硅化超顺磁氧化铁纳米颗粒标记人羊膜间充质细胞的效率及其对细胞增殖的影响

旨在探讨一种新型硅化超顺磁性氧化铁纳米颗粒标记人羊膜间充质细胞的最佳方法, 并检测其对细胞增殖的影响. 用不同浓度的Si-SPIO和多聚赖氨酸混合制备PLL-Si-SPIO复合物, 标记体外培养的hAMCs. 利用普鲁士蓝染色和透射电子显微镜等方法对Si-SPIO的标记情况进行分析鉴定. 分析Si-SPIO标记后1~4周铁颗粒在细胞内的维持与稳定. 应用MTS分析法探讨经Si-SPIO标记后hAMCs的增殖活性. Si-SPIO标记后的hAMCs移植到小鼠纹状体内1周, 鉴定Si-SPIO阳性细胞的存活与分布. 观察发现, hAMCs经Si-SPIO标记后细胞内可检测到大量铁颗粒, 铁颗粒能在细胞内维持4周以上. Si-SPIO标记具有浓度依赖性, 最适浓度为20 µg/mL; 较低浓度的Si-SPIO对细胞增殖活力没有显著影响. 移植到小鼠脑内1周后可见Si-SPIO阳性细胞. 结果可知, 浓度为20 µg/mL的Si-SPIO标记hAMCs可获得良好的标记效果, 并且不影响细胞的增殖活力.     

荧光磁性纳米粒子标记间充质干细胞靶向胃癌研究

目的:探索一种高效的早期胃癌诊断体系.方法:荧光磁性纳米粒子与间充质干细胞共培养,不同时间点检测干细胞存活率.利用荧光显微镜、普鲁士蓝染色、透射电子显微镜等方法观测干细胞被标记情况;建立裸鼠的胃癌模型,并将标记后的干细胞尾静脉注射入裸鼠体内,14d后用动物成像仪和核磁共振成像仪检测.结果:荧光磁性纳米粒子在低于 100μg/mL浓度下对间充质干细胞没有毒性作用,荧光磁性纳米粒子与干细胞共培养6h后,荧光显微镜、普鲁士蓝染色、透射电子显微镜检测结果显示粒子被内吞后定位于细胞质中,动物实验显示通过检测肿瘤部位的荧光信号和核磁信号,被标记的干细胞能够准确定位到胃癌的发生部位.结论:这种双模式检测体系为胃癌的早期诊断与治疗提供一种新的方法.     

纳米铁标记骨髓间质干细胞的MR研究

探讨超顺磁性氧化铁纳米粒子(Superparamagnefic iron oxide,SPIO)体外标记大鼠骨髓间质干细胞(mesenchymal stem cells,MSCs)的磁共振(magnetic resonance,MR)的成像特征.选取第5代细胞进行SPIO标记,其标记浓度为28 mg/L,选取不同的标记细胞数量,使用1.5 TMR进行T1WISE、T2WIFSE、T2WFGR扫描,测量不同扫描序列标记细胞管的信号强度改变,并进行统计学分析.细胞标记率为92%,细胞存活率为97%,MR成像显示,随着细胞数量的增多,标记细胞信号呈线性减低趋势.MR成像能敏感地显示SPIO标记的骨髓间质干细胞.     

骨髓间充质干细胞可塑性研究

骨髓间充质干细胞（bone marrow mesenchymal stem cells,MSCs）具有跨系统、甚至跨胚层分化的特性,称为MSCs的可塑性（plasticity）,成为细胞工程、再生医学中的主要选择细胞。但随着对成体干细胞可塑性质疑的出现,使MSCs是否具有转分化能力,存在着极大的分歧。对MSCs可塑性是否真正存在的进一步明确,越加显得急切和重要,也对干细胞基础理论及其临床应用具有指导性意义。     

