磷酸二氢钾对根尖牙乳头干细胞成牙及成骨向分化能力的影响

Effect of KH2 PO4 on the odonto- and osteogenic differentiation potential of human stem cells from apical papillae

目的探讨磷酸二氢钾（KH2PO。）对根尖牙乳头干细胞（stem cells fromapical papilla，SCAP）成牙与成骨向分化能力的影响，以期为组织工程牙再生、骨再生及l临床运用KH：PO。提供实验依据。方法通过酶消化法分离培养人SCAP，将SCAP分别培养于常规培养液d一最低基础培养基及含1．8mmol／LKH：PO。的d一最低基础培养基常规培养液中，分为空白对照组和实验组（KH：PO。刺激组）。通过碱性磷酸酶（alkaline phosphotase，ALP）活性检测、茜素红染色实验及实时定量反转录聚合酶链反应（reverse transcription PCR，RT—PCR）和蛋白质印迹法检测SCAP体外成牙及成骨向分化的能力。结果ALP活性检测结果显示，3d时实验组ALJP活性[（0．370±0．013）Sigma单位·min-1·mg-1]显著高于空白对照组[（0．2845±0．0084）Sigma单位·min-1·mg-1]，差异有统计学意义（P〈0．01）；培养5、7d后茜素红染色结果显示实验组形成了明显的矿化结节，实验组5、7d时钙离子浓度[分别为（0．539．4-0．007）、（1．617±0．042）μg／g]均显著高于空白对照组[（0．138±0．037）μg／g]，P〈0．01。RT—PCR及蛋白质印迹法检测结果显示：实验组SCAP培养3、7d后ALP、核心结合蛋白因子2、成骨相关转录因子、骨钙蛋白及牙本质涎磷蛋白的基因及蛋白表达均显著高于空白对照组（P〈0．01，P〈0．05）。结论1．8mmol／LKH2PO。可以明显促进人SCAP体外成牙及成骨能力。

Objective To determine the effects of KH2PO4 on the odonto- and osteogenic differentiation potential of human stem cells from apical papillae （SCAP） in vitro. Methods SCAP were isolated and cultured respectively in alpha minimum essential medium （α-MEM） or ct-MEM containing 1.8 mmol/L KHzPO4. Alkaline phosphotase（ALP） activity, alizarin red staining, real-time reverse transcription polymerase chain reaction（RT-PCR） and Western blotting were used to examine the odonto and osteogenic potential of SCAP in the two media. Results SCAP cultured in ct-MEM containing 1.8 mmol/L KHzPO4 exhibited a higher ALP activity [ （0. 370 ± 0. 013） Sigma unit ~ min-1 mg-1] at day 3 than control group [ （0. 285 ＋ 0. 008） Sigma unit min-1 mg-l] and KH2PO4-treated SCAP formed more calcified nodules at day 5 [ （0. 539 ±0. 007） μg/g] and day 7 [ （ 1. 617±0. 042） g/g] than those in normal medium[ （0. 138 0. 037） μg/g,P 〈0. 01]. The expression of odonto- and osteogenic markers were significantly up-regulated after the stimulation of KH2PO4 at day 3 and 7 respectively, as compared with control group. Conclusions 1.8 mmo]/L KH2PO4 can promote the odonto and osteogenic differentiation potential of human SCAP.