摘要
目的研究Oct-4在小鼠骨髓间充质干细胞(BMSCs)定向诱导分化中的甲基化状态。方法全骨髓贴壁筛选法培养小鼠BMSCs,通过形态学观察及免疫细胞化学进行鉴定。骨块接种培养法培养小鼠成骨细胞,采用碱性磷酸酶(AKP)及茜素红进行鉴定。诱导小鼠BMSCs向成骨细胞分化,采用间接免疫荧光染色和RT-PCR法观察Oct-4在小鼠BMSCs诱导分化前后的表达情况,甲基化特异性PCR检测Oct-4在小鼠BMSCs诱导分化前后的甲基化状态。结果分离培养的小鼠BMSCs在体外能大量增殖,形成形态均一的细胞集落,培养细胞的CD29、c-Kit和CD44呈阳性表达,CD34呈阴性表达。诱导10d后的细胞及从小鼠颅骨中分离、培养出的成骨细胞,AKP及茜素红染色呈阳性。间接免疫荧光染色和RT-PCR结果显示,Oct-4在小鼠BMSCs定向诱导分化中表达下调。小鼠BMSCs定向诱导分化中Oct-4基因启动子区的CpG岛出现了甲基化。结论成功在体外培养了小鼠BMSCs及小鼠成骨细胞,Oct-4基因可能参与了成体干细胞多潜能性的维持,Oct-4基因在小鼠BMSCs定向诱导分化中的表达下调可能是其启动子区CpG岛甲基化的缘故。
Objective To investigate the methylation Oct-4 in orientation-induced differentiation in bone marrow mesenchymal stem cells （BMSCs）. Methods Mice BMSCs were isolated and purified from bone marrow by adherent culture, and then identified hy morphology and immunoeytoehemistry, Mouse osteoblastie cells were cultured by bone fragments inoculation, and then identified by alkaline phosphatase （AKP） staining and alizarin red staining. BMSCs were induced to differentiate into osteoblasts in vitro. Indirect immunofluores- cenee staining and reverse transcription polymerase chain reaction （RT-PCR） were used to detect the expressions of Oct-4 in BMSCs before and after induction. The methylation status of Oct-4 gene in mouse BMSCs was ex- plored by a methylation-specific PCR before and after induction. Results The isolated mice BMSCs massively proliferated in vitro and formed cell colones with uniform morphology. Positive expressions of CD29, c-Kit, and CD44 and negative expression of CD34 were found in the isolated cells. After 10 days＇ induction,both AKP and the alizarin red were positive in cells and osteoblastic cells isolated from mice skull bones. The indirect immunoin- fluorescence staining and RT-PCR also showed that the Oct-4 expression in the directed differentiation of mouse BMSCs was down-regulated. The CpG island of Ore-4 gene promoter in mouse BMSCs became methylated during the induced differentiation. Conclusions Mice BMSCs and osteoblasts were successfully cultured in vitro in this study. Oct-4 may be involved in the maintenance of adult stem cell pluripotency. The down-regulated expression of Oct-4 gene in mouse BMSCs during the directed differentiation may contribute to the methylation of CpG island in Otc-4 gene promoter.
出处
《中国医学科学院学报》
CAS
CSCD
北大核心
2013年第3期275-280,共6页
Acta Academiae Medicinae Sinicae
关键词
OCT-4
小鼠骨髓间充质干细胞
分化
甲基化
Oct-4
bone marrow-derived mesenehymal stem ceils
differentiation
methylation
