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类型 基础研究 预答辩日期 2018-03-23
开始(开题)日期 2016-03-25 论文结束日期 2017-10-21
地点 东南大学丁家桥校区江苏省生物材料与器件重点实验室三楼会议室 论文选题来源 973、863项目     论文字数 7.37 (万字)
题目 氧化铁纳米颗粒协助促进间充质干细胞成骨分化效应研究
主题词 氧化铁纳米颗粒,间充质干细胞,成骨分化,基因组学;长链非编码RNAs
摘要 组织与器官的损伤后修复与功能重建是医学领域研究的重大课题,随着干细胞技术和生物材料科学的发展,再生医学与组织工程领域备受关注。组织工程通过将种子细胞引入受损部位,在支架材料的引导与支持下,促进缺损部位的再生与修复。间充质干细胞(Mesenchymal stem cells, MSCs)是一种多能干细胞,具有多向分化潜能,在不同的诱导条件下能够分化成多种间质细胞,如成骨细胞、脂肪细胞、成肌细胞等。MSCs存在于多种组织中,并且在组织的损伤修复过程中起着重要的作用,其中骨髓来源MSCs的成骨分化是骨缺损后修复重建过程中成骨细胞的重要来源。 已有大量研究报道磁场能促进骨损伤的修复,同时近年来国内外有关磁性骨支架材料的研究进展迅速,然而目前关于磁性材料是否能影响、如何影响MSCs的分化能力知之甚少。在本文的研究中,我们使用本课题组自主研发的、可用于临床的超顺磁性氧化铁纳米颗粒(Iron oxide nanoparticles, IONPs),研究其对人骨髓来源MSCs的作用及相关机理,期望为氧化铁纳米颗粒用于骨组织再生修复治疗的进一步研究提供指导。 具体来说,主要工作包括以下几个方面: (1) 氧化铁纳米颗粒对MSCs的毒性研究 发现氧化铁纳米颗粒与MSCs共孵育的72小时内,随着时间推进颗粒逐渐进入细胞,且主要分布于内吞体或溶酶体内;定量分析发现,MSCs对于氧化铁纳米颗粒的摄入具有时间依赖性和浓度依赖性。急性毒性方面,使用CCK-8实验检测孵育24小时后细胞活力,结果表明100、200和 400 μg/mL的氧化铁纳米颗粒使细胞活力分别下降了5.69%、7.45%和8.65%;长时毒性方面,100 μg/mL 氧化铁纳米颗粒孵育21天后乳酸脱氢酶(Lactic dehydrogenase, LDH)释放仅增加了2.87%,且细胞凋亡率维持在5%以下。 (2) 氧化铁纳米颗粒对MSCs作用的基因组学研究 基于前期细胞相容性实验结果,使用了100 μg/mL 氧化铁纳米颗粒与MSCs共孵育7天,对细胞基因组表达水平的变化进行基因芯片分析,并与目前已知数据库进行比对。检测出2092个上调的编码基因和1631个下调的编码基因,以此为据可以研究氧化铁纳米颗粒与MSCs的复杂作用机制。通过对表达水平显著变化的基因进行DAVID分析,得到了Gene Ontology基因功能注释和KEGG信号传导途径受到纳米材料的影响规律,也印证了基因表达差异是细胞对氧化铁纳米颗粒的应答调控过程。其中基因功能注释富集结果显示,氧化铁纳米颗粒可能会协助MSCs的成骨分化。 (3) 氧化铁纳米颗粒协助促进MSCs成骨分化的研究 不同浓度氧化铁纳米颗粒与MSCs共孵育,7至14天后测定细胞碱性磷酸酶(Alkaline phosphatase, ALP)活性,21天后对细胞外基质钙化结节进行染色,结果表明100 μg/mL 氧化铁纳米颗粒能够协助促进MSCs成骨分化。细胞形貌的扫描电镜(Scanning electron microscopy, SEM)与分化标志物免疫印迹实验进一步证实了这一效应。此外,通过与铁离子和包覆材料PSC的对照,结果显示这种促进作用来自于完整的颗粒性质。在KEGG信号通路的分析中,发现了MAPK(Mitogen-activated protein kinase)信号通路可能是影响氧化铁纳米颗粒协助成骨分化效应的重要因素,并对此通路的激活及与成骨分化的关系进行了研究,发现氧化铁纳米颗粒能够激活MAPK信号通路从而促进MSCs成骨分化。 (4) 长链非编码RNAs参与氧化铁纳米颗粒协助MSCs成骨分化研究 进一步使用长链非编码(Long non-coding RNAs, lncRNAs)芯片分析了氧化铁纳米颗粒协助促进MSCs成骨分化后lncRNAs表达变化,发现了411个上调的lncRNAs和605个下调的lncRNAs。对差异lncRNAs和mRNAs的共表达分析,并分析了对显著差异的共表达mRNAs的DAVID信号通路,发现lncRNAs可能主要通过影响RTKs/MAPK 和BMPs/Smads信号通路、肌动蛋白重排和细胞磁场感知而参与氧化铁纳米颗粒促进的MSCs成骨分化。 (5) LncRNA-INZEB2调控氧化铁纳米颗粒协助成骨分化效应的机制研究 验证了芯片结果中差异表达显著的lncRNAs的表达水平,发现了lncRNA-LOC105373660的表达随着孵育时间逐渐增加,序列比对后发现其转录本位于ZEB2基因2号内含子的反向重复序列,故将其命名为INZEB2。降低它的表达会明显抑制氧化铁纳米颗粒的协助促进成骨分化效应,这说明INZEB2的存在对于维持氧化铁纳米颗粒促进的成骨表型至关重要。ZEB2是抑制Smads激活的成骨分化的重要蛋白,继续证实了下调ZEB2可以恢复降低INZEB2所抑制的成骨分化,证明INZEB2是通过ZEB2调控氧化铁纳米颗粒促进的MSCs成骨分化。进一步地,发现; INZEB2会通过影响ZEB2转录本的可变剪切抑制ZEB2蛋白表达水平,从而重新激活被ZEB2所抑制的Smads依赖的成骨分化。
英文题目 Osteogenic differentiation facilitation effect of iron oxide nanoparticles in mesenchymal stem cells
英文主题词 Iron oxide nanoparticles, Mesenchymal stem cells, Osteogenic differentiation, Genomics, long non-coding RNAs, Nanobiological effects.
英文摘要 Regeneration and functional reconstruction of injured tissues and organs in one of the major issues in biomedical research, regenerative medicine and tissue engineering attracts much concerns with the progress in stem cells sciences and biomaterials technologies. Tissue engineering can promote the regeneration and repair of the defect site by introducing the seed cells into the damaged parts under the guiding and supporting of scaffold biomaterials. MSCs is a kind of pluripotent stem cell with multiple differentiation potential, which can be differentiated