大鼠肝移植术后受体骨髓间充质干细胞输注肝内示踪与标记方法比较

目的采用羧基荧光素二醋酸盐琥珀酰亚胺酯（carboxyfluorescin diacetate succinimidyl ester,CFSE）与溴化脱氧尿嘧啶核苷（5-bromo-2-deoxyuridine,Brdu）两种细胞标记物观测肝移植术后受体骨髓间充质干细胞（MSCs）肝内分布情况,并对两种方法进行比较。方法在已构建的DA-Lewis大鼠原位肝移植模型及提取培养Lewis大鼠MSCs的基础上分别用CFSE和Brdu对Lewis来源的MSCs进行标记．术中经门静脉输注．通过荧光显微镜和免疫组织化学法观测术后2mo内各时间点受体肝组织内MSCs的分布情况。结果CFSE细胞标记率约为（94．1±1．4）％。受体肝组织第1、7、14天内有CFSE标记的MSCs聚集,对照组第5天肝组织内发布的CFSE标记MSCs消失。Brdu细胞标记率约为（90．3±3．5）％。受体肝组织第14天、1个月、2个月可见Brdu阳性的MSCs分布,对照组相应时间点肝组织内未发现Brdu阳性的MSCs。结论CFSE和Brdu均为MSCs的良好标记物。CFSE标记细胞后荧光迅速减弱,适用于短期示踪。Brdu标记细胞后可维持较长时间．适用于长期示踪。两种标记方法均是MSCs较为简单有效的方法,实验显示受体MSCs大部分分布于移植肝脏。     

绿色荧光蛋白转基因小鼠来源肌卫星细胞的体外培养及体内示踪

目的:从绿色荧光蛋白转基因小鼠中分离培养肌卫星细胞(MSCs)并进行体内示踪.方法:利用差速贴壁结合克隆分选方法,分离了MSCs,并于体外进行培养传代、鉴定及分化.检测所获MSCs的生长曲线及细胞周期并与来源于野生型鼠的同代MSCs进行比较.将绿色荧光蛋白标记的MSCs注射到裸鼠胫前肌,于注射后当时、注射后1周、2周、3周和4周利用二维荧光成像平台进行体内示踪.结果:MSCs被成功分离、传代及鉴定.来源于绿色荧光蛋白(GFP)标记或未标记小鼠MSCs的生长曲线、细胞周期及肌原性分化等无差别.MSCs注射后4周内可以动态观察到注射部位的绿色荧光信号并获得组织学证实.结论:来源于GFP转基因小鼠的MSCs在生长和增殖特性上与未转基因来源的MSCs相似,在体内可以通过二维荧光成像平台进行可靠的、无创性的示踪.     

PKH26标记大鼠骨髓间质干细胞的实验研究

目的建立PKH26标记大鼠骨髓间质干细胞（MSCs）的方法，并探讨标记MSCs的基本生物学活性。方法大鼠MSCs按PKH26标记程序进行标记后培养，采用激光共聚焦显微镜、流式细胞分析仪等观察细胞生长状态和荧光标记活性变化；应用RT-PCR检测标记后MSCs的GAPDH、nucleostemin及Bmi-1基因的表达；选用碱性磷酸酶染色、Von Kossa染色及骨形成蛋白3（BMP3）基因表达分析等技术，观察标记后MSCs体外分化成骨细胞的特性。结果PKH26标记后细胞呈红色荧光，荧光的强度随着PKH26浓度的增加而递增，并与传代时间和代数相关；标记后细胞生长状态良好，基本生长特性如传代培养和生长曲线无明显改变；细胞GAPDH、nucleostemin及Bmi-1基因表达未见改变；标记MSCs诱导后ALP、Von Kossa染色阳性，呈现典型的成骨细胞形态和生物学特征，并表达BMP3基因。结论PKH26可稳定标记大鼠骨髓MSCs并能传代培养，标记后细胞形态、生长活力及多向分化潜能等无明显影响，该技术可用于追踪MSCs转归、可塑性及干细胞移植方面的实验研究。     

