Oct-4在骨髓间充质干细胞定向诱导分化中的甲基化研究

发布时间：2018-03-22 04:34

  本文选题：Oct-4　切入点：骨髓间充质干细胞　出处：《石河子大学》2009年硕士论文　论文类型：学位论文

【摘要】：目的:体外分离、培养小鼠骨髓间充质干细胞(mouse Bone marrow mesenchymal stem cells,mBMSCs),并诱导其向成骨细胞方向分化,检测Oct-4在mBMSCs诱导分化前后的表达及甲基化状态的改变,为进一步研究Oct-4在BMSCs增殖分化中的作用提供研究思路。 方法:采用全骨髓贴壁筛选法体外培养、扩增mBMSCs。用CD29、CD44、c-Kit以及CD34免疫细胞化学染色鉴定mBMSCs。取第3代小鼠BMSCs,用含10%FBS,0.1μmol/L地塞米松,50μg/ml维生素C和10mmol/Lβ?甘油磷酸钠的高糖DMEM培养基定向诱导mBMSCs向成骨细胞分化,并用碱性磷酸酶(alkaline phosphatase, AKP)染色、茜素红染色等方法进行鉴定,同时用体外分离培养的成骨细胞作阳性对照。采用RT-PCR及间接免疫荧光染色方法,从mRNA和蛋白水平检测Oct-4在mBMSCs成骨诱导分化前后的表达;运用甲基化特异PCR检测Oct-4在mBMSCs诱导分化前后的甲基化状态。 结果:mBMSCs接种24h后已大量贴壁并伸展开;48h后贴壁细胞明显增多并分裂增殖,贴壁细胞呈梭形或成纤维细胞样形态。传代培养后,细胞形态更加均一,增殖加快,呈涡旋样生长。免疫细胞化学染色和间接免疫萤光染色结果显示第3代mBMSCs CD29、CD44、c-Kit表达阳性,CD34表达阴性。第3代mBMSCs在成骨诱导诱导剂诱导4天后细胞形态变化,随着诱导时间的延长,细胞逐渐增大,细胞形态由长梭形逐渐变为多角形。mBMSCs诱导10d后大多数细胞和体外培养成骨细胞形态相似,均呈AKP和茜素红阳性反应。RT-PCR结果与间接免疫荧光染色结果同时显示,第3代的mBMSCs有Oct-4表达,而向成骨方向诱导10d后的mBMSCs及小鼠成骨细胞均未见Oct-4表达;MSP结果显示,mBMSCs在向成骨方向诱导10天后,Oct-4基因的启动子区的CpG岛出现甲基化,而未诱导的第3代mBMSCs为非甲基化状态。 结论: 1.在本实验室成功的建立了小鼠成骨细胞培养体系。 2.成功分离培养mBMSCs,并诱导其向成骨细胞分化,观察到Oct-4在mBMSCs定向诱导分化中表达下调。 3.首次观察到在mBMSCs定向诱导分化中Oct-4基因的甲基化状态发生了改变,提示Oct-4基因在mBMSCs定向诱导分化中的表达下调可能与其启动子区甲基化有关。
[Abstract]:Objective: to isolate and culture mouse Bone marrow mesenchymal stem stem mBMSCs, and to induce the differentiation of mouse Bone marrow mesenchymal stem cells into osteoblasts, and to detect the expression of Oct-4 before and after mBMSCs induction and the changes of its methylation state. To further study the role of Oct-4 in the proliferation and differentiation of BMSCs. Methods: mBMSCs were amplified by whole bone marrow adherent screening method in vitro. MBMSCs were identified by CD29 CD4C4c-Kit and CD34 immunocytochemical staining. BMSCs of the third generation of mice were obtained, and vitamin C and 10mmol/L 尾 were determined by 50 渭 g/ml vitamin C and 50 渭 g/ml of dexamethasone containing 10s and 0.1 渭 mol/L dexamethasone. The differentiation of mBMSCs into osteoblasts was induced by high glucose DMEM medium of sodium glycerophosphate. The differentiation of mBMSCs into osteoblasts was identified by alkaline phosphatase staining and alizarin red staining. At the same time, osteoblasts isolated and cultured in vitro were used as positive control. The expression of Oct-4 before and after osteogenic differentiation of mBMSCs was detected by RT-PCR and indirect immunofluorescence staining from the level of mRNA and protein. Methylation specific PCR was used to detect the methylation of Oct-4 before and after differentiation induced by mBMSCs. Results after 24 hours of inoculation, a large number of adherent cells were proliferated and proliferated, and the adherent cells were fusiform or fibroblast-like. After passage, the morphology of the cells was more uniform and the proliferation was accelerated. The results of immunocytochemical staining and indirect immunofluorescence staining showed that the third generation of mBMSCs CD29 + CD4c-Kit positive expression of CD34 was negative, the third generation of mBMSCs was induced by osteoblast inducer for 4 days, and the cell morphology changed with the prolongation of induction time. After 10 days of induction, most of the cells were similar to those of osteoblasts cultured in vitro. The results of AKP and alizarin red positive reaction. RT-PCR and indirect immunofluorescence staining showed that most of the cells were similar to those of osteoblasts cultured in vitro, and the results of RT-PCR and indirect immunofluorescence staining showed that most of the cells were similar to those of osteoblasts cultured in vitro. Oct-4 expression was found in the mBMSCs of the third generation, but no Oct-4 expression was found in the mBMSCs and mouse osteoblasts after 10 days of osteogenic induction. The results showed that there was methylation in the CpG island of the promoter region of Oct-4 gene 10 days after induction to the osteogenic direction. The uninduced third generation mBMSCs was demethylated. Conclusion:. 1. The mouse osteoblast culture system was successfully established in our laboratory. 2. MBMSCs were successfully isolated and cultured and induced to differentiate into osteoblasts. The down-regulated expression of Oct-4 was observed in mBMSCs induced differentiation. 3. It was observed for the first time that the methylation status of Oct-4 gene was changed during mBMSCs directed differentiation, suggesting that the down-regulation of Oct-4 gene expression in mBMSCs induced differentiation might be related to the promoter methylation.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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