基质细胞衍生因子1对人牙髓干细胞增殖、迁移和成牙本质能力的影响

Effects of stromal cell-derived factor-1 on proliferation,migration,and odontoblastic differentiation of human dental pulp stem cells

目的:对比基质细胞衍生因子1(stromal cell-derived factor-1,SDF-1)和粒细胞集落刺激因子(granulocyte colony-stimulating factor,G-CSF)对人牙髓干细胞(dental pulp stem cell,DPSC)的体外增殖、迁移和成牙本质能力的影响。方法:分别在培养基中加入100μg/L SDF-1或100μg/L G-CSF,采用细胞计数试剂盒(cell counting kit-8,CCK-8)和集落形成实验(colony-forming unit,CFU)检测SDF-1和G-CSF对DPSC增殖的影响;采用划痕实验和Transwell迁移实验检测两者对DPSC迁移能力的影响;对DPSC进行成牙本质诱导,通过碱性磷酸酶(alkaline phosphatase,ALP)染色、测定ALP活性、茜素红染色和real-time RT-PCR检测成牙本质相关基因的表达,以检测两者对DPSC体外成牙本质能力的影响。结果:SDF-1和G-CSF能够轻度提高DPSC的增殖及集落形成能力,但差异无统计学意义。加入SDF-1或G-CSF的实验组划痕汇合速率明显高于对照组(P<0.01),但两种因子间差异无统计学意义。Transwell迁移实验中,对照组每视野的迁移细胞数量为(5.0±1.4)个,SDF-1组每视野的迁移细胞数量为(24.3±6.8)个,G-CSF组每视野的迁移细胞数量为(11.8±3.3)个,各组间差异有统计学意义(P<0.05)。经成牙本质诱导后,实验组细胞ALP染色加深,ALP活性上升,矿化结节形成数量增加,成牙本质相关基因的表达均显著高于对照组。结论:SDF-1对DPSC的增殖能力影响不显著,但能明显提高DPSC的迁移能力和成牙本质分化能力,效果优于G-CSF。

Objective ： To compare the effects of stromal cell-derived factor-1 （ SDF-1 ） and granulocyte colony-stimulating factor （G-CSF） on proliferation, migration, and odontoblastic differentiation of human dental pulp stem cell （DPSC） in vitro. Methods： DPSCs were cultured in vitro and treated with either 100 μg/L SDF-1 or 100 μg/L G-CSF. Cell counting kit-8 （ CCK-8 ） and colony-forming unit （CFU） were used to detect the effect of SDF-1 and G-CSF on the proliferation ability of DPSC. Cell migration of DPSC was determined by wound healing assay and Transwell migration assay. The effects of SDF-1 and G-CSF on odontoblastic differentiation of DPSC were evaluated by alkaline phosphatase （ALP） staining, ALP activity and alizarin red S staining. The expression of odontoblastic-related genes such as dentin ma- trix protein 1 （DMP-1） and dentin sialophosphoprotein （DSPP） were quantified by real-time RT-PCR. Results： SDF-1 and G-CSF promoted the proliferation of DPSC slightly, but the difference was not statis- tically significant. Wound healing assay showed that SDF-1 and G-CSF promoted cell migration of DPSC significantly （P 〈 0.01 ）, but there was no significant difference between the two factors. In Transwell migration assay, the number of migrated cells of the control group was 5.0± 1.4 per sight, while the SDF-1 group was 24.3 ± 6.8 per sight and the G-CSF group was 11.8 ±3.3 per sight, suggesting that cell migration of DPSC was improved significantly after being treated with SDF-1 or G-CSF, and SDF-1 was more effective than G-CSF （ P 〈 0.05 ）. Significantly greater odontoblastic differentiation potential was found in SDF-1 group and G-CSF group based on the ALP staining. Higher ALP activity, more mineralization nodule formation and higher expressions of DMP-1 and DSPP were also found after SDF-1 or G-CSF treatment. Conclusion ： SDF-1 had no significant effect on the proliferation of DPSC, but couldsignificantly promote cell migration and odontoblastic differentiation of DPSC. Its effect on DPSC was better than G-CSF.