Apical bud上皮诱导脂肪间充质干细胞向成牙本质样细胞分化的研究

摘要

目的：通过体外及体内实验探讨大鼠切牙Apical bud上皮细胞诱导大鼠脂肪来源间充质干细胞向成牙本质细胞分化的能力。方法：分离培养大鼠脂肪间充质干细胞、Apical bud上皮细胞,制备Apical bud上皮条件诱导液并体外对ADSCs诱导7d ,通过实时定量PCR方法检测成牙本质关键基因DMP-1及DSPP的表达。制备ADSCs及Apical bud上皮重组细胞团进行大鼠肾被膜下移植，8W后取材，分别采用HE染色、Masson三色法和免疫组化方法对移植生成物进行组织学检测。结果：Apical bud上皮诱导ADSCs后DMP-1及DSPP mRNA表达水平显著高于对照组。体内移植8W后HE染色可见管样牙本质和骨样牙本质样结构，Masson三色法可以观察到有绿色的牙本质样结构，免疫组化检测中CK14染色阳性，DSPP染色阳性。结论：Apical bud上皮细胞在体外能够诱导ADSCs向成牙本质细胞分化，且细胞重组在体内可以构建出牙本质样结构。%Objective:To study the effect of rat apical bud in differentiation of ADSC into odontoblast-like cells in vitro and in vivo.Methods:Rat ADSCs and apical bud were isolated and then ADSCs were cultured with conditioned medium (from rat incisor apical bud cells). After 7 days of induction, the gene expression of DMP-1 and DSPP were analyzed by real time PCR. ADSCs mixed with prepared apical bud and the reconstituted cell mass were implanted under rat renal capsules for up to 8 weeks, analyzed with histological and immunohistochemical evaluation.Results:Compared with the control group, apical bud conditioned medium treatment significantly induced the mRNA expression level of DMP-1 and DSPP in ADSCs. Histological examination of the product of reconstituted cells mass of apical bud epithelium and ADSCs showed positive results. Hematoxylin and eosin (H&E) staining showed tube-like dentin and bone-like dentin. Masson's trichrome staining showed green dentin-like structure. Immunohistochemidtry of CK14 and DSPP were both positive.Conclusion:Induced by apical bud epithelium conditioned medium, rat ADSCs can differentiate into odontoblast-like cells in vitro. Moreover,reconstituted cells mass of apical bud epithelium and ADSCs can construct dentin-like structure in vivo.