瘦素对人根尖乳头干细胞成骨/成牙本质相关基因表达的影响

The effects of leptin on osteogenesis/odontogenic related gene expression of human apical papillary stem cells

目的探讨瘦素(leptin)对人根尖乳头干细胞(human stem cells from the apical papilla,hSCAPs)增殖及成骨/成牙本质相关基因表达的影响,为临床年轻恒牙根尖持续发育的研究提供实验基础。方法采用人根尖乳头组织块培养法获得hSCAPs,以免疫荧光染色法、Western blot和qRT-PCR分别检测hSCAPs中leptin及其受体OBRb蛋白及基因的表达。实验组用质量分数为0.1μg/mL的leptin(0.1μg/mL组)和1.5μg/mL的leptin(1.5μg/mL组)分别刺激hSCAPs,对照组仅加入α-MEM培养基,CCK-8、流式细胞仪法、qRT-PCR分别检测各组hSCAPs增殖情况及成骨/成牙本质相关基因:碱性磷酸酶(alkaline phosphatase,ALP)、牙本质基质蛋白-1(dentin matrix protein-1,DMP-1)和牙本质涎磷蛋白(dentin sialophosphoprotein,DSPP)、骨钙素(osteocalcin,OCN)mRNA的表达。结果 hSCAPs表达leptin及其受体OBRb的蛋白及基因。与对照组相比,实验组细胞增殖能力及S期细胞比例均高于对照组(P <0.05),且其中1.5μg/mL组高于0.1μg/mL组(P <0.05)。3 d和7 d时,0.1μg/mL组、1.5μg/mL组hSCAPs的ALP、DMP-1、DSPP mRNA的表达量高于对照组,且1.5μg/mL组高于0.1μg/mL组,差异有统计学意义(P <0.05),14 d时0.1μg/mL组、1.5μg/mL组hSCAPs的ALP、DMP-1、DSPP mRNA的表达量明显低于对照组(P <0.05);0.1μg/mL组、1.5μg/mL组OCN mRNA的表达量于7 d、14 d时高于对照组,且1.5μg/mL组均高于0.1μg/mL组,差异有统计学意义(P <0.05),于14 d达到峰值。结论 Leptin可促进hSCAPs增殖,并上调hSCAPs成骨/成牙本质相关基因的表达。

Objective To investigate the effects of leptin on the proliferation of stem cells from human stem cells from the apical papilla(hSCAPs)and the expression of osteogenic/dentinogenic genes in vitro to provide an experimental basis for the sustainable development of young permanent teeth.Methods The tissue block method was used to isolate and culture hSCAPs from the apical papilla of the immature third permanent molar.The expression of leptin and OBRb in hSCAPs was detected using immunocytofluorescence staining,western blotting and reverse transcription polymerase chain reaction(RT-PCR)analysis.The hSCAPs was treated with 0.1μg/mL of leptin(0.1μg/mL group)or 1.5μg/mL of leptin(1.5μg/mL group)at different time points.The control group was treated with alpha-MEM medium.Cell proliferation was measured using the CCK8 assay and cell cycle analysis.QRT-PCR was used to detect the expression of related osteoblast/odontogenic genes for alkaline phosphatase(ALP),dentin matrix protein-1(DMP-1),dentin sialophosphoprotein(DSPP),and osteocalcin(OCN)mRNA.The differences between the treatment groups and the control group were analyzed statistically using one-way ANOVA followed by Bonferroni analysis.Results The expression of both leptin and OBRb were found in hSCAPs.Compared with the control group,the cell proliferation capacity and S phase cells in the treatment groups were higher than those in the control group,with the 1.5μg/mL group displaying higher levels than 0.1μg/mL group,and the treated hSCAPs demonstrated a higher proliferation rate and a higher expression of ALP,DSPP,and DMP-1 from day 3 to day 7,with the 1.5μg/mL group displaying higher levels than 0.1μg/mL group,and the difference was statistically significant(P<0.05),at day 7.The treated hSCAPs demonstrated a lower expression of ALP,DSPP,and DMP-1.Compared with the control group,the treated hSCAPs demonstrated a higher expression of OCN from day 7 to day 14,with significantly higher expression in the 1.5μg/mL group compared to the 0.1μg/mL group.Conclusion Leptin may promote cell proliferation and upregulate the expression of relative osteogenic/dentinogenic genes.