人牙髓细胞与牙周韧带细胞多向分化能力的比较

目的 比较人牙髓细胞(dental pulp cells,DPC)和牙周韧带细胞(periodontal ligamentcells,PDLC)的多向分化能力,揭示其干细胞成分特征,为开展干细胞介导的生物牙根再生奠定实验基础.方法 酶消化法分离培养人DPC和PDLC,流式细胞术检测STRO-1的表达.诱导细胞成牙本质及成骨分化、成脂分化和成软骨分化,von Kossa染色、抗骨钙素(osteocalcin,OCN)和牙本质涎蛋白(dentin sialoprotein,DSP)免疫组化染色、油红O染色、阿新蓝染色、抗Ⅱ型胶原免疫组化染色以及实时荧光定量反转录聚合酶链反应(RT-PCR)等检测DPC和PDLC的多向分化.结果 DPC和PDLC体外呈克隆样生长,STRO-1阳性率分别是(16.5%±4.2%)和(11.6%±1.1%).100%的DPC和83.3%的PDLC样本可多向分化.细胞诱导分化后,OCN、牙本质涎磷蛋白(dentinsialophosphoprotein,DSPP)、过氧化物酶体激活物增生受体2(peroxisomal proliferator activated receptorgamma 2,PPARγ2)、脂蛋白脂酶(lipoprotein lipase,LPL)和Ⅱ型胶原mRNA表达上调,与诱导前相比差异均有统计学意义(P<0.001),DPC和PDLC间OCN和PPARγ2基因上调倍数的差异有统计学意义(P<0.001).结论 人DPC和PDLC的间充质干细胞比例和多向分化能力相似.

Multilineage differentiation of human dental pulp cells and periodontal ligament cells in vitro

Objective To compare the multilineage differentiation potential of human dental pulp cells (DPC) and periodontal ligament cells (PDLC) in vitro, and to identify the stem cell characteristics. Methods Human DPC and PDLC were isolated by enzymatic digestion. STRO-1 expression was investigated by flow cytometry. Cells were induced to odontogenic/osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation. The multilineage differentiation capacities of DPC and PDLC were evaluated by yon Kossa stain, anti-osteocalcin (OCN) and anti-dentin sialoprotein (DSP) immunocytochemistry, oil red O stain, Alcian blue stain, anti-collagen type Ⅱ immunocytochemistry, and real time RT-PCR. Results Colony formation was observed in DPC and PDLC, with STRO-1 positive rate of ( 16. 5% ± 4. 2% ) and ( 11.6% ± 1.1% ) respectively. Multilineage differentiation was demonstrated in 100% of DPC samples in contrast to 83.3% of PDLC samples. OCN, dentinsialophosphoprotein ( DSPP),peroxisome-proliferator-activated receptor gamma 2 (PPARγ2), lipoprotein lipase (LPL) and collagen type Ⅱ mRNA levels in DPC and PDLC were significantly upregulated after induction (P <0. 001 ). There were significant differences in the ratio of upregulation of OCN and PPARγ2 mRNAs between DPC and PDLC (P <0. 001 ). Conclusions DPC and PDLC contain similar proportion of mesenchymal stem cells and possess comparable multilineage differentiation capacities.