RNA结合蛋白NOVA1调控间充质干细胞成脂分化的作用研究

发布时间：2018-01-17 14:05

  本文关键词：RNA结合蛋白NOVA1调控间充质干细胞成脂分化的作用研究　出处：《北京协和医学院》2017年博士论文　论文类型：学位论文

  更多相关文章： 间充质干细胞 成脂分化 神经-肿瘤腹侧抗原1 未折叠蛋白应答反应 多聚嘧啶束结合蛋白1 PPARγ信号通路

【摘要】：研究背景间充质干细胞(mesenchymal stem cells,MSCs)是一类中胚层来源的具有自我更新和多向分化潜能的成体干细胞,因其组织来源广泛、免疫原性低并具免疫调节作用而成为临床上最具应用前景的一类干细胞。RNA结合蛋白(RNA binding proteins,RBPs)能够在转录后水平调控基因表达,进而影响细胞增殖、分化、迁移与凋亡等一系列生物学特性。NOVA1(neuro-oncological ventral antigen 1)最初被认为是神经特异性的RBP,对神经细胞的存活以及发育至关重要,参与神经突触蛋白基因的选择性剪接;随后,NOVA1在肿瘤细胞和棕色脂肪发育中的作用也相继被发现。我们的前期研究显示,在人MSCs成脂分化过程中NOVA1表达升高,而NOVA1在MSCs成脂分化中的作用及其调控机制有待深入研究。研究MSCs成脂分化机制对于我们了解脂肪组织的发育、治疗脂肪细胞分化失衡的疾病具有重要的理论与临床意义。研究目的1.明确脂肪间充质干细胞(Adipose-derived mesenchymal stem cells,ADSCs)与骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)正常培养及成脂分化过程中NOVA1的动态表达变化,并通过敲减和过表达实验明确NOVA1对MSCs凋亡、迁移以及成脂分化的影响。2.探讨NOVA1调节MSCs成脂分化的分子机制。3.探讨影响NOVA1基因表达的相关因素。研究方法和结果1.MSCs及其成脂分化过程中RBPs的表达变化方法:采用油红O染色与茜素红染色以及流式细胞鉴定人脂肪组织中分离的ADSCs;通过Real-time PCR检测成脂分化过程中12个RBPs的表达;采用Real-time PCR检测人MSCs向成骨、成肌分化过程中NOVA1的表达;采用免疫荧光或免疫组化的方法对ADSCs成脂分化过程和人腹部皮下脂肪组织中NOVA1蛋白表达进行检测;采用Real-time PCR检测大鼠各组织中Nova1的表达。结果:分离得到人ADSCs高表达CD44、CD73、CD90、CD105等间充质干细胞表面标志物,不表达CD45、CD34、CD11b、CD19和HLA-DR。ADSCs成脂诱导过程中,hnRNPH1和PTBP1在诱导7天表达显著升高;hnRNPH2、RBM38、NOVA1在诱导3天和7天表达均显著升高,其中NOVA1升高程度最为明显,而FOXC2 在诱导 3 天和 7 天表达降低。TIA1、TIAL1、hnRNPF、hnRNPA1、hnRNPM、FOX2在ADSCs成脂分化过程中未发生明显变化。MSCs向成骨、成肌方向分化过程中未检测到NOVA1的表达发生明显变化;免疫荧光显示,未诱导的ADSCs中NOVA1主要在细胞质中表达,有明显的核质表达区别,而在成脂诱导中,细胞质和细胞核中表达均升高,核质表达差别不明显;人腹部皮下脂肪组织中NOVA1主要在成熟脂肪细胞的细胞核中表达;基质血管成份中NOVA1的表达远低于成熟脂肪组织;比较大鼠心脏、骨骼肌、脂肪、肝、肺、肾等组织,发现在脂肪组织中Nova1表达最高,骨骼肌中次之,肾中表达最低。2.NOVA1对MSCs凋亡和迁移以及成脂分化的影响方法:NOVA1敲减后采用流式细胞术检测ADSCs凋亡情况并利用划线法检测其迁移能力。为了研究NOVA1对MSCs分化能力的影响,通过在MSCs中敲减和过表达NOVA1并进行成脂诱导,在不同时间点采用Real-time PCR的方法检测成脂标志基因的表达,诱导7天采用油红O染色观察脂滴形成情况。结果:NOVA1敲减后ADSCs凋亡水平增加,迁移能力下降。敲减NOVA1抑制MSCs成脂分化,过表达NOVA1后MSCs成脂分化能力增加。过表达NOVA1后即使不添加成脂诱导液,7天后成脂分化标志物ADIPOQ的表达仍升高,但未见脂滴形成。3.NOVA1在MSCs成脂分化中的作用机制方法:两组ADSCs分别感染NOVA1敲减(shNOVA1)及对照(Scramble)慢病毒后,收取RNA并命名为shNOVA1和Scramble,另取两组ADSCs感染NOVA1敲减及对照慢病毒后成脂诱导3天,收取RNA并命名为shNOVA1_A3和Scramble_A3,对四组样本进行全转录本表达谱芯片检测以及差异基因GO分析和通路富集(pathway)分析;采用Western blot的方法对敲减NOVA1后成脂分化过程中未折叠蛋白应答(unfolded protein response,UPR)相关蛋白表达进行验证;在成脂诱导液中添加250nM的内质网应激激动剂毒胡萝卜素(thapsigargin,TG)处理7天,与未添加TG组比较脂滴的形成以及成脂分化标志基因的表达,western blot检测UPR相关蛋白的表达;STRING网站对能够与NOVA1相互作用的蛋白进行预测,通过IP实验对预测到的蛋白进行验证,并通过在ADSCs中敲减该蛋白后成脂诱导,检测成脂分化标志基因的表达与脂滴的形成。结果:通过对Scramble_A3 vs Scramble的差异基因比较分析,发现参与脂质代谢和脂质生物合成相关基因表达差异最为明显。对123个参与脂质代谢的基因进行聚类分析,发现在成脂诱导过程中明显升高或降低的基因在敲减NOVA1后升高或降低的程度减弱。提示NOVA1参与ADSCs成脂诱导过程中脂质代谢基因的表达调控。进一步对shNOVAl_A3 vs Scramble_A3的差异基因进行通路富集(pathway)分析,发现参与Unfolded Protein Response的基因表达差异最明显,其次是PPAR信号通路。Western blot显示参与UPR信号通路的基因在敲减NOVA1后被激活;激活内质网应激信号抑制ADSCs成脂分化。此外,通过NOVA1结合蛋白预测以及IP结果证实NOVA1能够与PTBP1相互结合,并且ADSCs成脂诱导7天PTBP1表达升高,敲减PTBP1抑制ADSCs成脂分化。4.影响MSCs成脂分化过程中NOVA1基因表达的相关因素方法:采用不同浓度的Pilocarpine和NPY处理ADSCs,在1天、3天时采用Real-time PCR的方法检测NOVA1的表达变化;不同的成脂诱导成分10-6MDEX、0.5mM IBMX、10 μM Insulin、200 μM吲哚美辛、1μM罗格列酮、完全成脂诱导液以及添加10μM PPARy的拮抗剂GW9662的吲哚美辛、罗格列酮、完全成脂诱导液处理ADSCs,在1天、3天、7天时采用Real-time PCR的方法检测NOVA1的表达变化。结果:不同浓度的Pilocarpine和NPY以及DEX和insulin对ADSCs中NOVA1的表达未见影响,IBMX在处理1天时诱导NOVA1表达升高,3天和7天影响不明显;吲哚美辛在诱导3天和7天时可显著促进NOVA1表达升高;而这种促进作用会被GW9662所抑制。研究结论1.ADSCs中表达多种RBPs,在其成脂分化过程中,hnRNPH1、PTBP1、hnRNPH2、RBM38、FOXC2、NOVA1等RBPs表达发生变化,NOVA1变化差异最明显,而NOVA1在ADSCs成骨以及成肌分化过程中表达无显著变化,说明NOVA1在干细胞分化中的表达具谱系特异性;NOVA在脂肪组织中的表达较在其它组织中高。提示NOVA1在脂肪分化中发挥特异性作用。2.NOVA1敲减促进ADSCs凋亡,抑制其迁移。敲减NOVA1抑制ADSCs和BMSCs成脂分化过程中成脂相关基因的表达与脂滴的形成,过表达NOVA1则有促进作用,说明NOVA1参与调控MSCs成脂分化;但单纯过表达NOVA1不能激活MSCs的成脂分化。3.NOVA1通过作用于脂质代谢相关基因以及UPR信号影响MSCs成脂分化,并且NOVA1可能与PTBP1作为复合体发挥作用。4.NOVA1基因启动子区域存在PPARy应答元件,PPARy配体吲哚美辛和罗格列酮能够促进NOVA1的表达,这种促进作用可被PPARγ拮抗剂GW9662抑制,说明PPARy信号通路参与调控NOVA1的表达。
