100 for 30 minutes on ice. Cellular alkaline phosphatase（ALP） activity was assayed with p-nitrophenyl phosphate as the substrate. The enzyme activity was expressed as p-nitrophenyl produced per milligram of protein [bicinchoninic acid（BCA） protein assay kit]. To examine mineral nodule formation,the cultured cells were fixed in 4% paraformaldehyde and washed in water,and the mineralization of the extracellular matrix was assayed by 1% alizarin red S staining and elution of staining was examined as optical density（D） under microplate reader. The mean difference was considered significant at 0. 05 and 95% confidence interval. Results： The DPSCs had typical fibroblast morphology and could form mineral nodules after being cultured with osteo/odontoblstic induction medium for 14 days. 6 μg/L rh TGF-β1significantly promoted the DPSCs proliferation on the3 rd and 5th days. After the incubation of osteo/odontoblastic induction medium,the DPSCs with the6 μg/L rh TGF-β1increased ALP activities compared with the control; D values in the 6 μg/L rh TGF-β1group was 0. 31 ± 0. 03,while the control group was 0. 02 ± 0. 01（P 0. 05）. The total protein content in the 6 μg/L rh TGF-β1group was（2 775. 46 ± 83. 54） mg/L,and the control group was（1 432. 20 ±110. 83） mg/L（P 0. 05）. To eliminate the cells proliferation influence,relative ALP activities,which was defined as the total ALP divided by the total protein content,the 6μg/L rh TGF-β1group was 6 times higher than the control group. Alizarin red S staining showed increased mineral nodule formation in the rh TGF-β1group. The elution of staining under microplate reader also showed more optical density in the6 μg/L rh TGF-β1-treated cells（0. 83 ± 0. 02） than that in the control groups（0. 55 ± 0. 05,P 0. 05）. Conclusion： 6 μg/L rh TGF-β1could significantly promote DPSCs proliferation and odontoblastic differentiation in vitro.