微环境对体外间充质干细胞向心肌样细胞分化的作用

目的研究心肌组织中何种细胞对骨髓间充质干细胞(MSCs)向心肌样细胞定向分化起决定性作用。方法分离培养骨髓MSCs、心肌细胞(CM)和内皮细胞(EC)。MSCs经BrdU标记后与CM、EC分别共培养和单独培养,通过形态学、免疫细胞化学和双重免疫细胞化学鉴定。结果分离培养的MSCs、CM和EC纯度高于95%。标记MSCs与CM共培养后,细胞具有心肌细胞样形态。4周时,有44%的细胞BrdU与肌动蛋白(sarcomeric actin)或连接蛋白-43(connexin-43)双重免疫细胞化学染色阳性。与EC共培养以及单独培养时,MSCs形态变化不明显,无双染阳性细胞出现。结论CM对骨髓MSCs向心肌样细胞的定向分化具有重要作用。     

移植骨髓间充质干细胞减轻脂多糖诱导的小鼠急性肺损伤

目的 观察移植骨髓间充质干细胞 （MSCs） 对脂多糖 （LPS） 诱导小鼠急性肺损伤 （ALI）的治疗修复作用。方法 全骨髓培养法培养小鼠骨髓MSCs;细胞免疫化学染色鉴定MSCs特异表面标记;小鼠咽后壁吸入LPS制造小鼠肺损伤;尾静脉注射引入MSCs;称重计算肺水肿指数;肺组织切片HE染色观察组织病理改变;ELISA检测肺泡灌洗液和肺组织匀浆中IL-1β含量;Brdu（5-Bromo-2-Deoxyuridine）标记供体MSCs,免疫组织化学染色及双染色观察移植细胞的迁移和分化状态。结果 培养的MSCs细胞表面标记CD44阳性,而造血系表面标记CD34阴性。吸入LPS后,小鼠出现典型的肺损伤病理改变,肺水肿指数和肺组织匀浆IL-1β含量明显增加。标记的MSCs移植入同种异体的肺损伤小鼠,其肺部出现标记的MSCs,并表达上皮细胞标志抗原-细胞角蛋白（CK）。治疗后小鼠的肺水肿指数和肺组织匀浆IL-1β含量下降。结论 外源性MSCs移植到肺损伤小鼠体内,可迁移至肺损伤部位,并表达上皮细胞标志;减轻肺水肿程度,减少炎症因子释放。     

神经生长因子转染骨髓间充质干细胞诱导分化成神经样细胞

目的探讨神经生长因子(NGF)转染大鼠骨髓间充质干细胞(MSCs)后的表达及其对MSCs分化成神经样细胞的影响。方法在体外低密度扩增大鼠骨髓MSCs。应用基因重组技术,构建pEGFP-NGF真核表达质粒,并将其转染至MSCs中。免疫荧光标记法检测中间神经丝蛋白(NF-M)和胶质纤维酸性蛋白(GFAP)的表达。在荧光显微镜下观察MSCs阳性荧光的表达率、细胞的形态变化和类型。结果MSCs阳性荧光的表达率约为30%。转染后,MSCs呈现多突起,且几个突起之间互相连接成网状,似神经元样的形态。免疫荧光标记法鉴定其中一部分细胞表达NF-M,另一部分细胞表达GFAP。结论转染的MSCs可表达NGF并在含有NGF的微环境中分化成神经样细胞。     

超顺磁性氧化铁纳米粒子和CM-DiI荧光双标记骨髓间充质干细胞

背景:细胞移植疗效监测取决于有效的标记方法,从而能够对移植细胞进行示踪。超顺磁性氧化铁纳米粒子(superparamagnetic iron oxide,SPIO)是一种较为理想的新型磁共振对比剂,用SPIO标记细胞可实现对移植细胞进行活体示踪。而荧光活性染料CM-DiI无细胞毒性,不影响细胞的生长,适合标记和示踪细胞。目的:观察SPIO及CM-DiI对骨髓间充质干细胞的标记及示踪效果。方法:全骨髓法培养猪骨髓间充质干细胞,用含50mg/L铁浓度的SPIO及CM-DiI标记骨髓间充质干细胞,将双标后的骨髓间充质干细胞经冠状动脉注入猪心肌梗死模型。4周后取心脏组织行冰冻切片观察。结果与结论:SPIO及CM-DiI在体外标记骨髓间充质干细胞效率高,几乎达100%。经冠状动脉移植4周后心肌组织可找到双标记的骨髓间充质干细胞,结果提示SPIO及CM-DiI双标记骨髓间充质干细胞体内示踪效果好。     