[Abstract]:The research background of mesenchymal stem cells (mesenchymal stem cells, MSCs) is a kind of mesodermal self-renewal and multilineage differentiation potential of adult stem cells, because of its organization wide source, low immunogenicity and immunomodulatory effects become clinically a kind of the most promising stem cell.RNA binding protein (RNA binding proteins, RBPs) expression in the post transcriptional regulation genes, thereby affecting cell proliferation, differentiation, migration and apoptosis and a series of biological characteristics of.NOVA1 (neuro-oncological ventral antigen 1) was initially thought to be a neural specific RBP on neuronal cell survival and development is crucial, alternative splicing in neural synaptic protein gene; subsequently, the role of NOVA1 in tumor cells and brown fat in the development have been found. Our previous studies have shown that, as a NOVA1 table fat during the differentiation process of human MSCs Increased, while NOVA1 in MSCs needs further study in the role of lipid differentiation and regulation mechanism of MSCs differentiation mechanism. Understanding of adipose tissue development for us, has important theoretical and clinical significance of treatment of fat cell differentiation imbalance diseases. Objective: 1. clear adipose derived mesenchymal stem cells (Adipose-derived mesenchymal stem cells ADSCs), and bone marrow mesenchymal stem cells (Bone marrow mesenchymal stem cells, BMSCs) expression of the dynamic changes of NOVA1 normal culture and adipogenic differentiation process, and through the knockdown and overexpression of the clear NOVA1 on MSCs apoptosis, migration and adipogenic differentiation of.2. NOVA1 MSCs.3. the molecular mechanism of lipid regulation the differentiation of the factors related to the expression of NOVA1 gene. The research methods and results of 1.MSCs and RBPs expression changes in the process of differentiation by oil red O staining and Alizarin Separation of pigment red staining and flow cytometry in human adipose tissue by ADSCs; Real-time PCR to detect the expression of 12 RBPs differentiation process; the detection of human MSCs Real-time PCR to the bone, NOVA1 expression during muscle differentiation; using the method of immunofluorescence or immunohistochemistry to detect the expression of NOVA1 protein lipid differentiation and abdominal subcutaneous fat tissue of ADSCs; the expression of Nova1 Real-time in each tissue PCR detection in rats. Results: isolated ADSCs high expression of CD44, CD73, CD90, CD105 and other mesenchymal stem cell surface markers and the expression of CD45, CD34, CD11b, CD19 and HLA-DR.ADSCs fat induced process, hnRNPH1 and PTBP1 were significantly increased in 7 days induced expression; hnRNPH2, RBM38, NOVA1 in the induction of 3 days and 7 days were significantly increased, which increased the level of NOVA1 was the most obvious, while FOXC2 induced by 3 days and 7 days decreased .TIA1, TIAL1, hnRNPF, hnRNPA1, hnRNPM, FOX2 in ADSCs did not change significantly in the process