Jia W Q, Zhao Y M, Ge L H
Department of Pediatric Dentistry, Peking University School and Hospital of Stomatology & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China.
Beijing Da Xue Xue Bao Yi Xue Ban. 2017 Aug 18;49(4):680-684.
To explore suitable concentration of recombinant human transforming growth factor β1 (rhTGF-β) usage and study the effect of rhTGF-β on differentiation of dental pulp stem cells (DPSCs).
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 rhTGF-β were 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 or 14 days with rhTGF-β supplemented 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 phosphate-buffered 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.
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 rhTGF-β significantly promoted the DPSCs proliferation on the 3rd and 5th days. After the incubation of osteo/odontoblastic induction medium, the DPSCs with the 6 μg/L rhTGF-β1 increased ALP activities compared with the control; D values in the 6 μg/L rhTGF-β group 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 rhTGF-β group 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 rhTGF-β group was 6 times higher than the control group. Alizarin red S staining showed increased mineral nodule formation in the rhTGF-β group. The elution of staining under microplate reader also showed more optical density in the 6 μg/L rhTGF-β-treated cells (0.83±0.02) than that in the control groups (0.55±0.05, P<0.05).
6 μg/L rhTGF-β could significantly promote DPSCs proliferation and odontoblastic differentiation in vitro.
探讨重组人转化生长因子β1（rhTGF-β）的合适使用浓度，并研究rhTGF-β对牙髓干细胞（DPSCs）分化的影响。
从18 - 25岁人群的健康第三磨牙中分离出DPSCs，并按照说明书在体外进行培养。向培养基中添加不同浓度（1、6、10μg/L）的rhTGF-β，通过CCK - 8（细胞计数试剂盒 - 8）检测法检测DPSCs的增殖情况，然后选择合适的浓度。对于分化，将DPSCs与添加了含有10 nmol/L地塞米松、10 mmol/Lβ - 甘油磷酸钠、50 g/L抗坏血酸磷酸酯、10 nmol/L 1,25 - 二羟基维生素D3和10%胎牛血清的成骨/成牙本质细胞诱导培养基的rhTGF-β一起孵育7或14天。然后用磷酸盐缓冲盐水洗涤细胞3次，并在冰上用1% Triton X - 100超声处理30分钟。以对硝基苯磷酸为底物测定细胞碱性磷酸酶（ALP）活性。酶活性以每毫克蛋白质产生的对硝基苯酚表示[二辛可宁酸（BCA）蛋白质检测试剂盒]。为检测矿化结节形成，将培养的细胞用4%多聚甲醛固定并用水洗涤，通过1%茜素红S染色检测细胞外基质的矿化情况，并在酶标仪下检测染色洗脱的光密度（D）。差异均值在0.05和95%置信区间被认为具有显著性。
DPSCs具有典型的成纤维细胞形态，在用成骨/成牙本质细胞诱导培养基培养14天后可形成矿化结节。6μg/L rhTGF-β在第3天和第5天显著促进DPSCs增殖。在用成骨/成牙本质细胞诱导培养基孵育后，与对照组相比，添加6μg/L rhTGF-β1的DPSCs的ALP活性增加；6μg/L rhTGF-β组的D值为0.31±0.03，而对照组为0.02±0.01（P<0.05）。6μg/L rhTGF-β组的总蛋白含量为（2775.46±83.54）mg/L，对照组为（1432.20±110.83）mg/L（P<0.05）。为消除细胞增殖影响，将相对ALP活性定义为总ALP除以总蛋白含量，6μg/L rhTGF-β组比对照组高6倍。茜素红S染色显示rhTGF-β组矿化结节形成增加。酶标仪下染色洗脱也显示，6μg/L rhTGF-β处理的细胞（0.83±0.02）的光密度高于对照组（其为0.55±0.05，P<0.05）。
6μg/L rhTGF-β可在体外显著促进DPSCs增殖和成牙本质细胞分化。