into many stromal cells under different induce conditions, such as osteoblasts, adiocytes, myocytes, et al. MSCs exist in multiple tissues and play an important role during the tissue damage repair process. Among them, bone marrow derived MSCs is an important source of osteoblasts in bone reconstruction process. Recently, many studies have reported that magnetic field can facilitate the repair of bone injury, meanwhile, research progress of magnetic bone scaffold materials developed rapidly. However, whether magnetic materials can affect or how they influence MSCs differentiation ability are poorly understand. In this dissertation, we employed the clinical approved IONPs developed by our research group to study the nanobiological effects to human bone marrow derived MSCs and the associated mechanisms, which is expect to provide guidance for the further study of IONPs in the treatment of bone tissue regeneration. In detail, main work in the dissertation including these points: (1) Cytotoxicity study of IONPs in MSCs IONPs was gradually uptaken by cells within the co-incubation of 72 hours, and mainly distributed in the endosomes or lysosomes. The uptake of IONPs was both time- and dose-depent after quantitative analysis. For acute cytotoxicity, CCK-8 assay was performed to determine the cell viability after 24 hours co-incubation, cell viability decreased by 5.69%, 7.45% and 8.65% respectively under the concentration of 100, 200 and 400 μg/mL. For long-term cytotoxicity, the LDH leakage increased only by 2.87%, and the apoptotic rates were below 5% after co-incubation of 21 days. (2) Genomics study of IONPs interaction with MSCs The variation of gene expression in IONPs-treated cells was analyzed by employing gene microarray tests. After blasting to the known database, 2092 up-regulated coding genes and 1631 down-regulated coding genes were found, which was the bases to study the complex interaction mechnisms between IONPs and MSCs. After bioinformatics analysis, the influence rules of gene functional annotations and KEGG signaling transduction pathways were obtained, which showed IONPs might promote osteogenic differentiation of MSCs. (3) IONPs promote osteogenic differentiation of MSCs IONPs with different concentration were co-incubate with MSCs. The results showed 100 μg/mL IONPs can improve the cellular ALP activity after 7 to 14 days, and the extracellular matrix calcification nodules can stained red after 21 days. SEM detection was further performed to observe the morphological variation, and western blot to detect the differentiation marker, which confirmed the osteogenic differentiation facilitation effect of IONPs. Moreover, by contrast with the ferric ion and coating material PSC, this facilitation effect was owing to the whole particle. MAPK signaling pathway is enriched in KEGG pathways analysis, which suggest this pathway might be a potential key factor in regulating IONPs-promoted osteogenic differentiation. After detection of phosphorylated kinases, results suggested that this pathway was activated during IONPs treatment, and then subsequently promote osteogenic genes transcription. (4) Participation of lncRNAs in IONPs-promoted osteogenic