SPIO标记脂肪干细胞移植治疗大鼠脑梗死的磁共振示踪成像研究

目的通过采用多聚左旋赖氨酸（PLL）与超顺磁氧化铁纳米微粒（SPIO）的复合物对脂肪干细胞（ADSCs）体外进行标记,观察SPIO标记ADSCs脑内移植治疗大鼠脑梗死的分布和迁徙情况。方法显微镜下直接结扎大脑中动脉方法制备大鼠脑梗死模型36只,随机分成缺血未干预对照组（12只）、磁性标记ADSCs移植组（12只）和未磁性标记ADSCs移植组（12只）,采用立体定向方法脑内移植。对移植后大鼠的神经系统行为和运动功能进行评估,病理组织化学染色观察ADSCs在体内的分布情况,并用磁共振成像的方法在体观察ADSCs的分布,并与病理结果进行对比。结果移植后3周神经系统行为学评分显示移植组动物明显改善,ADSCs脑内移植后3周MRT2WI显示移植区低信号改变并通过胼胝体向病灶迁移。病理组织检查显示磁共振低信号改变区可见普鲁士蓝染色阳性细胞。结论移植ADSCs可以有效地促进大鼠脑梗死后神经行为功能的恢复,MRI可用于在体评价经SPIO和PLL复合物标记的ADSCs细胞移植后在体内的分布、迁移过程。     

超顺磁性氧化铁标记骨髓间充质干细胞移植治疗兔脊髓损伤的磁共振活体示踪

目的通过蛛网膜下腔置管,将超顺磁性氧化铁(SPIO)标记后的骨髓间充质干细胞(MSCs)移植入兔脊髓损伤模型,观察脊髓损伤MR活体示踪移植细胞的可行性。方法制作兔SCI模型,并在蛛网膜下腔置管以备移植。将实验用大白兔随机分为三组:A组为移植SPIO标记细胞;B组为移植未标记细胞;C组不移植细胞只注射PBS液做对照组。在细胞移植后3d、7d、14d、21d,进行MR活体示踪,并做病理学检测进行对照。结果 SPIO标记的MSCs移植入脊髓损伤模型后7d,MR扫描的T2WI上脊髓损伤区域出现点状低信号影;14d后T2WI上脊髓损伤区域的点状低信号影增多,21d后T2WI上脊髓损伤区域的点状低信号影减少。脊髓损伤区域组织切片行普鲁士蓝染色,发现局部组织上出现大量含蓝色铁颗粒的细胞,其细胞变化规律与MR示踪结果一致。结论蛛网膜下腔移植的SPIO标记MSCs可定向迁移到脊髓损伤区域,利用MR可对移植细胞进行活体示踪。     

磁性氧化铁纳米粒子造影剂在MRI应用进展

近年来,磁性氧化铁纳米粒子（SPIO）已有广泛的体内应用：如磁共振成像,细胞标记、干细胞祖细胞示踪及组织修复,肿瘤生物探针检测,移植排斥反应的监测等方面,本文综述了USPIO的化学合成、表面修饰以及它们的生物医学应用进展。     