of differentiation of.MSCs into bone, muscle differentiation in the process to detect the expression of NOVA1 changed significantly; immunofluorescence showed that induced ADSCs in NOVA1 is mainly expressed in the cytoplasm, obvious nucleo cytoplasmic expression the difference, in adipogenic induction, increased expression in the cytoplasm and the nucleus, cytoplasmic expression difference was not significant; abdominal subcutaneous adipose tissue NOVA1 mainly expressed in the mature fat cell nucleus; the expression of NOVA1 in stromal vascular component is much lower than that of mature adipose tissue; rat heart, skeletal muscle, fat, liver, lung, kidney and other tissues, the highest expression of Nova1 in adipose tissue and skeletal muscle in the kidney was the lowest.2.NOVA1 on MSCs apoptosis and migration and effect method of adipogenic differentiation: knockdown of NOVA1 by flow cytometry Detection of ADSCs apoptosis and detect its migration ability by crossed method. In order to study the effect of NOVA1 on MSCs differentiation ability, through in MSCs knockdown and overexpression of NOVA1 and adipogenic induction, at different time points by using the method of Real-time PCR detection of the expression of adipogenic marker gene, induced by oil red 7 days O staining was used to observe the formation of lipid droplet increased levels of apoptosis. Results: ADSCs knockdown of NOVA1 migration decreased. Knockdown of NOVA1 inhibits adipogenic differentiation of MSCs and expression of NOVA1 MSCs after adipogenic differentiation ability increased after overexpression of NOVA1. Even if it does not add adipogenic liquid, 7 days after the expression of adipogenic differentiation marker ADIPOQ still increased, but no lipid droplet formation in the.3.NOVA1 MSCs method of mechanism of adipogenic differentiation in two groups were infected with ADSCs knockdown of NOVA1 (shNOVA1) and control (Scramble) lentivirus, charged RNA and named as shNOVA1 and Scramble, another two Knockdown of NOVA1 in ADSCs infection group and control lentivirus after adipogenic induction for 3 days, charge RNA and named as shNOVA1_A3 and Scramble_A3, four groups of samples for full transcript microarray detection and differential gene GO analysis and pathway enrichment analysis (pathway); the method of Western blot NOVA1 after the knock reduction into protein response the unfolded adipogenic differentiation process (unfolded protein response, UPR) to verify the expression of endoplasmic reticulum stress related protein; add 250nM in adipogenic medium agonist thapsigargin (thapsigargin, TG) for 7 days, and without the addition of TG group and lipid droplet formation and adipogenic differentiation marker gene expression. The expression of Western blot detection of UPR related protein; STRING site to predict to proteins that interact with NOVA1 through the IP experiment of the predicted protein was verified, and through ADSCs knockdown of the protein after adipogenic induction The guide, detection of adipogenic differentiation marker gene expression and lipid droplet formation. Results: the difference of Scramble_A3 gene vs Scramble comparative analysis, found in genes related to lipid metabolism and lipid biosynthesis in the expression of the most obvious differences. The clustering analysis of 123 genes involved