兔骨髓间充质干细胞来源的心肌(样)细胞的诱导分化研究

目的体外诱导骨髓间充质干细胞(Mesenchymal stem cells,MSCs)向肌源性细胞分化,探索诱导后的MSCs移植于心肌梗死区的存活和分化情况。方法提取、分离、培养兔的MSCs。经5-氮胞苷诱导后,进行免疫组化,电镜观察。4',6二乙酞基-2-苯基吲哚(DAPI)标记MSCs,建立兔心肌梗死模型。实验动物随机分两组:实验组(n=10)在心梗区域注入经诱导后的MSCs;对照组(n=10)在心梗区域注入不含MSCs的培养液。移植4周后,进行病理标本观察和免疫组化检测。结果5-氮胞苷诱导MSCs4周,部分细胞表达肌钙蛋白T(troponin T),电镜观察到肌丝形成。MSCs在体外用DAPI标记,用荧光显微镜观察细胞发蓝色荧光。移植4周后,在实验组中用荧光显微镜观察可见梗死区组织标本中可见DAPI标记带蓝色荧光的供体细胞核,移植细胞表达troponin T。结论MSCs经5-氮胞苷诱导后可向心肌细胞转化。移植细胞可在心肌存活,并向心肌细胞(样)转化。     

兔髓核细胞诱导大鼠骨髓间充质干细胞分化为髓核细胞的研究

目的 研究体外新西兰大白兔髓核细胞(nucleus pulposus cells)与SD大鼠骨髓间充质干细胞(mes-enchymal stem cells,MSCs)共培养时,兔髓核细胞与大鼠MSCs直接和间接接触对MSCs分化为髓核细胞的影响.方法 DAPI (4‘,6-二咪基-2‘-苯吲哚盐酸)标记原代髓核细胞后,分别与第三代MSCs按接触组和非接触组(Transwell培养系统)共培养.每隔24小时应用免疫荧光观察MSCs的形态学变化,并用RT-PCR方法检测Ⅱ型胶原和可凝集蛋白多糖(Aggrecan)的表达.结果 直接接触培养组中可见分化的MSCs形态和功能接近髓核细胞;非直接接触组的MSCs未见变化.结论 髓核细胞与MSCs的直接接触,是诱导MSCs分化为髓核细胞的重要因素.     

心肌细胞共培养诱导骨髓间充质干细胞向心肌样细胞分化

目的在体外以新生大鼠心肌细胞（CM）与骨髓间充质干细胞（MSCs）共同培养的方式模拟心肌微环境,研究MSCs分化为心肌细胞的机制。方法分离大鼠MSCs在体外培养纯化后进行细胞标记,将已标记的MSCs分别与搏动的CM、停止搏动的CM以及心肌细胞条件培养液混合培养。分别在共培养后第4、5天用免疫荧光染色检测MSCs细胞中的心肌特异性肌钙蛋白T（Troponin T）。结果与搏动的CM共培养后第4天MSCs出现自发收缩,与CM同步搏动并表达Troponin T,而在抑制心肌细胞搏动或缺乏与心肌直接接触的情况下MSCs未出现上述变化。结论说明在与CM直接接触的前提下,CM对MSCs的机械牵拉刺激为诱导MSCs分化为心肌细胞的必须条件。单纯的心肌细胞条件培养液则非关键因素。了解MSCs分化为心肌细胞的机制对于寻找适宜的细胞移植条件有重要指导意义。     