心脏MRI在体示踪移植细胞的可行性研究

目的：寻找一种能够对移植细胞进行在体示踪的标记方法,为细胞移植的动物和临床实验中细胞的存留、迁移提供重要手段。方法：从中华小型猪髂骨处抽取骨髓,体外培养扩增MSCs。将SPIO和MSCs共同孵育培养36h。普鲁士蓝染色评价细胞的标记效率;通过MTT比色实验评价SPIO对细胞生长能力的影响;台盼蓝染色检验标记后细胞的活性;使用Costar Transwell方法评价铁离子对细胞迁移能力的影响;用细胞分化诱导液培养标记后的细胞评价其向成脂肪细胞和成骨细胞的分化能力。在体内实验中将SPIO标记或未标记的自体MSCs注射到心肌或冠状动脉内,通过心脏磁共振检查对移植细胞进行在体示踪观察。在细胞移植后不同时期取材动物心脏切片进行普鲁士蓝染色观察移植细胞。结果：MSCs经铁离子标记后普鲁士蓝染色阳性率在98%以上,可见蓝染颗粒位于细胞浆内,标记细胞电镜切片观察可见高密度铁颗粒位于细胞浆内。MTT比色实验发现随着培养液中SPIO浓度的增加细胞增殖能力没有明显改变;台盼蓝染色显示标记后98％的细胞保持活性;细胞经SPIO标记后仍可保持原有的细胞形态,可继续培养、传代。细胞迁移试验发现细胞标记后对SDF-1和VEGF诱导的迁移能力没有明显的减低。铁离子标记后细胞仍可向成脂肪细胞和成骨细胞分化。注射到心肌或冠状动脉内的SPIO标记的MSCs可通过心脏磁共振检查进行在体示踪,动态观察显示SPIO标记细胞在磁共振影像上表现为低密度灶影或信号缺失,并且在移植后4周仍可显影。病理学检查可以看到移植细胞呈普鲁士蓝染色阳性,并和影像学有很好的一致性。结论：临床使用的SPIO磁共振对比剂可以安全、有效的标记MSCs。利用心脏磁共振对移植的标记细胞可以实现移植细胞的在体示踪观察。此标记方法结合心脏磁共振技术为移植细胞的在体示踪和细胞存留、迁移的研究提供了良好的手段。     

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-Dil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-Dil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50 mg/L铁浓度的SPIO及CM-Dil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-Dil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-Dil双标记骨髓间充质干细胞体内示踪效果好.     

荧光磁性纳米粒子标记三维培养系统中的脂肪间充质干细胞

背景：荧光磁性纳米粒子具有量子点和磁粒子的双重特质,生物相容性好,能够被胞吞入细胞质高效地标记细胞。目的：验证荧光磁性纳米粒子标记人脂肪间充质干细胞的可行性。方法：抽脂术抽取健康人脂肪组织,采用Ⅰ型胶原酶消化法分离,贴壁培养纯化人脂肪间充质干细胞。取第6代脂肪间充质干细胞与荧光磁性纳米粒子孵育过夜,采用普鲁士蓝染色、激光共聚焦显微镜检测体外标记细胞情况,荧光成像系统观察荧光磁性纳米粒子标记的干细胞体内示踪效果。结果与结论：普鲁士蓝染色显示荧光磁性纳米粒子以蓝色颗粒的形式分散于脂肪间充质细胞胞质中。激光共聚焦显微镜观察显示人脂肪间充质干细胞的细胞核被Hoechest33258染成蓝色,细胞质被标记成绿色;荧光成像结果表明标记的人脂肪间充质干细胞具有很好的成像效果。结果提示荧光磁性纳米粒子可以作为示踪剂在体外标记人脂肪间充质干细胞,为脂肪间充质干细胞的移植转化研究提供新方法。     

蛛网膜下腔移植SPIO标记的MSCs在大鼠脊髓损伤模型的MR活体示踪研究

目的 将超顺磁性氧化铁纳米粒子(SPIO)标记的骨髓间充质干细胞BMSCs通过蛛网膜下腔置管移植到脊髓损伤大鼠模型,以磁共振成像(MRI)观察其迁移和分布.方法 用携带绿色荧光蛋白的腺病毒(AD5/F35-EGFP)和SPIO标记分离纯化后的BMSCs,免疫荧光和普鲁士蓝染色显示标记效果.制作大鼠脊髓损伤模型,并将蛛网膜下腔置管成功的10只SD大鼠随机分为2组,将标记细胞(实验组,n＝5)和未标记细胞(对照组,n＝5)经蛛网膜下腔移植到模型鼠.在移植前、移植后3、7、14天用3T MRI对移植细胞进行活体示踪,并与损伤脊髓组织切片GFP表达进行对照.结果 AD5/F35-EGFP和SPIO可以高效地标记BMSCs,标记细胞表达绿色荧光,普鲁士蓝染色显示BMSCs胞质内出现蓝色铁颗粒,标记对细胞增殖活力没有明显的影响.蛛网膜下腔移植标记细胞到大鼠脊髓损伤模型,MRI显示损伤区域逐渐增强的T2低信号,组织切片荧光检查与MRI结果一致,未标记细胞组MRI无明显低信号改变.结论 SPIO纳米颗粒可有效标记BMSCs.蛛网膜下腔移植的BMSCs可迁移到脊髓损伤区域;利用MRI可对移植细胞进行活体示踪.     