in lipid metabolism were found to increase or decrease in fat in the process of induction of genes increased or decreased in NOVA1 knockdown after weaken. Expression and regulation of lipid metabolism in ADSCs suggests that NOVA1 induced adipogenic gene in the process. Further differences in groups to the shNOVAl_A3 Scramble_A3 vs (pathway) for pathway enrichment analysis, found that the expression of Protein Response gene in Unfolded the most obvious difference, followed by PPAR.Western blot showed that the signal pathway involved in UPR signaling pathway was activated in NOVA1 gene knockdown; endoplasmic reticulum stress activated signal suppression ADSCs Adipogenic differentiation. In addition, the prediction of NOVA1 binding protein and IP results show that NOVA1 can combine with PTBP1 and ADSCs expression increased 7 days induced by PTBP1 knockdown of PTBP1 lipid, inhibit the adipogenic differentiation of ADSCs.4. MSCs into factors related to expression of NOVA1 gene in the differentiation of fat methods: different concentrations of Pilocarpine and NPY ADSCs, on the 1 day, the NOVA1 expression method of Real-time PCR by 3 days; different adipogenic component 10-6MDEX, 0.5mM IBMX, 10 M Insulin, 200 M indomethacin, 1 M rosiglitazone, completely antagonist GW9662 fat induced liquid and adding 10 M PPARy of indomethacin rosiglitazone, completely adipogenic liquid treatment ADSCs, in 1 days, 3 days, the NOVA1 expression of PCR by Real-time method for 7 days. Results: different concentrations of Pilocarpine and NPY, DEX and insulin on ADSCs in the NOVA1 table No effect of IBMX, NOVA1 in the 1 day of treatment induced increased expression of 3 days and 7 days, the effect was not obvious; indomethacin can significantly promote the expression of NOVA1 increased in the induction of 3 days and 7 days; and this effect will be inhibited by GW9662. The expression of a variety of RBPs 1.ADSCs in the conclusion of the study, in the process of adipogenic differentiation in hnRNPH1, PTBP1, hnRNPH2, RBM38, FOXC2, NOVA1 and RBPs expression changes, the most obvious difference in change in NOVA1, NOVA1 and ADSCs in osteoblast and myogenic differentiation was no significant change, indicating that NOVA1 expression of stem cell differentiation with lineage specific; the expression of NOVA in adipose tissue than in in the other tissues. It suggests that NOVA1 might play a specific role of.2.NOVA1 in adipose cell differentiation knockdown promotes ADSCs apoptosis, and inhibit their migration. Knockdown of NOVA1 inhibited ADSCs and BMSCs adipogenic differentiation process of adipogenic gene expression and lipid droplet formation, overexpression of N OVA1 will have a role in promoting, indicating that NOVA1 is involved in the regulation of adipogenic differentiation of MSCs; but the simple expression of NOVA1 could not activate MSCs adipogenic differentiation of.3.NOVA1 through effects on lipid metabolism related genes UPR and MSCs signal affects the adipogenic differentiation, and NOVA1 and PTBP1 may play a role as a complex.4.NOVA1 gene promoter PPARy response element in promoter region, PPARy ligand indomethacin and rosiglitazone can promote the expression of NOVA1, this effect can be PPAR gamma antagonist GW9662 inhibited the expression of PPARy signaling pathway involved in the regulation of NOVA1.

