毛囊区神经嵴干细胞成骨分化诱导的实验研究

Osteogenic differentiation of neural crest stem cells from mouse hair follicles in vitro

目的分析毛囊区神经嵴干细胞(neural crest stem cells,NCSCs)体外成骨分化能力及相关分子调控机制,探讨其用于骨再生修复的可行性。方法与成骨前体细胞MC3T3-E1比较分析,在DMEM/F12培养基中加入成骨诱导液,进行成骨分化诱导,通过免疫细胞化学、碱性磷酸酶(alkaline phosphatase,ALP)及茜素红染色,观察NCSCs成骨分化能力;在成骨诱导1、2、4、7周不同时段,RT-PCR方法分别检测Runx2、Osterix、转录活化因子4(activating transcription factor4,Atf4)、骨桥素(os-teopontin,OPN)、骨钙素(osteocalcin,OCN)等相关成骨分化调控基因的表达特点。结果免疫细胞化学检测显示,成骨特异性标记物Runx2、OPN、OCN均为阳性,但成骨分化能力低于MC3T3-E1细胞。RT-PCR结果发现,不同诱导时段,OPN、OCN及Atf4mRNA均表达,但Runx2早期高表达,晚期低表达,Osterix早、晚期表达,中期未见表达;而MC3T3-E1细胞在各期未见明显差异。NCSCs成骨诱导7周后,ALP和钙结节茜素红染色呈阳性。结论 NCSCs具有成骨分化的能力,初步探讨其分化机制,为体内实验进行颌骨或颅骨缺损的修复奠定基础。

Objective To investigate the osteogenic potency and mechanism of neural crest stem cells（NCSCs）from mouse hair follicles.Methods NCSCs were isolated from mouse whisker follicles.Osteogenic differentiation was induced by DMEM/F12 containing ascorbic acid,glycerol phosphate,and dexamethasone for 1,2,4 and 7 weeks respectively.Mineralization was visualized by alkaline phosphatase（ALP）and alizarin red staining.Genes expression of osteogenic differentiation,Runx2,Osterix,Atf4（activating transcription factor 4）,OPN（osteopontin）and OCN（osteocalcin）were detected by immunocytochemistry and RT-PCR.Meanwhile,Pro-osteoblast cell line,MC3T3-E1 was used as the comparative analysis.Results Immunocytochemistry staining demonstrated that Runx2、OPN and OCN gene expression of NCSCs were positive,but the positive rate of NCSCs were lower than that of MC3T3-E1.The results of RT-PCR showed that OPN,OCN and Atf4 genes were always expressed in 1,2,4 and 7 weeks,but Runx2 expression was high in the early period,low expression in the later period,and osterix was expressed in the early and later period,was absent from the intermediate period.Meanwhile,MC3T3-E1 always expressed these genes within induced culture period.The massive and active alkaline phosphatase production,and the sodium alizarinsulfonate dyeing of mineralized nodes presented after NCSCs were cultured with the induced medium for 7 weeks.Conclusions NCSCs from hair follicles had the potency of osteogenic differentiation,and could be applied in bone tissue engineering.