超顺磁氧化铁标记骨髓间充质干细胞移植治疗帕金森病大鼠模型的组织学研究

目的:研究应用超顺磁氧化铁(SPIO)和BrdU标记骨髓间充质干细胞(MSCs)移植治疗帕金森病(PD)大鼠模型,观察移植的MSCs在PD模型鼠脑内的迁移及分化,探讨MSCs移植治疗PD的可能性。方法:分离和培养大鼠骨髓间充质干细胞,一组MSCs用脂质体转染法进行SPIO标记,另一组MSCs用BrdU标记;制作PD大鼠模型,将不同标记的MSCs分别移植到PD大鼠右侧纹状体区,利用阿朴吗啡观察PD鼠的行为学变化。应用透射电镜和免疫荧光双标法观察移植后的MSCs增殖、迁徙和分化。结果:电镜显示,SPIO标记的MSCs呈类神经元形态。免疫酶标显示移植后4周和12周BrdU-MSCs组和SPIO-MSCs组的注射侧黑质区TH免疫反应阳性细胞较对照组注射侧黑质区有增多趋势,但无统计学差异。免疫荧光组化结果表明,MSCs移植后4周,在移植区可见少量BrdU和Nestin双标为阳性的神经前体细胞,12周时可见少量双标的神经细胞,未发现明显的BrdU和TH双标为阳性的细胞。移植组与对照组相比行为学(旋转次数)有明显差异(P<0.05)。结论:MSCs移植治疗能改善PD模型大鼠的行为学。MSCs进入PD模型大鼠脑内可存活并处于增殖状态,有广泛迁移并在局部微环境的作用下能向神经前体细胞和神经细胞转化,但数目较少。故其改善PD大鼠神经功能的机理值得进一步探讨。     

超顺磁性氧化铁标记骨髓间充质干细胞的磁共振成像研究

目的明确顺磁性氧化铁纳米粒子(SPIO)体外标记兔骨髓间充质干细胞(MSCs)的适当浓度和不同标记浓度对细胞的生物学活性影响,以及经MR成像的特征和可成像的最低标记细胞量等。方法分离、纯化、培养兔MSCs,体外不同浓度SPIO标记,荧光显微镜观察铁颗粒在细胞内的位置、不同标记浓度下的最佳孵育时间、不同标记浓度细胞形态的改变、细胞内铁颗粒的分布及标记率;测量并绘制未标记细胞和标记细胞的MTT生长曲线;选取适当浓度标记量,对不同细胞量组进行标记后MR成像,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。结果标记后的铁颗粒均位于细胞质内;20～50μgFe/ml培养液是SPIO标记干细胞的适当浓度阈值,超过50μgFe/ml培养液浓度可使细胞的变形增殖能力受到不同程度的抑制;在此浓度阈值标记后孵育18～24h即可有效标记细胞97%～100%;细胞对铁颗粒的吸收与细胞的数量、标记浓度和孵育时间呈正相关;SPIO标记的MSCs在T2WI尤其是FFE(T2WI)序列信号明显降低;在35μgFe/ml培养液标记浓度下,MR可成像的最低细胞量为5×104;35μgFe/ml培养液浓度时,标记细胞1×105和5×104MR成像可使T2WI、FFE图像信号均降低,而且没有使靶灶扩大的磁敏感伪影。结论SPIO可以简便标记MSCs并且在适当浓度下对MSCs的生物学活性没有影响,MRT2WI和T2WI序列可敏感显像磁性标记的干细胞。     