骨髓间充质干细胞标记及示踪技术的研究与进展

背景：骨髓间充质干细胞标记及示踪技术是干细胞移植治疗的研究热点之一。虽然近几年骨髓间充质干细胞标记及示踪技术有了很大进展,但仍存在很多问题有待进一步解决。 目的：对国内外骨髓间充质干细胞标记及示踪技术的研究与进展作一综述。 方法：应用计算机检索CNKI和Foreign Medical Journal Service数据库中1982-01/2011-10关于骨髓间充质干细胞标记及示踪技术的文章,在标题和摘要中以“骨髓间充质干细胞;标记方法;示踪技术”或“bone marrow,labeling,Tracer”为检索词进行检索。最终选择29篇文献进行综述。 结果与结论：骨髓间充质干细胞的示踪技术众多,主要有同位素示踪法、抗原标记法、荧光蛋白标记法、荧光染料标记法、核磁共振对比增强剂标记法、Lac-Z基因标记法、Y染色体标记法。每一种方法都有其优缺点,选择的标记方法应具有特异性强、灵敏度高、对细胞或机体影响小、标记和检测方法简单易行、标记时间合适等特点。种子细胞的示踪技术仍需要进一步深入研究。     

Highly efficient magnetic stem cell labeling with citrate-coated superparamagnetic iron oxide nanoparticles for MRI tracking

Tracking of transplanted stem cells is essential to monitor safety and efficiency of cell-based therapies. Magnetic resonance imaging (MRI) offers a very sensitive, repetitive and non-invasive in vivo detection of magnetically labeled cells but labeling with commercial superparamagnetic iron oxide nanoparticles (SPIONs) is still problematic because of low labeling efficiencies and the need of potentially toxic transfection agents. In this study, new experimental citrate-coated SPIONs and commercial Endorem and Resovist SPIONs were investigated comparatively in terms of in vitro labeling efficiency, effects on stem cell functionality and in vivo MRI visualization. Efficient labeling of human mesenchymal stem cells (MSCs) without transfection agents was only achieved with Citrate SPIONs. Magnetic labeling of human MSCs did not affect cell proliferation, presentation of typical cell surface marker antigens and differentiation into the adipogenic and osteogenic lineages. However, chondrogenic differentiation and chemotaxis were significantly impaired with increasing SPION incorporation. Transplanted SPION-labeled MSCs were visualized in vivo after intramuscular injection in rats by 7T-MRI and were retrieved ex vivo by Prussian Blue and immunohistochemical stainings. Though a careful titration of SPION incorporation, cellular function and MRI visualization is essential, Citrate SPIONs are very efficient intracellular magnetic labels for in vivo stem cell tracking by MRI.     

Intervertebral disc cell-mediated mesenchymal stem cell differentiation

Low back pain is one of the largest health problems in the Western world today, and intervertebral disc degeneration has been identified as a main cause. Currently, treatments are symptomatic, but cell-based tissue engineering methods are realistic alternatives for tissue regeneration. However, the major problem for these strategies is the generation of a suitable population of cells. Adult bone marrow-derived mesenchymal stem cells (MSCs) are undifferentiated, multipotent cells that have the ability to differentiate into a number of cell types, including the chondrocyte-like cells found within the nucleus pulposus (NP) of the intervertebral disc; however, no method exists to differentiate these cells in an accessible monolayer environment. We have conducted coculture experiments to determine whether cells from the human NP can initiate the differentiation of human MSCs with or without cell-cell contact. Fluorescent labeling of the stem cell population and high-speed cell sorting after coculture with cell-cell contact allowed examination of individual cell populations. Real-time quantitative polymerase chain reaction showed significant increases in NP marker genes in stem cells when cells were cocultured with contact for 7 days, and this change was regulated by cell ratio. No significant change in NP marker gene expression in either NP cells or stem cells was observed when cells were cultured without contact, regardless of cell ratio. Thus, we have shown that human NP and MSC coculture with contact is a viable method for generating a large population of differentiated cells that could be used in cell-based tissue engineering therapies for regeneration of the degenerate intervertebral disc.     

