人间充质干细胞成脂分化调控机制的研究

发布时间：2018-04-30 02:27

本文选题：间充质干细胞 + 成脂诱导分化　；参考：《北京协和医学院》2016年博士论文

【摘要】：背景：人骨髓间充质干细胞(bone marrow mesenchymal stem cells, BM-MSCs)是一类具有自我更新和多向分化潜能的成体干细胞,是构成骨髓微环境的主要细胞成分之一,在调控微环境中干细胞稳态和维持干细胞的功能中发挥重要作用。BM-MSCs分化能力的异常,通过改变其正常BM-MSCs的免疫调节能力和造血支持功能,从而在各种血液性疾病和相应免疫性疾病中发挥重要作用。Notch信号通路(Notch signaling pathway)是一条经典的跨膜信号通路,通过受体与配体结合,作用于PI3K/Akt通路在调控细胞生长、发育和凋亡中发挥重要作用,而PI3K/Akt/mTOR通路作为自噬(autophagy)的调控信号,是自噬发生中重要的靶点。此外,研究认为自噬的发生在细胞生长和分化中起着重要作用。因此,以PI3K/Akt/mTOR信号通路为切入点,探讨Notch信号与自噬发生在BM-MSCs成脂诱导分化过程中的作用,为阐述临床疾病中MSCs的异常分化提供理论基础。目的：研究Notch信号通路和自噬发生在正常骨髓间充质干细胞成脂诱导分化过程中的表达变化情况,并进一步阐明Notch信号和自噬发生在骨髓间充质干细胞成脂诱导分化中发挥作用可能涉及的机制,为研究间充质干细胞的成脂分化提供实验支持和理论依据。方法：(1)采用贴壁、传代培养方法获得正常人骨髓组织中BM-MSCs;通过成脂诱导分化和成骨诱导分化检测BM-MSCs多向分化潜能；流式细胞仪检测BM-MSCs免疫表型。 (2)应用Real-time PCR和Western blot检测BM-MSCs成脂诱导分化过程中Notch信号和自噬水平的表达变化情况。(3)筛选不同浓度Notch信号抑制剂DAPT(0μM、2.5 μM、5μM和10M)对BM-MSCs中Notch信号表达抑制情况。(4)在BM-MSCs成脂诱导分化过程中,加用Notch信号抑制剂DAPT(5μM),通过油红O染色和Real-time PCR检测成脂相关基因PPARy和C/EBPa mRNA表达变化,评价抑制Notch信号对BM-MSCs成脂分化能力的影响。(5)应用自噬抑制剂3-MA(5 mM)或氯喹(20μM)抑制BM-MSCs分化早期自噬发生,同时单用或联合应用DAPT,验证自噬发生对DAPT促BM-MSCs成脂分化能力产生影响。(6)采用Western blot检测BM-MSCs成脂分化过程中PI3K/Akt/mTOR信号通路中相应蛋白表达变化情况。(7)应用MDC染色、透射电镜和Western blot联合检测,探讨Notch信号抑制剂DAPT是否通过PI3K/Akt/mTOR信号对BM-MSCs自噬发生产生影响。(8)Western blot方法和共聚焦染色方法检测自噬抑制剂3-MA (5mM)或氯喹(20 μM)对DAPT促BM-MSCs自噬发生的逆转作用。结果： (1)成功分离培养获得高纯度的人BM-MSCs,正常BM-MSCs细胞形态呈长梭形或多角形,旋涡状生长；具有成脂诱导分化和成骨诱导分化能力；流式细胞仪检测BM-MSCs免疫表型,其高表达CD44、CD73、CD90、CD105和CD166,不表达CD14、CD19、CD34、CD45和HLA-DR。(2)BM-MSCs在成脂诱导培养体系中诱导分化14天,Notch受体NOTCH1、NOTCH2和NOTCH3 mRNA明显降低(P0.05),NOTCH4 mRNA有下降趋势,但无统计学意义(P0.05)；Notch配体DLL1、DLL3、DLL4、DLK1和DLK2 mRNA表达明显下降(P0.05),Jagged1和Jagged2 mRNA无明显变化(P0.05)；Notch信号依赖的转录因子HEY1下降明显(P0.05),HES1和HEY2无明显变化(P0.05)。(3)在BM-MSCs成脂诱导分化早期,自噬相关蛋白LC-3和Beclin 1表达量显著升高,表明在分化早期自噬水平增加,随着BM-MSCs成脂诱导时间延长,自噬水平逐渐降低。(4)5 μMDAPT能够有效抑制BM-MSCs中Notch信号的表达。(5)BM-MSCs成脂诱导分化过程中,单独加入Notch信号抑制剂DAPT(5 μM),或联合应用自噬抑制剂3-MA(5 mM)或氯喹(20μM),通过油红O染色和成脂相关基因PPARγ和C/EBPa mRNA检测证实,DAPT可能通过激活自噬发生而促进BM-MSCs成脂诱导分化。(6)PTEN-PI3K/Akt/mTOR通路在BM-MSCs成脂诱导分化过程中伴随成脂诱导进程发生时间相关性改变。 (7)Notch抑制剂DAPT通过作用于PTEN-PI3K/Akt/mTOR信号通路诱导BM-MSCs自噬的发生。结论：BM-MSCs成脂诱导分化过程中Notch信号表达降低,而细胞自噬水平在BM-MSCs成脂诱导分化早期激活；Notch信号抑制剂DAPT能够促进BM-MSCs成脂诱导分化,而抑制分化早期自噬的发生能够逆转DAPT的促成脂作用；DAPT抑制Notch信号通过作用于PTEN-PI3K/Akt/mTOR通路激活BM-MSCs自噬发生从而促进其成脂分化。背景：间充质干细胞(mesenchmal stem cells, MSCs)因其具有自我更新和多向分化潜能而被广泛应用于组织损伤修复和再生医学临床应用中。MSCs来源十分广泛,可从骨髓、脂肪、脐带和胎盘等众多组织中分离获得,其中人脐带间充质干细胞(human umbilical cord mesenchymal stem cells, hUC-MSCs)因其具有取材容易,体外扩增快且不涉及伦理问题等特点受到更多关注。成脂分化和成骨分化作为MSCs主要的分化方向,对于探索MSCs功能试验和临床研究具有重要意义。半乳糖凝集素(Galectins)作为p-半乳糖苷结合凝集素家族中的一员在调控细胞免疫、抗原识别、神经发生和血管生成中具有重要作用,最新研究认为家族中某些亚型参与了细胞分化和脂肪细胞生成,但是目前对于Galectins在hUC-MSCs成脂分化和成骨分化中的作用尚不明确。目的：检测Galectins家族成员在hUC-MSCs成脂分化和成骨分化过程中的表达变化情况,为进一步研究hUC-MSCs分化功能提供一定的理论基础。方法： (1)分离提纯人脐带间充质干细胞,通过流式细胞仪检测其间充质干细胞免疫表型,油红O染色、Von Kossa染色和茜素红染色观察其成脂分化和成骨分化能力,鉴定分离的hUC-MSCs;(2)RT-PCR半定量检测hUC-MSCs内Galectins各成员(Gal-1、Gal-2、Gal-3、Gal-4、Gal-7、Gal-8、Gal-9、Gal-10、Gal-12、Gal-13和Gal-14)表达变化情况,筛选出其高表达亚型；(3)Real-time PCR检测hUC-MSCs成脂分化和成骨分化过程中不同时间段(第0天、1天、3天、7天、14天和21天)Gal-1、Gal-3、Gal-8和Gal-9 mRNA表达变化情况； (4)收集hUC-MSCs成脂分化和成骨分化过程中不同时间段细胞培养上清,通过ELISA检测Gal-1和Gal-9分泌蛋白表达。