本文關(guān)鍵詞： 骨髓間充質(zhì)干細(xì)胞 KLF2 KLF15 成脂分化 成骨分化　出處：《北京協(xié)和醫(yī)學(xué)院》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景及意義成熟脂肪細(xì)胞、肌細(xì)胞及成骨細(xì)胞可以由共同的間充質(zhì)干細(xì)胞分化而來。這三個(gè)方向的分化進(jìn)程相互聯(lián)系、相互制約,在體內(nèi)呈現(xiàn)出動(dòng)態(tài)的平衡關(guān)系。研究發(fā)現(xiàn)有些轉(zhuǎn)錄因子處于不同方向分化調(diào)控網(wǎng)絡(luò)的交叉點(diǎn),其表達(dá)水平或與體內(nèi)各組織分化平衡狀態(tài)的維持密切相關(guān)。KLF轉(zhuǎn)錄因子家族(Kruppel-like factor family)成員KLF2和KLF15在脂肪組織中均有表達(dá),在成脂分化不同階段發(fā)揮重要作用。此外,KLF2還表達(dá)于肌肉、骨髓等部位,KLF15還表達(dá)于骨骼肌及骨組織,但它們在骨骼肌發(fā)育、骨組織形成中是否發(fā)揮重要作用還未見報(bào)道。目前關(guān)于KLF家族成員與細(xì)胞分化的相關(guān)數(shù)據(jù)多來自于具有單向分化潛能的細(xì)胞系,不利于將不同方向分化機(jī)制聯(lián)系并統(tǒng)一起來,而利用多向分化潛能的間充質(zhì)干細(xì)胞可以同步建立不同方向的體外分化模型,對(duì)KLF2和KLF15在各分化過程中的表達(dá)進(jìn)行分析,為進(jìn)一步研究KLF2和KLF15在成肌、成骨分化過程中的作用機(jī)制以及這三種分化過程的聯(lián)系提供依據(jù)。 目的研究轉(zhuǎn)錄因子KLF2和KLF15在人骨髓間充質(zhì)干細(xì)胞(human bone marrow mesenchymal stem cells, hBMSCs)成脂、成肌、成骨分化過程中的表達(dá)水平及變化趨勢,并通過與成脂相關(guān)因子PPARy、GLUT4、成骨相關(guān)因子RUNX2及成肌相關(guān)因子MYOD的表達(dá)模式進(jìn)行比較,探討這些因子之間可能存在的聯(lián)系。 方法采用密度梯度離心法分離人骨髓間充質(zhì)干細(xì)胞,將貼壁細(xì)胞傳代培養(yǎng),采用第四代細(xì)胞分別向成骨細(xì)胞、成肌細(xì)胞與脂肪細(xì)胞進(jìn)行誘導(dǎo),應(yīng)用茜素紅、油紅O染色和免疫熒光細(xì)胞化學(xué)方法對(duì)誘導(dǎo)分化的細(xì)胞進(jìn)行鑒定。通過熒光實(shí)時(shí)定量聚合酶鏈反應(yīng)(realtime PCR)檢測在誘導(dǎo)分化不同時(shí)間點(diǎn)KLF2、KLF15、PPARγ、GLUT、myoD和RUNX2的mRNA表達(dá)水平,通過免疫熒光細(xì)胞化學(xué)方法檢測在誘導(dǎo)分化不同時(shí)間點(diǎn)KLF2和KLF15蛋白的定位和豐度。 結(jié)果在特定誘導(dǎo)劑作用下hBMSCs可分化為脂肪細(xì)胞、成肌細(xì)胞與成骨細(xì)胞,經(jīng)鑒定均為陽性。 熒光實(shí)時(shí)定量PCR結(jié)果顯示KLF2在hBMSCs成脂、成肌分化早期表達(dá)水平均呈下降趨勢,在成骨分化中也有下降趨勢,早期不明顯；KLF15在成脂分化中期及成肌、成骨分化早期mRNA表達(dá)水平均表現(xiàn)為上升趨勢。免疫熒光染色支持定量PCR結(jié)果。 定向誘導(dǎo)hBMSCs成脂、成肌、成骨過程中分別檢測到相關(guān)標(biāo)志基因的上調(diào)。 結(jié)論在hBMSCs成脂、成肌、成骨分化過程中,轉(zhuǎn)錄因子KLF2和KLF15分別表現(xiàn)出明顯的變化,表明KLF2與KLF15的表達(dá)與骨髓間充質(zhì)干細(xì)胞成脂、成肌、成骨分化的啟動(dòng)和維持密切相關(guān)；兩者變化情況呈相反趨勢,提示KLF2與KLF15可能存在相互競爭。KLF15在成骨分化過程出現(xiàn)顯著上調(diào),提示KLF15可能參與成骨分化調(diào)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- 緯 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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

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