【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】：2017
【分类号】：R329.2

【相似文献】

相关期刊论文 前10条

1 陈茜;肖盼;陈剑;蔡小芳;蔡继业;;原子力显微镜对人羊膜间充质干细胞成脂分化的观察[J];生物技术通报;2010年06期

2 黄月琴;王松波;;骨髓间充质干细胞成肌和成脂分化的调控[J];中国生物化学与分子生物学报;2010年07期

3 张国华;屈长青;杨公社;;全反式维甲酸抑制猪脂肪间充质干细胞的成脂分化[J];中国生物化学与分子生物学报;2007年06期

4 潘志宏;韩宁;;P糖蛋白抑制对体外培养骨髓间充质干细胞成脂分化的影响[J];复旦学报(医学版);2010年04期

5 范正伟;张晾;潘杰;;TNF-α缺失小鼠前脂肪细胞成脂分化特征[J];济南大学学报(自然科学版);2013年03期

6 吴伟;季菊玲;王奎栋;李智耀;龚雨琴;季煜华;;去乙酰化酶抑制剂TSA对间充质干细胞C3H10T1/2增殖和成脂分化的影响[J];暨南大学学报(自然科学与医学版);2011年03期

7 王奎栋;李智耀;吴伟;龚雨琴;季煜华;;SAHA抑制间充质干细胞C3H10T1/2的增殖与成脂分化[J];中国生物化学与分子生物学报;2012年06期

8 王新鲁;姬云涛;屈长青;;Rho激酶抑制剂Y-27632对C3H10T1/2细胞增殖与成脂分化的作用[J];中国生物化学与分子生物学报;2013年07期

9 张金超;刘丹丹;孙静;张大威;申世刚;杨梦苏;;Dy~(3+)对小鼠骨髓基质细胞成骨和成脂分化及成骨细胞横向分化为脂肪细胞的影响[J];科学通报;2008年22期

10 龚雨琴;季煜华;李智耀;王奎栋;;去乙酰化酶抑制剂TSA通过激活ERK和p38抑制间充质干细胞成脂分化[J];中国生物化学与分子生物学报;2012年01期

相关会议论文 前4条

1 王玉;朱国英;王建平;李旭芳;翟江龙;;辐射对大鼠骨髓间充质干细胞增殖及向成骨和成脂分化的影响[A];中华医学会第七次全国骨质疏松和骨矿盐疾病学术会议论文汇编[C];2013年