differentiation LncRNAs microarray was performed to analyze the variation of lncRNAs expression in IONPs-treated cells, and 411 up-regulated lncRNAs and 605 down-regulated lncRNAs were identified. To obtain more comprehensive information about lncRNAs regulation in IONPs-promoted osteogenic differentiation for MSCs, the coding-noncoding co-expression network was constructed. After functional annotation of co-expressed coding genes, it could be concluded that lncRNAs would influence RTKs/MAPK pathway, BMPs/Smads pathway, magnetic response and cytoskeleton rearrangement to regulate osteogenic differentiation via their co-expressed coding genes. (5) Mechanism of INZEB2 in regulating IONPs-promoted osteogenic differentiation Among the differentially expressed lncRNAs, LOC105373660 was up-regulated during IONP exposure. Using the sequence and genomic location for BLAST searches, the putative transcript is transcribed from the partial opposite strand of ZEB2’s intron 2, therefore, it was named for INZEB2 (intronic ZEB2). Meanwhile, knockdown of INZEB2 inhibited the osteogenesis phenotype of IONPs treated MSCs, and caused a significant increase of ZEB2 protein level which was reduced by IONPs treatment. Furthermore, we demonstrated that INZEB2 interacted with the intron 2 of ZEB2 pre-mRNA which leading to the splicing of its complementary sequence within this intron. As a result, a premature translation termination occurred and viable ZEB2 protein level was reduced. Therefore, TGF-β/BMPs/Smads-mediated osteogenic differentiation was reactivated. Taken together, these results illustrated INZEB2 responds to IONPs and modulates ZEB2 expression by influencing its pre-mRNA splicing, which results in the facilitation of osteognenic differentiation in MSCs.
学术讨论
主办单位时间地点报告人报告主题
东南大学生物与医学纳米技术研究组 2014.11.8 江苏省生物材料与器件重点实验室 王琪炜 非编码RNA及其调控方式
东南大学生物与医学纳米技术研究组 2015.9.19 江苏省生物材料与器件重点实验室 王琪炜 Study on biological effects of nano-materials based on gene expression profile analysis
东南大学生物与医学纳米技术研究组 2015.10.28 江苏省生物材料与器件重点实验室 王琪炜 Ferumoxytol促进间充质干细胞成骨分化及潜在的后续应用
东南大学生物与医学纳米技术研究组 2015.11.7 江苏省生物材料与器件重点实验室 王琪炜 Controlling stem cells for regeneration
东南大学生物与医学纳米技术研究组 2016.9.24 江苏省生物材料与器件重点实验室 王琪炜 Magnetoreceptors-----Bases for magnetic perception
南京市第一医院 2016.11.9 南京市第一医院骨科实验室 王琪炜 磁性纳米颗粒应用于骨修复的研究
中科院大连化学物理研究所 2017.4.14 大连化物所1810组 王琪炜 基于蛋白质组学的磁生物效应----相关基础及科学问题
东南大学生物与医学纳米技术研究组 2017.5.10 扬州大学比较医学中心 王琪炜 生物磁感应与量子效应
     
学术会议
会议名称时间地点本人报告本人报告题目
中国干细胞第五届年会 2015.11.13 重庆,中国 Response of MAPK pathway to magnetic nanoparticles in vitro treatment promotes osteogenic differentiation of hBMSCs
The 16th International Conference on Biomedical Engineering 2016.12.8 新加坡 Whole genome expression analysis reveals osteogenic differentiation promotion effect of iron oxide nanoparticles on mesenchymal stem cells
2017中国生物医学工程大会 2017.4.20 北京,中国
     
代表作
论文名称
Response of MAPK pathway to iron oxide nanoparticles in vitro treatment promotes osteogenic
Magnetic iron oxide nanoparticles accelerate osteogenic differentiation of mesenchymal stem cells vi
 
答辩委员会组成信息
姓名职称导师类别工作单位是否主席备注
华子春 正高 教授 博导 南京大学
胡勤刚 正高 教授 博导 南京市口腔医院
冯继锋 正高 教授 博导 江苏省肿瘤医院
章非敏 正高 教授 博导 南京医科大学
张宇 正高 教授 博导 东南大学
      
答辩秘书信息
姓名职称工作单位备注
张淼 其他 讲师 东南大学