MRI活体示踪心肌梗死大鼠骨髓间充质干细胞的移植

背景：干细胞移植的疗效和安全性评估均需要对体内干细胞的存活、分布、迁徙、增殖及分化进行连续监测。目的：观察MRI示踪超顺磁性氧化铁标记的骨髓间充质干细胞在缺血心肌组织的分布、迁徙情况。方法：直接贴壁法分离和培养大鼠骨髓间充质干细胞,获得的细胞进行免疫鉴定。以新型超顺磁性氧化铁标记骨髓间充质干细胞,体外MRI成像确定其体内示踪的可行性。标记后锥虫蓝拒染试验、MTT比色试验分别检测标记细胞的活力、增殖情况。60只SD大鼠随机分为3组,制备大鼠心肌梗死模型2周后再次开胸移植含标记骨髓间充质干细胞的PBS混合液、含未标记骨髓间充质干细胞的PBS混合液和等量PBS。于移植后第1天、第3周行MRI检查,动态观察移植细胞的分布、迁徙,并根据MRI图像定位选择性行CD90免疫组化检查。结果与结论：骨髓间充质于细胞标记后,普鲁士蓝染色见胞浆内蓝色铁颗粒,标记效率为99％,标记细胞与未标记细胞间锥虫蓝拒染率、MTT吸光度差异无显著性意义。体外MRI可检测到标记细胞,并在T2WI及T2W／FFE序列上呈低信号。细胞移植1d后,在T2WI及T2W／FFE序列上可见标记细胞在梗死心肌边缘呈类圆形低信号;移植3周后,移植区域信号边界模糊,范围扩大,对比度降低。CD90免疫组化检测证实移植细胞可由梗死边缘向梗死区域迁徙。结果可见新型超顺磁性氧化铁可成功对大鼠骨髓间充质干细胞进行标记,细胞标记后可被MRI检测。     

猪骨髓间充质干细胞超顺磁性氧化铁标记方法的实验研究

目的：探索超顺磁性氧化铁纳米颗粒（SPIO）标记猪骨髓间充质干细胞（MSCs）的合适方案。方法：分离培养猪MSCs,用不同SPIO浓度、不同共孵育时间进行标记,根据标记效率指标（普鲁士蓝染色、细胞铁浓度测定、细胞透射电镜检查）和标记毒性指标（台盼蓝排除试验、CCK-8试验）确定适宜的标记条件,然后对不同标记细胞进行磁共振（MR）体外成像。结果：（1）随着SPIO浓度的增加,MSCs细胞内平均含铁量相应升高;细胞涂片普鲁士蓝染色也显示细胞内蓝染颗粒相应增加。当SPIO浓度为25、50μg·mL-1时,细胞存活率和细胞活力与无SPIO时差异无统计学意义（P〉0.05）;但当SPIO浓度增加到100μg·mL-1时,CCK-8试验显示细胞活力下降（P〈0.01）;当SPIO浓度增加到200μg·mL-1时,细胞存活率和细胞活力均下降（P〈0.05）;（2）设定SPIO浓度为50μg.mL-1,随共孵育时间增加,细胞内含铁量逐渐升高（各组间比较均P〈0.01）;当标记时间为0、2、4、8、16、24h时,细胞存活率和细胞活力并差异无统计学意义（P〉0.05）;但当标记时间进一步延长到48h时,细胞存活率有所降低（P〈0.05）;（3）按＂50μg/ml×24h方案＂标记后,1.5TMR成像仪体外MRT2＊ WIflash检测阈值为7.5×104个细胞。结论：50μg·mL-1×24h方案是猪骨髓间充质干细胞比较合适的磁探针标记方案。     

In vivo quantitative assessment of cell viability of gadolinium or iron‐labeled cells using MRI and bioluminescence imaging

In cell therapy, noninvasive monitoring of in vivo cell fate is challenging. In this study we investigated possible differences in R₁, R₂ or R₂* relaxation rate as a measure of overall cell viability for mesenchymal stem cells labeled with Gd‐liposomes (Gd‐MSCs) or iron oxide nanoparticles (SPIO‐MSCs). Cells were also transduced with a luciferase vector, facilitating a correlation between MRI findings and cell viability using bioluminescence imaging (BLI). Viable Gd‐MSCs were clearly distinguishable from nonviable Gd‐MSCs under both in vitro and in vivo conditions, clearly differing quantitatively (ΔR₁ and ΔR₂) as well as by visual appearance (hypo‐ or hyperintense contrast). Immediately post‐injection,viable Gd‐MSCs caused a substantially larger ΔR₂ and lower ΔR₁ effect compared with nonviable MSCs. With time, the ΔR₁ and ΔR₂ relaxation rate showed a good negative correlation with increasing cell number following proliferation. Upon injection, no substantial quantitative or visual differences between viable and nonviable SPIO‐MSCs were detected. Moreover, nonviable SPIO‐MSCs caused a persisting signal void in vivo, compromising the specificity of this contrast agent. In vivo persistence of SPIO particles was confirmed by histological staining. A large difference was found between SPIO‐ and Gd‐labeled cells in the accuracy of MR relaxometry in assessing the cell viability status. Gd‐liposomes provide a more accurate and specific assessment of cell viability than SPIO particles. Viable Gd cells can be differentiated from nonviable Gd cells even by visual interpretation. These findings clearly indicate Gd to be the favourable contrast agent in qualitative and quantitative evaluation of labeled cell fate in future cell therapy experiments. Copyright © 2012 John Wiley & Sons, Ltd.     