Human mesenchymal stem cell differentiation to NP-like cells in chitosan-glycerophosphate hydrogels

Intervertebral disc (IVD) degeneration is one of the major causes of low back pain. As current clinical treatments are aimed at restoring biomechanical function and providing symptomatic relief, interest in methods focused on biological repair has increased. Several tissue engineering approaches using different cell types and hydrogels/scaffolds have been proposed. Owing to the unsuitable nature of degenerate cells for tissue engineering attention has focused on the use of mesenchymal stem cells (MSCs). Additionally, while rigid scaffolds have been demonstrated to allow MSC differentiation to the chondrocyte-like cells of the IVD, hydrogels are being increasingly studied as they allow minimally invasive implantation without extensive damage to the IVD. Here, we have studied the temperature-sensitive hydrogel chitosan-glycerophosphate (C/Gp), seeded with human MSCs and cultured for 4 weeks in standard medium. We have analysed the gene and protein expression profile of the MSCs and compared it to that of both nucleus pulposus (NP) cells and articular chondrocytes cultured in C/Gp. Gene expression analysis for chondrocytic-cell marker genes demonstrated differentiation of MSCs to a phenotype which showed similarities to both articular chondrocytes and NP cells. Conventional PCR demonstrated a lack of expression of osteogenic marker genes and the hypertrophic marker gene type X collagen. MSCs also secreted both proteoglycans and collagens in a ratio, which more closely resembled that of NP cells than articular chondrocytes. These results therefore suggest that MSC-seeded C/Gp gels could be used clinically for the regeneration of the degenerate human IVD.     

Expression of transferrin receptor and ferritin following ferumoxides-protamine sulfate labeling of cells: implications for cellular magnetic resonance imaging

Ferumoxides-protamine sulfate (FE-Pro) complexes are used for intracellular magnetic labeling of cells to non-invasively monitor cell trafficking by in vivo MRI. FE-Pro labeling is non-toxic to cells; however, the effects of FE-Pro labeling on cellular expression of transferrin receptor (TfR-1) and ferritin, proteins involved in iron transport and storage, has not been reported. FE-Pro-labeled human mesenchymal stem cells (MSCs), HeLa cells and primary macrophages were cultured from 1 week to 2 months and evaluated for TfR-1 and ferritin gene expression by RT-PCR and protein levels were determined using Western blots. MTT (proliferation assay) and reactive oxygen species (ROS) analysis were performed. FE-Pro labeling of HeLa and MSCs resulted in a transient decrease in TfR-1 mRNA and protein levels. In contrast, Fe-Pro labeling of primary macrophages resulted in an increase in TfR-1 mRNA but not in TfR-1 protein levels. Ferritin mRNA and protein levels increased transiently in labeled HeLa and macrophages but were sustained in MSCs. No changes in MTT and ROS analysis were noted. In conclusion, FE-Pro labeling elicited physiological changes of iron metabolism or storage, validating the safety of this procedure for cellular tracking by MRI.     

Effects of hindlimb unloading on ex vivo growth and osteogenic/adipogenic potentials of bone marrow-derived mesenchymal stem cells in rats

The goal of this study was to determine the effects of hindlimb unloading (HU) on the ex vivo growth and the osteogenic potential of mesenchymal stem cells (MSCs) from the femurs of rats. Microgravity was simulated by 28-day HU in male Sprague-Dawley (SD) rats, and the bone marrow (BM) was collected from hindlimb femurs of HU or control (CTL) rats. MSCs were isolated from BM and cultured for eight passages. Then MSCs at passages 2, 4, and 8 were induced for osteogenesis or adipogenesis. The results revealed that HU decreased the osteogenic potential of MSCs and also decreased the expression of osteoblast gene marker mRNAs in cells induced by osteogenic conditions. Meanwhile, the expression of Runx2 mRNA and the phosphorylation of ERK were also decreased. There were no significant differences of osteoblast gene marker and Runx2 mRNA expression between cells induced from different passages of MSCs in UH rats. Under adipogenic conditions, HU increased both the adipogenic potential of MSCs and the expression of adipocytic gene marker mRNAs in induced cells. HU also increased the expression of PPAR gamma 2 mRNA, but with no effect on the phosphorylation of p38MAPK. The adipogenic potential of MSCs and the expression of adipocytic gene marker mRNAs in induced cells decreased along with cell cultures under normal gravity. This suggests that the normal gravity during in vitro MSC culture and the centrifugal force produced during cell harvest after each passage could decrease the adipogenic potential of MSCs, but could not reverse the effect of HU on the osteogenic potential of MSCs.     

