维甲酸对小型猪牙周膜干细胞成骨诱导能力的比较研究

The comparative study of the osteogenic induction ability of retinoic acid on small pig periodontal ligament stem cells

目的分离、培养、鉴定小型猪牙周膜干细胞(PDLSCs)并使用不同浓度的维甲酸(RA)对其进行成骨方向诱导,观察各浓度RA诱导后小型猪PDLSCs的碱性磷酸酶活性、相关成骨基因表达的差异。方法采用组织块法获得小型猪PDLCs,有限稀释法纯化小型猪PDLSCs,免疫荧光法检测STRO-1、免疫细胞化学法检测波形蛋白、角蛋白鉴定小型猪PDLSCs。分别使用0.5μm、1μm、2μm RA对小型猪PDLSCs进行成骨诱导,14 d后行ALP染色,诱导21 d后,茜素红染色检测矿化结节。诱导14 d后对比各组细胞ALP活性。Real-time PCR定量分析各组RA诱导小型猪PDLSCs第21天ALP、OPN、OCN、Col I基因表达情况。结果纯化后小型猪PDLSCs STRO-1,波形蛋白阳性表达,角蛋白阴性表达,各组RA诱导14 d后,ALP染色阳性,诱导21 d后各诱导组细胞茜素红染色阳性。ALP活性检测显示,小型猪PDLSCs诱导至第14天,ALP活性随RA浓度增高而增高(P<0.001)。Real-time PCR分析成骨相关基因表达结果显示,ALP、OCN基因表达与RA浓度呈剂量依赖关系。结论0.5μm、1μm、2μm三种浓度的RA均可诱导小型猪PDLSCs分化为成骨样细胞,并且其成骨诱导的能力在一定范围内随着RA浓度增高而增强,可以认为RA可能是一种更优秀的成骨诱导剂。

Objective To isolate,culture and identify miniature swine periodontal ligament stem cells(PDLSCs),and induce their osteogenic differentiation with different concentrations of retinoic acid(RA),and to observe the differences in alkaline phosphatase activity and the expression of relevant osteogenic genes in porcine PDLSCs induced by various RA concentrations.Methods Porcine PDLCs were harvested by using tissue block method,PDLSCs were purified using a limited dilution method.Immunofluorescence had been used to detect the expression of STRO-1 and immunocytochemistry to detect the expression of vimentin and cytokeratin of porcine PDLSCs.Osteogenic induction of PDLSCs was performed using 0.5μm,1μm,and 2μm RA.The cells were stained by ALP stain kit on the 14th days.Mineralized nodules was stained by Alizarin red S on the 21th days.Activity of ALP was detected on 14th day.The expression of osteoblastic-associated genes(ALP,OPN,OCN,Col I)was analyzed by Real-time PCR on the 21st day.Results After purification,miniature swine PDLSCs expressed STRO-1 and vimentin,and the result of cytokeratin was negative.PDLSCs induced by RA showed positive expression of ALP on the 14th day.Alizarin red S were stained positive.ALP activity increased with increasing RA concentration after procine PDLSCs induction until day 14(P<0.001).After 21 days of osteogenic induction,the Real-time PCR analysis of the osteogenesis-related gene expression results showed that,the genes expression of ALP and OCN were up-regulated by RA in a dose-dependent manner.Conclusion 0.5μm,1μm,2μm of RA can induce differentiation of miniature swine PDLSCs into osteogenic cells,and its osteogenic ability to induce is enhanced with increasing RA concentration in a certain range,it can be considered that RA may be a better osteogenic inducer.