MRI体外示踪SPIO标记人脐带间充质干细胞的实验研究

目的:研究超顺磁性纳米铁颗粒(superparamagnetic ironoxide particles,SPIO)体外标记人脐带间充质干细胞(HuCMScs)及MRI成像示踪的可行性.方法:从人胎儿脐带中分离培养、扩增脐带间充质干细胞(HUCMSCs),分别采用0 μg/ml,25μg/ml,50μg/ml浓度的SPIO标记0.5×106,1×106,2×106和10×106 HUCMSCs.普鲁士蓝染色和透射电镜鉴定细胞内铁颗粒情况,并用3.0T MR/离体扫描T1WI,T2WI,GRE/300序列成像,测定细胞群信号.结果:不同数量的HUCMSCs与0 μg/ml,25 μg/ml,50 μg/ml浓度的SP10共同培养18小时,普鲁士蓝染色发现标记的细胞随标记浓度的升高染色程度逐渐加深.透射电镜检查显示细胞内含致密铁颗粒.离体MRI不同序列测定不同浓度SPIO标记相同数量的细胞群,GRE/30°和T2WI测定的各组之间均有统计学差别,501μg/ml与25μg/ml组分别与0μg/ml组之间有显著统计学差别(P<0.05);同一浓度SPIO标记人脐带间充质干细胞,信号强度与细胞数量有关(P<0.05).结论:SPIO可以标记人脐带间充质干细胞,应用MRI可以对其进行体外示踪和监测.     

HLA-G参与骨髓间充质干细胞的抑制作用机制探讨

目的：探讨人骨髓间充质干细胞（MSC）在活体肾移植供受者间的混合淋巴细胞培养（MLR）中的作用机制,证明人白细胞抗原（HLA）-Ⅰ类分子HLA-G参与了MSC的免疫调节作用。方法：从人骨髓中分离培养MSC,采用形态学观察及流式细胞术分析鉴定后,加入活体肾移植供受者间的MLR,观察MSC的HLA-G表达及对T细胞增殖的影响,淋巴细胞增殖实验采用MTT法。结果：MSC细胞表面和胞浆内均表达HLA-G,流式细胞术分析细胞表面HLA-G平均表达率为37.3%,胞浆内HLA-G平均表达率为65.1%,ELISA法检测细胞培养上清中可溶性HLA-G含量为18.9ng/mL。将MSC和培养上清液加入活体肾移植供受者间的MLR体系中,均使得T细胞抑制率增高;而加入HLA-G特异性抗体,MSC的抑制率降低。结论：MSC表面和胞浆内均有HLA-G表达,在其培养上清中检测到由MSC分泌的可溶性HLA-G5;MSC表达和分泌的HLA-G是其发挥抑制功能的关键因素之一。为MSC在预防器官移植术后排斥反应的临床应用提供了理论依据。     

骨髓间充质干细胞的研究进展

骨髓间充质干细胞是存在于骨髓中的具有高度自我更新能力和多向分化潜能的干细胞群体 ,具有支持造血、多向分化潜能以及在细胞和基因工程中具有潜在应用前景等特点 ,将在医学上具有重要的临床应用价值。     

SD大鼠骨髓间充质干细胞钙离子波动的探究

本实验旨在通过化学药物促进或抑制骨髓间充质干细胞(BMMSCs)增殖以研究胞内Ca2+浓度在此过程中的变化规律.同步化于G1期的MSCs分别用10%胎牛血清(FBS)、15 ng/mL表皮生长因子(EGF)做短期(1 h)或持续(32 h)刺激,或用10 μg/mL丝裂霉素C(Mi C)短期(1 h)刺激和刺激2.5 h后除去,检测胞内Ca2+浓度变化.结果 表明在增殖相关的化学信号瞬时刺激下,MSCs胞内Ca2+信号瞬时增高,然后回复到一个稳态水平:促增殖时维持高稳态水平,抑增殖则低;持续的抑制导致胞内Ca2+波动弱.     