结果：(1)所分离hUC-MSCs高表达D29、CD73、CD90、CD105、CD44、CD166和HLA-ABC,不表达CD14、CD31、CD45、CD133和HLA-DR,且在体外诱导条件下,具有向脂肪细胞和成骨细胞成分化能力。(2)hUC-MSCs细胞表达Galectins家族中Gal-1、Gal-3、Gal-7、Gal-8、Gal-9、Gal-10、Gal-12和Gal-13亚型,其中Gal-1、Gal-3、Gal-8和Gal-9 mRNA表达量较高。(3)Gal-1 mRNA和Gal-9 mRNA在hUC-MSCs成脂分化和成骨分化过程中随诱导时间进程逐渐降低；而Gal-3mRNA和Gal-8 mRNA在hUC-MSCs成脂分化和成骨分化过程中随诱导时间进程逐渐升高。 (4)ELISA检测提示Gal-1和Gal-9可能通过外分泌作用在hUC-MSCs成脂分化和成骨分化过程中发挥作用。结论：Galecins (Gal-1、Gal-3、Gal-8和Gal-9)在hUC-MSCs成脂分化和成骨分化过程中表达变化明显,其中Gal-1和Gal-9可能通过外分泌发挥作用,为进一步研究Galectins在hUC-MSCs分化中的作用提供了理论基础。
[Abstract]:Background: human bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BM-MSCs) are adult stem cells with self renewal and pluripotent differentiation potential. It is one of the main cell components that constitute the microenvironment of bone marrow. It plays an important role in regulating the homeostasis of stem cells and maintaining the function of stem cells in the micro environment and plays an important role in the differentiation of.BM-MSCs. Abnormality of ability, by changing the immune regulation ability and hematopoietic support function of normal BM-MSCs, plays an important role in various blood diseases and corresponding immune diseases. The.Notch signaling pathway (Notch signaling pathway) is a classic transmembrane signaling pathway, through the binding of receptor to ligand, and the role of PI3K/Akt pathway in modulation. The cell growth, development and apoptosis play an important role, and the PI3K/Akt/mTOR pathway is an important target for autophagy (autophagy). In addition, it is considered that the occurrence of autophagy plays an important role in cell growth and differentiation. Therefore, the PI3K/Akt/mTOR signal pathway is used as the breakthrough point to explore the Notch signal and self. The role of phagocytosis in BM-MSCs lipid induced differentiation provides a theoretical basis for explaining the abnormal differentiation of MSCs in clinical diseases. Objective: To study the changes in the expression of Notch signaling pathway and autophagy during the induced differentiation of normal bone marrow mesenchymal stem cells (MSCs), and to further elucidate the occurrence of Notch signals and autophagy in bone. The mechanism of action may be involved in the lipid induced differentiation of MSCs to provide experimental support and theoretical basis for the study of lipid differentiation of mesenchymal stem cells. Methods: (1) BM-MSCs in normal human bone marrow tissues was obtained by adherence and passage culture, and BM-MSCs multidirection was detected by lipid induced differentiation and osteogenic differentiation. Differentiation potential; flow cytometry was used to detect BM-MSCs immunophenotype. (2) the expression of Notch signal and autophagy level in BM-MSCs induced lipid induced differentiation was detected by Real-time PCR and Western blot. (3) the inhibition of DAPT (0 mu M, 2.5 mu M, 5 micronM and 4) on the expression inhibition of Notch signal inhibitors. (4) In the process of BM-MSCs lipid induced differentiation, Notch signal inhibitor DAPT (5 mu M) was used to detect the expression changes of lipid related genes PPARy and C/EBPa mRNA through oil red O staining and Real-time PCR, and the effect