人骨髓间充质干细胞定向分化过程中转录因子KLF2和KLF15的表达研究

发布时间：2018-01-31 19:27

本文关键词： 骨髓间充质干细胞 KLF2 KLF15 成脂分化成骨分化　出处：《北京协和医学院》2012年硕士论文　论文类型：学位论文

【摘要】：研究背景及意义成熟脂肪细胞、肌细胞及成骨细胞可以由共同的间充质干细胞分化而来。这三个方向的分化进程相互联系、相互制约,在体内呈现出动态的平衡关系。研究发现有些转录因子处于不同方向分化调控网络的交叉点,其表达水平或与体内各组织分化平衡状态的维持密切相关。KLF转录因子家族(Kruppel-like factor family)成员KLF2和KLF15在脂肪组织中均有表达,在成脂分化不同阶段发挥重要作用。此外,KLF2还表达于肌肉、骨髓等部位,KLF15还表达于骨骼肌及骨组织,但它们在骨骼肌发育、骨组织形成中是否发挥重要作用还未见报道。目前关于KLF家族成员与细胞分化的相关数据多来自于具有单向分化潜能的细胞系,不利于将不同方向分化机制联系并统一起来,而利用多向分化潜能的间充质干细胞可以同步建立不同方向的体外分化模型,对KLF2和KLF15在各分化过程中的表达进行分析,为进一步研究KLF2和KLF15在成肌、成骨分化过程中的作用机制以及这三种分化过程的联系提供依据。目的研究转录因子KLF2和KLF15在人骨髓间充质干细胞(human bone marrow mesenchymal stem cells, hBMSCs)成脂、成肌、成骨分化过程中的表达水平及变化趋势,并通过与成脂相关因子PPARy、GLUT4、成骨相关因子RUNX2及成肌相关因子MYOD的表达模式进行比较,探讨这些因子之间可能存在的联系。方法采用密度梯度离心法分离人骨髓间充质干细胞,将贴壁细胞传代培养,采用第四代细胞分别向成骨细胞、成肌细胞与脂肪细胞进行诱导,应用茜素红、油红O染色和免疫荧光细胞化学方法对诱导分化的细胞进行鉴定。通过荧光实时定量聚合酶链反应(realtime PCR)检测在诱导分化不同时间点KLF2、KLF15、PPARγ、GLUT、myoD和RUNX2的mRNA表达水平,通过免疫荧光细胞化学方法检测在诱导分化不同时间点KLF2和KLF15蛋白的定位和丰度。结果在特定诱导剂作用下hBMSCs可分化为脂肪细胞、成肌细胞与成骨细胞,经鉴定均为阳性。荧光实时定量PCR结果显示KLF2在hBMSCs成脂、成肌分化早期表达水平均呈下降趋势,在成骨分化中也有下降趋势,早期不明显；KLF15在成脂分化中期及成肌、成骨分化早期mRNA表达水平均表现为上升趋势。免疫荧光染色支持定量PCR结果。定向诱导hBMSCs成脂、成肌、成骨过程中分别检测到相关标志基因的上调。结论在hBMSCs成脂、成肌、成骨分化过程中,转录因子KLF2和KLF15分别表现出明显的变化,表明KLF2与KLF15的表达与骨髓间充质干细胞成脂、成肌、成骨分化的启动和维持密切相关；两者变化情况呈相反趋势,提示KLF2与KLF15可能存在相互竞争。KLF15在成骨分化过程出现显著上调,提示KLF15可能参与成骨分化调控。
[Abstract]:Background and significance mature adipocytes, myocytes and osteoblasts can differentiate from common mesenchymal stem cells. There is a dynamic equilibrium relationship in vivo. Some transcription factors are found to be at the crossroads of differentiation regulatory networks in different directions. Its expression level is closely related to the maintenance of differentiation equilibrium in vivo. KLF transcription factor family (Kruppel-like factor family). Both KLF2 and KLF15 were expressed in adipose tissue. In addition, KLF2 is also expressed in muscle, bone marrow and other parts are also expressed in skeletal muscle and bone tissue, but they develop in skeletal muscle. Whether bone tissue plays an important role in bone formation has not been reported. At present, most of the data about KLF family members and cell differentiation are derived from cell lines with unidirectional differentiation potential. It is not conducive to linking and unifying the differentiation mechanism in different directions, but the differentiation model in vitro can be established simultaneously by using the multi-directional differentiation potential of mesenchymal stem cells. The expression of KLF2 and KLF15 in the process of differentiation was analyzed in order to further study the expression of KLF2 and KLF15 in myogenesis. The mechanism of osteogenic differentiation and the relationship between the three differentiation processes are provided. Objective to study the expression of transcription factors KLF2 and KLF15 in human bone marrow mesenchymal stem cells (BMSCs). Human bone marrow mesenchymal stem cells. The expression level and change trend of hBMSCs in the process of adipogenic, myogenic and osteogenic differentiation, and the expression of GLUT4 in the process of osteogenesis were analyzed by PPA Ryan GLUT4. The expression patterns of osteoblast-associated factors (RUNX2) and myogenic related factors (MYOD) were compared to explore the possible relationship between these factors. Methods Human bone marrow mesenchymal stem cells were isolated by density gradient centrifugation. Adherent cells were cultured and induced to osteoblasts, myoblasts and adipocytes by 4th passage cells. Alizarin red was used. Identification of differentiated cells by Oil Red O staining and Immunofluorescence Cytochemistry. Real-time Polymerase chain reaction (PCR). KLF2 was detected at different time points of induction and differentiation. The mRNA expression levels of GLUTUM-myoD and RUNX2 in KLF15 PPAR- 纬 were detected. The localization and abundance of KLF2 and KLF15 proteins at different time points of differentiation were detected by immunofluorescence cytochemistry. Results hBMSCs could differentiate into adipocytes, myoblasts and osteoblasts under the action of specific inducer. The results of real-time quantitative PCR showed that the expression of KLF2 in hBMSCs was decreased in the early stage of myogenic differentiation and decreased in the early stage of osteogenic differentiation, but it was not obvious at the early stage. The expression of KLF15 increased in the middle stage of adipogenic differentiation, the early stage of osteogenic differentiation and the expression of mRNA. Immunofluorescence staining supported the results of quantitative PCR. The up-regulation of related marker genes was detected in the process of directed induction of hBMSCs fat-forming, musculogenesis and osteogenesis. Conclusion during the process of adipogenesis, myogenesis and osteogenic differentiation of hBMSCs, the transcription factors KLF2 and KLF15 show significant changes respectively. The results showed that the expression of KLF2 and KLF15 was closely related to the initiation and maintenance of bone marrow mesenchymal stem cells (MSCs) adipogenesis, myogenesis and osteogenic differentiation. The changes of KLF15 and KLF15 may be significantly up-regulated in the process of osteogenic differentiation, suggesting that KLF15 may be involved in the regulation of osteogenic differentiation.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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