人根尖牙乳头干细胞条件培养基体外诱导成牙分化的研究

Conditional Medium of Stem Cells from Apical Papilla in the Vitro Induction of Odontogenic Differentiation

目的探讨人根尖牙乳头干细胞(conditional medium of stem cells from apical papilla,SCAP-CM)条件培养基诱导牙髓细胞成牙分化的作用。方法从人新鲜拔除的离体牙中,分离培养根尖牙乳头干细胞,收集其在常氧和低氧环境下培养的条件培养基[SCAP-CM(N)和SCAP-CM(H)]。用浓缩后的SCAP-CM(N)、SCAPCM(H)处理牙髓细胞,检测牙髓细胞的碱性磷酸酶活性,以qRT-PCR检测成牙相关基因Runx2、ALP、DSPP、OCN及DMP-1的表达,以WesternBlot检测牙本质涎蛋白的表达。结果在加入成骨诱导培养基的前提下,7天后SCAP-CM(H)组的碱性磷酸酶活性明显提高,SCAP-CM(N)组其次,而空白对照组的碱性磷酸酶活性最低。在基因水平上,相比空白对照组,SCAP-CM(N)和SCAP-CM(H)组的成牙相关基因Runx2、ALP、DSPP在mRNA水平上的表达增加。在蛋白水平上,SCAP-CM(H)和SCAP-CM(N)组DSP表达较其他组有所增加。结论SCAP-CM在体外具有一定的诱导成牙本质的能力,具有协同促进矿化的能力,在再生性牙髓治疗中具有潜在的应用价值。

Objective To investigate the effect of the conditional medium of stem cells from apical papilla(SCAPCM)on odontogenic differentiation.Methods Stem cells from apical papilla(SCAP)were isolated from freshly-extracted human teeth and were cultured in vitro;the conditional media(CM)of SCAP were given isolated culture in normoxic en⁃vironment[SCAP-CM(N)]or hypoxic environment[SCAP-CM(H)];the human dental pulp cells(DPC)were treated with concentrated SCAP-CM(N)and concentrated SCAP-CM(H);the alkaline phosphatase activity of dental pulp cells was evaluated;the expressions of the odontogenesis-related genes of Runx2,ALP,DSPP,OCN and DMP-1 were detect⁃ed by quantitative reverse transcription polymerase chain reaction(qRT-PCR)and the expression of DSP protein was detected by Western Blot method.Results 7 days after the addition of osteogenic induction mediu,the alkaline phospha⁃tase activity of the DPCs treated with SCAP-CM(H)increased te most,then wasthat of the DPCs treated with SCAP-CM(N)while the DPCs in blank control group was the lowest;comparing with the blank control,the expressions of Runx2,ALP and DSPP at mRNA level increased in the DPCs treated with SCAP-CM(N)and SCAP-CM(H)as well as the ex⁃pression of DSP at protein level.Conclusions SCAP-CM is of certain function in odontogenic differentiation induction and mineralization in vitro,which is of potential clinical value in dental pulp regeneration.