人VEGF基因转染大鼠骨髓间充质干细胞的实验研究

为建立hVEGF165基因转染大鼠间充质干细胞的方法．采用密度梯度离心-贴壁培养法获Wistar大鼠BMMSC,并测定其生长曲线和表面标志CD34、CD44、CD45及SH3,然后向成骨细胞及脂肪细胞诱导分化;用脂质体介导pcDNA3.1-hVEGF165转染BMMSC,观察转染后细胞形态和生长情况的变化,通过RT-PCR、Western和ELISA鉴定VEGF在细胞中的表达情况．经培养的大鼠BMMSC,CD44、SH3检测为阳性．CD45、CD34阴性,可诱导分化为成骨细胞和脂肪细胞;经RT-PCR、Western和ELISA检测证实阳离子脂质体能成功地将hVEGF165基因转染至大鼠BMMSC中,并获得有效的表达．真核表达栽体pcDNA3.1-hVEGF165在BMMSC中获有效表达,为VEGF基因转染BMMSC移植对心梗后大鼠心功能及心室重构的影响提供了实验依据．     

骨髓间充质干细胞向神经细胞分化的研究

无论是在体外实验、还是在体内实验,MSCs都可以向中枢神经系统(CNS)神经细胞分化,但争议颇多。因为功能性神经元不仅要具有典型神经元的形态、特异性标记,还要求具有可兴奋性、能和其他神经元形成突触联系、产生突触电位等,所以对于骨髓间充质干细胞是否能诱导出真正具有功能的神经元存在很大分歧。在此对MSCs向神经细胞诱导分化研究的现况、存在的问题及发展前景给以综述。     

Dose-Response of Superparamagnetic Iron Oxide Labeling on Mesenchymal Stem Cells Chondrogenic Differentiation: A Multi-Scale In Vitro Study

Aim

The aim of this work was the development of successful cell therapy techniques for cartilage engineering. This will depend on the ability to monitor non-invasively transplanted cells, especially mesenchymal stem cells (MSCs) that are promising candidates to regenerate damaged tissues.

Methods

MSCs were labeled with superparamagnetic iron oxide particles (SPIO). We examined the effects of long-term labeling, possible toxicological consequences and the possible influence of progressive concentrations of SPIO on chondrogenic differentiation capacity.

Results

No influence of various SPIO concentrations was noted on human bone marow MSC viability or proliferation. We demonstrated long-term (4 weeks) in vitro retention of SPIO by human bone marrow MSCs seeded in collagenic sponges under TGF-β1 chondrogenic conditions, detectable by Magnetic Resonance Imaging (MRI) and histology. Chondrogenic differentiation was demonstrated by molecular and histological analysis of labeled and unlabeled cells. Chondrogenic gene expression (COL2A2, ACAN, SOX9, COL10, COMP) was significantly altered in a dose-dependent manner in labeled cells, as were GAG and type II collagen staining. As expected, SPIO induced a dramatic decrease of MRI T2 values of sponges at 7T and 3T, even at low concentrations.

Conclusions

This study clearly demonstrates (1) long-term in vitro MSC traceability using SPIO and MRI and (2) a deleterious dose-dependence of SPIO on TGF-β1 driven chondrogenesis in collagen sponges. Low concentrations (12.5–25 µg Fe/mL) seem the best compromise to optimize both chondrogenesis and MRI labeling.     

Dose Dependent Side Effect of Superparamagnetic Iron Oxide Nanoparticle Labeling on Cell Motility in Two Fetal Stem Cell Populations

Multipotent stem cells (SCs) could substitute damaged cells and also rescue degeneration through the secretion of trophic factors able to activate the endogenous SC compartment. Therefore, fetal SCs, characterized by high proliferation rate and devoid of ethical concern, appear promising candidate, particularly for the treatment of neurodegenerative diseases. Super Paramagnetic Iron Oxide nanoparticles (SPIOn), routinely used for pre-clinical cell imaging and already approved for clinical practice, allow tracking of transplanted SCs and characterization of their fate within the host tissue, when combined with Magnetic Resonance Imaging (MRI). In this work we investigated how SPIOn could influence cell migration after internalization in two fetal SC populations: human amniotic fluid and chorial villi SCs were labeled with SPIOn and their motility was evaluated. We found that SPIOn loading significantly reduced SC movements without increasing production of Reactive Oxygen Species (ROS). Moreover, motility impairment was directly proportional to the amount of loaded SPIOn while a chemoattractant-induced recovery was obtained by increasing serum levels. Interestingly, the migration rate of SPIOn labeled cells was also significantly influenced by a degenerative surrounding. In conclusion, this work highlights how SPIOn labeling affects SC motility in vitro in a dose-dependent manner, shedding the light on an important parameter for the creation of clinical protocols. Establishment of an optimal SPIOn dose that enables both a good visualization of grafted cells by MRI and the physiological migration rate is a main step in order to maximize the effects of SC therapy in both animal models of neurodegeneration and clinical studies.     

