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。
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。
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。
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。
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。
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.