of inhibiting Notch signal on lipid differentiation ability of BM-MSCs was evaluated. (5) the application of autophagic inhibitor (5) or chloroquine (20 mu) to inhibit the early differentiation Autophagy occurs at the same time, at the same time using single or combined use of DAPT to verify the effect of autophagy on the ability of DAPT to promote BM-MSCs differentiation. (6) Western blot was used to detect the changes in the expression of corresponding proteins in the PI3K/Akt/mTOR signaling pathway of BM-MSCs in the process of lipid differentiation. (7) MDC staining, transmission electron microscopy and Western blot combined detection, and Notch on Notch Whether the signal inhibitor DAPT affects the autophagy of BM-MSCs through the PI3K/Akt/mTOR signal. (8) Western blot method and confocal staining method are used to detect the reversal effect of autophagy inhibitor 3-MA (5mM) or chloroquine (20 mu M) on the autophagy of DAPT BM-MSCs. Results: (1) high purity human BM-MSCs, normal BM-MSCs thin is obtained. Cell morphology was long spindle shaped or polygonal, vortexed, with lipid induced differentiation and osteogenic differentiation, and BM-MSCs immunophenotype was detected by flow cytometry, which expressed high expression of CD44, CD73, CD90, CD105 and CD166, and did not express CD14, CD19, CD34, CD45 and HLA-DR. (2) induction and differentiation for 14 days in the lipid induced culture system. H1, NOTCH2 and NOTCH3 mRNA decreased significantly (P0.05), and NOTCH4 mRNA declined, but there was no statistical significance (P0.05). (P0.05) (3) the expression of autophagy related protein LC-3 and Beclin 1 increased significantly at the early stage of BM-MSCs induced differentiation, indicating that the autophagy level increased at the early stage of differentiation, and the autophagy level gradually decreased with the prolongation of BM-MSCs induction time. (4) 5 micron MDAPT could effectively inhibit the expression of Notch signal in BM-MSCs. (5) BM-MSCs induced differentiation process. Notch signal inhibitor DAPT (5 mu M), or combined use of autophagy inhibitor 3-MA (5 mM) or chloroquine (20 micron M), was confirmed by oil red O staining and lipid related genes PPAR y and C/EBPa mRNA. DAPT may promote lipoid induced differentiation by activating autophagy. (6) the pathway is induced by lipid induced differentiation. (7) Notch inhibitor DAPT induced BM-MSCs autophagy by acting on the PTEN-PI3K/Akt/mTOR signaling pathway. Conclusion: the expression of Notch signal in BM-MSCs induced differentiation is reduced, and the level of autophagy is activated at the early stage of BM-MSCs induced differentiation; Notch letter Inhibitor DAPT can promote BM-MSCs induced differentiation, and inhibition of autophagy at early differentiation can reverse the effect of DAPT on lipid action; DAPT inhibits Notch signal to activate BM-MSCs autophagy by activating the PTEN-PI3K/Akt/mTOR pathway to promote its lipid differentiation. Background: mesenchymal stem cells (Mesenchmal stem cells, MSCs) .MSCs is widely used in the clinical application of tissue injury repair and regenerative medicine, which is widely used in tissue injury