Towards whole-body imaging at the single cell level using ultra-sensitive stem cell labeling with oligo-arginine modified upconversion nanoparticles

Mesenchymal stem cells (MSCs) have shown great potential in regenerative medicine. Sensitive and reliable methods for stem cell labeling and in vivo tracking are thus of great importance. Herein, we report the use of upconversion nanoparticles (UCNPs) as an exogenous contrast agent to track mouse MSCs (mMSCs) in vivo. To improve the labeling efficiency, oligo-arginine is conjugated to polyethylene glycol (PEG) coated UCNPs to enhance the nanoparticles uptake by mMSCs. Systematic in vitro tests reveal that the proliferation and differentiation of mMSCs are not notably affected by UCNP-labeling, suggesting that the labeled cells are able to maintain their stem cell potency. No apparent exocytosis is found in our in vitro labeling experiment by using a transwell culture system over a course of 10 days, indicating the potential capability of using our UCNP-labeling method for long-term stem cell tracking. To demonstrate the tracking sensitivity of our stem cell labeling approach, UCNP-labeled mMSCs are subcutaneously transplanted into mice and imaged using an in vivo upconversion luminescence (UCL) imaging system. As few as ~10 cells labeled with UCNPs are detected in vivo, evidencing a remarkable improvement in detection sensitivity of our UCNP-labeled hMSCs compared with other stem cell labeling techniques using conventional exogenous agents. We further track UCNP-labeled mMSCs after intravenous injection, and observe the translocation of mMSCs from lung where they initially accumulate, to liver, a phenomenon consistent to previous reports. Our results highlight the promise of using UCNPs as a new type of ultra-sensitive probes for labeling and in vivo tracking of stem cells at nearly the single cell level.     

两个不同骨髓干细胞亚群体外成肝潜能的比较研究

目的研究与比较骨髓造血干细胞(hematopoieticstemcells,HSCs)和间充质干细胞(mesen-chymalstemcells,MSCs)肝分化潜能。方法常规法分离培养大鼠骨髓MSCs。以Thy-1.1为标志,免疫磁珠法(magenicactivatedcellsorting,MACS)分离纯化HSCs。探寻MSCs、HSCs的诱导条件,从形态学、RT-PCR和免疫细胞化学法鉴定肝分化结果。结果MACS分选后Thy-1.1 细胞纯度94.20%,细胞活力99.62%。纤维状MSCs诱导后呈圆形,表达白蛋白及其mRNA,不表达甲胎球蛋白。诱导的HSCs为多角形,白蛋白表达逐渐减少,甲胎球蛋白表达逐渐增强。结论MACS方法能有效分选大鼠骨髓Thy-1.1 干细胞群。MSCs和HSCs二者具有不同的肝系分化潜能。MSCs在HGF/FGF-4的作用下表达成熟肝细胞的特异性基因,分化为成熟肝细胞样细胞;HSCs在HGF/FGF-1/FGF-2的作用下表达早期肝特异性基因,分化为肝干细胞样细胞。     

Meflin defines mesenchymal stem cells and/or their early progenitors with multilineage differentiation capacity

Mesenchymal stem cells (MSCs) are the likely precursors of multiple lines of mesenchymal cells. The existence of bona fide MSCs with self-renewal capacity and differentiation potential into all mesenchymal lineages, however, has been unclear because of the lack of MSC-specific marker(s) that are not expressed by the terminally differentiated progeny. Meflin, a glycosylphosphatidylinositol-anchored protein, is an MSC marker candidate that is specifically expressed in rare stromal cells in all tissues. Our previous report showed that Meflin expression becomes down-regulated in bone marrow-derived MSCs cultured on plastic, making it difficult to examine the self-renewal and differentiation of Meflin-positive cells at the single-cell level. Here, we traced the lineage of Meflin-positive cells in postnatal and adult mice, showing that those cells differentiated into white and brown adipocytes, osteocytes, chondrocytes and skeletal myocytes. Interestingly, cells derived from Meflin-positive cells formed clusters of differentiated cells, implying the in situ proliferation of Meflin-positive cells or their lineage-committed progenitors. These results, taken together with previous findings that Meflin expression in cultured MSCs was lost upon their multilineage differentiation, suggest that Meflin is a useful potential marker to localize MSCs and/or their immature progenitors in multiple tissues.