2 刘丽忠;任茂文;黄凯;;人脂肪来源基质细胞原代培养和成脂分化[A];第四届华东六省一市整形外科学术会议暨2007年浙江省整形、美容学术会议论文汇编[C];2007年

3 邹玲玲;鲁红云;刘红;李玲玲;舒晓春;孙辽;谢丹红;;GLP-1对人脂肪干细胞增殖、成脂分化及脂代谢的影响[A];中华医学会第十二次全国内分泌学学术会议论文汇编[C];2013年

4 王平;杨光;李远栋;吴纯标;;荣筋片影响SAMP6鼠成骨-成脂分化关键调控因子的实验研究[A];第二十届全国中西医结合骨伤科学术研讨会、第二届中国医师协会中西医结合医师分会骨伤科学术年会、第十九届浙江省中西医结合骨伤科专业委员会学术年会论文汇编[C];2013年

相关重要报纸文章 前1条

1 记者 白毅;骨髓间充质干细胞成骨与成脂分化平衡调控机制研究获进展[N];中国医药报;2009年

相关博士学位论文 前10条

1 高红伟;NCoR在骨髓间充质干细胞成脂分化中分子调控机制的实验研究[D];山东大学;2016年

2 宋宝全;人间充质干细胞成脂分化调控机制的研究[D];北京协和医学院;2016年

3 李文凯;嘌呤受体P2X7和P2Y2在大鼠骨髓间充质干细胞成骨和成脂分化中的作用[D];华中科技大学;2016年

4 董平;RNA结合蛋白NOVA1调控间充质干细胞成脂分化的作用研究[D];北京协和医学院;2017年

5 贾兵兵;环腺苷酸信号通路调控人脂肪间充质干细胞成脂分化的作用研究[D];浙江大学;2012年

6 江书忠;猪脂肪沉积关键基因的筛选及锌指蛋白KLF13的功能研究[D];华中农业大学;2014年

7 李大虎;CKIP-1负控间充质干细胞成脂分化研究[D];中国人民解放军军事医学科学院;2012年

8 李海芳;β-肾上腺素受体在小鼠MSCs成骨、成脂分化中的作用[D];清华大学;2010年

9 肖静;p204在3T3-L1细胞成脂分化过程中的表达及功能研究[D];清华大学;2010年

10 胡丽;IGF1通过PPARγ-Axin2-Wnt信号通路促进内源性脂肪干/前体细胞成脂分化[D];华中科技大学;2014年

相关硕士学位论文 前10条

1 李晓敏;健脾活骨方不同部位对体外骨髓间充质干细胞分化调节作用的研究[D];中国中医科学院;2015年

2 葛志敏;甲状旁腺激素对大鼠骨髓间充质干细胞成脂分化的影响[D];山西医科大学;2015年

3 杜蕾蕾;极低频电磁场对MSCs的作用及其机制研究[D];南京大学;2013年

4 曹美霞;过表达RhoA基因对C3H10T1/2细胞成脂分化作用的研究[D];安徽大学;2016年

5 黄艳;基于生物信息技术构建成脂分化分子调控网络数据查询和分析平台[D];西北农林科技大学;2016年

6 叶勖;TLR2/TLR4活化相关微小RNA在人骨髓间充质干细胞迁移、分化中的作用[D];安徽医科大学;2016年

7 张小强;杜仲提取物5-羟甲基-2-糠醛诱导细胞成脂分化的信号途径研究[D];南京中医药大学;2016年

8 郜一飞;植物提取物Embelin对小鼠肥胖的影响及机制研究[D];天津医科大学;2016年

9 叶晶晶;胞外钙离子对猪BMSCs增殖和成脂分化的影响及作用机制[D];华南农业大学;2016年

10 梁媛;反-9,反-12-十八碳二烯酸对脂肪干细胞成脂分化的影响及其机制研究[D];大连工业大学;2016年

，

本文编号：1436554