repair and regenerative medicine. It can be isolated from many tissues such as bone marrow, fat, umbilical cord and placenta. The human umbilical cord mesenchymal stem cells (human umbilical cord mesenchymal stem cells, hUC-MSCs) have a wide range of sources. More attention has been paid to the characteristics of easy extraction, rapid expansion in vitro and without ethical problems. Lipid differentiation and osteogenic differentiation as the main differentiation direction of MSCs is of great significance for exploring MSCs functional tests and clinical studies. As a member of the family of p- galactoside binding lectin, a member of galactoagglutinin (Galectins) is well regulated. Cellular immunity, antigen recognition, neurogenesis and angiogenesis are important. Recent studies suggest that some subtypes of the family have been involved in cell differentiation and adipocyte formation, but the role of Galectins in hUC-MSCs differentiation and osteogenic differentiation is unclear. The expression changes in differentiation and osteogenic differentiation provide a theoretical basis for further study of hUC-MSCs differentiation. Methods: (1) isolation and purification of human umbilical cord mesenchymal stem cells, using flow cytometry to detect the immunophenotype of mesenchymal stem cells, oil red O staining, Von Kossa staining and alizarin red staining to observe the lipid fractions. Differentiation and identification of hUC-MSCs, and (2) RT-PCR semi quantitative detection of the expression changes in hUC-MSCs Galectins members (Gal-1, Gal-2, Gal-3, Gal-4, Gal-7, Gal-8, Gal-9, Gal-10, Gal-9, etc.), and to screen out their high expression subtypes; (3) The expression of Gal-1, Gal-3, Gal-8 and Gal-9 mRNA in the interval (zeroth days, 1 days, 3 days, 7 days, 14 days and 21 days); (4) to collect the cell culture supernatant in the process of lipid differentiation and osteogenesis, and to detect the expression of Gal-1 and Gal-9 secreted protein by ELISA. Results: (1) hUC-MSCs high expression D29, CD73, CD73, CD73 6 and HLA-ABC, do not express CD14, CD31, CD45, CD133 and HLA-DR, and have the ability to differentiate into adipocytes and osteoblasts in vitro. (2) hUC-MSCs cells express Gal-1, Gal-3, Gal-7, Gal-8, Gal-7, and subtypes in Galectins family. Gal-9 mRNA gradually decreased with the induction time during the differentiation and osteogenic differentiation of hUC-MSCs, while Gal-3mRNA and Gal-8 mRNA increased with the induction time in the process of lipid differentiation and osteogenic differentiation of hUC-MSCs. (4) ELISA detection suggested that Gal-1 and Gal-9 may be divided into hUC-MSCs fat differentiation and osteogenic differentiation through external secretions. Conclusion: the expression of Galecins (Gal-1, Gal-3, Gal-8 and Gal-9) changes obviously in the process of lipid differentiation and osteogenesis differentiation of hUC-MSCs, in which Gal-1 and Gal-9 may play a role through exocrine, which provides a theoretical basis for further study of the role of Galectins in hUC-MSCs differentiation.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R329.2

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