Effect of Labeling with Iron Oxide Particles or Nanodiamonds on the Functionality of Adipose-Derived Mesenchymal Stem Cells

Stem cells are increasingly the focus of translational research as well as having emerging roles in human cellular therapy. To support these uses there is a need for improved methods for in vivo cell localization and tracking. In this study, we examined the effects of cell labeling on the in vitro functionality of human adipose-derived mesenchymal stem cells. Our results provide a basis for future in vivo studies investigating implanted cell fate and longevity. In particular, we investigated the effects of two different particles: micron-sized (∼0.9 µm) fluorescently labeled (Dragon Green) superparamagnetic iron oxide particles (M-SPIO particles); and, carboxylated nanodiamonds of ∼0.25 µm in size. The effects of labeling on the functionality of adipose-derived MSCs were assessed by in vitro morphology, osteogenic and adipogenic differentiation potential, CD marker expression, cytokine secretion profiling and quantitative proteomics of the intra-cellular proteome. The differentiation and CD marker assays for stem-like functionality were not altered upon label incorporation and no secreted or intra-cellular protein changes indicative of stress or toxicity were detected. These in vitro results indicate that the M-SPIO particles and nanodiamonds investigated in this study are biocompatible with MSCs and therefore would be suitable labels for cell localization and tracking in vivo.     

Superparamagnetic Iron Oxide Nanoparticles Labeling of Bone Marrow Stromal (Mesenchymal) Cells Does Not Affect Their “Stemness”

Superparamagnetic iron oxide nanoparticles (SPION) are increasingly used to label human bone marrow stromal cells (BMSCs, also called “mesenchymal stem cells”) to monitor their fate by in vivo MRI, and by histology after Prussian blue (PB) staining. SPION-labeling appears to be safe as assessed by in vitro differentiation of BMSCs, however, we chose to resolve the question of the effect of labeling on maintaining the “stemness” of cells within the BMSC population in vivo. Assays performed include colony forming efficiency, CD146 expression, gene expression profiling, and the “gold standard” of evaluating bone and myelosupportive stroma formation in vivo in immuncompromised recipients. SPION-labeling did not alter these assays. Comparable abundant bone with adjoining host hematopoietic cells were seen in cohorts of mice that were implanted with SPION-labeled or unlabeled BMSCs. PB+ adipocytes were noted, demonstrating their donor origin, as well as PB+ pericytes, indicative of self-renewal of the stem cell in the BMSC population. This study confirms that SPION labeling does not alter the differentiation potential of the subset of stem cells within BMSCs.     

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe₃O₄) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.     

间充质干细胞过滤分离器制备人羊膜间充质干细胞的研究

自然存在的间充质干细胞数量少,限制了其研究应用。依靠自主发明的间充质干细胞过滤分离器,分离制备了人羊膜间充质干细胞,并对制备的干细胞进行了三维培养扩增。结果表明,制备的干细胞形态长势良好,并能诱导分化为类胰岛样组织。与常规方法相比,干细胞收获率提高了8倍以上,且细胞活性状态良好。间充质干细胞过滤分离器可以批量制备高质量的各种间充质干细胞,有利于高效率地建设各种间充质干细胞库,以促进间充质干细胞的研究应用。     

Novel Positively Charged Nanoparticle Labeling for In Vivo Imaging of Adipose Tissue-Derived Stem Cells

Stem cell transplantation has been expected to have various applications for regenerative medicine. However, in order to detect and trace the transplanted stem cells in the body, non-invasive and widely clinically available cell imaging technologies are required. In this paper, we focused on magnetic resonance (MR) imaging technology, and investigated whether the trimethylamino dextran-coated magnetic iron oxide nanoparticle -03 (TMADM-03), which was newly developed by our group, could be used for labeling adipose tissue-derived stem cells (ASCs) as a contrast agent. No cytotoxicity was observed in ASCs transduced with less than 100 µg-Fe/mL of TMADM-03 after a one hour transduction time. The transduction efficiency of TMADM-03 into ASCs was about four-fold more efficient than that of the alkali-treated dextran-coated magnetic iron oxide nanoparticle (ATDM), which is a major component of commercially available contrast agents such as ferucarbotran (Resovist), and the level of labeling was maintained for at least two weeks. In addition, the differentiation ability of ASCs labeled with TMADM-03 and their ability to produce cytokines such as hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2), were confirmed to be maintained. The ASCs labeled with TMADM-03 were transplanted into the left kidney capsule of a mouse. The labeled ASCs could be imaged with good contrast using a 1T MR imaging system. These data suggest that TMADM-03 can therefore be utilized as a contrast agent for the MR imaging of stem cells.     

磁纳米颗粒标记脂肪干细胞向软骨分化及体外MRI示踪的实验研究

目的:探讨磁纳米颗粒(magnetic iron oxide particles,MIOP)体外标记脂肪间充质干细胞(ASCs)向软骨分化及MRI示踪的可行性。方法:从小鼠脂肪组织中分离培养、扩增脂肪间充质干细胞(ASCs),流式鉴定细胞表型后,分别采用不同浓度(25μg/mL,50μg/mL)的MIOP标记ASCs并向软骨细胞诱导分化。普鲁士蓝染色和透射电镜(TEM)鉴定细胞内磁纳米铁颗粒分布情况,应用3.0T MRI体外检测标记软骨细胞MRI信号。结果:从脂肪组织中可以分离获得大量高表达CD90、CD105、Sca-1的ASCs,不同浓度(25μg/mL,50μg/mL)的MIOP与ASCs共同孵育24小时后,普鲁士蓝染色发现ASCs随MIOP浓度的增加,蓝染程度逐渐加深且标记的ASCs可以向软骨细胞分化;TEM证实细胞内分布大量的黑色纳米铁颗粒。体外MRI T2序列证实随着MIOP浓度(25μg/mL,50μg/mL)的增加MRI信号值逐渐减低且具有统计学差异(P0.05)。结论:MIOP可以标记ASCs向软骨分化,体外应用MRI可以对其进行示踪。     

Efficient In Vitro Labeling Rabbit Bone Marrow-Derived Mesenchymal Stem Cells with SPIO and Differentiating into Neural-Like Cells

Mesenchymal stem cells (MSCs) can differentiate into neural cells to treat nervous system diseases. Magnetic resonance is an ideal means for cell tracking through labeling cells with superparamagnetic iron oxide (SPIO). However, no studies have described the neural differentiation ability of SPIO-labeled MSCs, which is the foundation for cell therapy and cell tracking in vivo. Our results showed that bone marrow-derived mesenchymal stem cells (BM-MSCs) labeled in vitro with SPIO can be induced into neural-like cells without affecting the viability and labeling efficiency. The cellular uptake of SPIO was maintained after labeled BM-MSCs differentiated into neural-like cells, which were the basis for transplanted cells that can be dynamically and non-invasively tracked in vivo by MRI. Moreover, the SPIO-labeled induced neural-like cells showed neural cell morphology and expressed related markers such as NSE, MAP-2. Furthermore, whole-cell patch clamp recording demonstrated that these neural-like cells exhibited electrophysiological properties of neurons. More importantly, there was no significant difference in the cellular viability and [Ca²⁺]i between the induced labeled and unlabeled neural-like cells. In this study, we show for the first time that SPIO-labeled MSCs retained their differentiation capacity and could differentiate into neural-like cells with high cell viability and a good cellular state in vitro.