Magnetic labeling of non‐phagocytic adherent cells with iron oxide nanoparticles: a comprehensive study

Small particles of iron oxide (SPIO) and ultrasmall particles of iron oxide (USPIO), inducing a strong negative contrast on T₂ and T₂*‐weighted MR images, are the most commonly used systems for the magnetic labeling of cultured cells and their subsequent detection by magnetic resonance imaging (MRI). The purpose of this work is to study the influence of iron incubation concentration, nanoparticle size and nanoparticle coating on the magnetic labeling and the viability of non‐phagocytic adherent cells in culture. The magnetic labeling of 3T6 fibroblasts was studied by T₂‐weighted MRI at 4.7 T and by dosing—or cytochemical revealing—of iron through methods based on Perl's Prussian blue staining. Cells were incubated for 48 h with increasing iron concentrations of SPIO (25–1000 µg Fe/ml Endorem®). Sinerem®, a USPIO (20–40 nm) coated with neutral dextran, and Resovist® (65 nm), a SPIO bearing an anionic carboxydextran coating, were compared with Endorem® (dextran‐coated, 80–150 nm) as magnetic tags. The iron loading of marrow stromal cell primary cultures (MSCs) isolated from rat femurs was compared with that of 3T6 fibroblasts. The SPIO‐labeling of cells with Endorem® was found to be dependent on the iron incubation concentration. MSCs, more sparsely distributed in the culture, exhibited higher iron contents than more densely populated 3T6 fibroblast cultures. A larger iron loading was achieved with Resovist® than with Endorem®, which in turn was more efficient than Sinerem® as a magnetic tag. The magnetic labeling of cultured non‐phagocytic adherent cells with iron oxide nanoparticles was thus found to be dependent on the relative concentration of the magnetic tag and of the cells in culture, on the nanoparticle size, and on the coating type. The viability of cells, estimated by methods assessing cell membrane permeability, was not affected by magnetic labeling in the conditions used in this work. Copyright © 2008 John Wiley & Sons, Ltd.     

Superparamagnetic iron oxide nanoparticles as a means to track mesenchymal stem cells in a large animal model of tendon injury

The goal of this study was to establish an SPIO‐based cell‐tracking method in an ovine model of tendonitis and to determine if this method may be useful for further study of cellular therapies in tendonitis in vivo. Functional assays were performed on labeled and unlabeled cells to ensure that no significant changes were induced by intracellular SPIOs. Following biosafety validation, tendon lesions were mechanically (n = 4) or chemically (n = 4) induced in four sheep and scanned ex vivo at 7 and 14 days to determine the presence and distribution of intralesional cells. Ovine MSCs labeled with 50 µg SPIOs/mL remained viable, proliferate, and undergo tri‐lineage differentiation (p < 0.05). Labeled ovine MSCs remained detectable in vitro in concentrated cell numbers as low as 10 000 and in volumetric distributions as low as 100 000 cells/mL. Cells remained detectable by MRI at 7 days, as confirmed by correlative histology for dually labeled SPIO+/GFP+ cells. Histological evidence at 14 days suggested that SPIO particles remained embedded in tissue, providing MRI signal, although cells were no longer present. SPIO labeling has proven to be an effective method for cell tracking for a large animal model of tendon injury for up to 7 days post‐injection. The data obtained in this study justify further investigation into the effects of MSC survival and migration on overall tendon healing and tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

Transplantation of MSCs Overexpressing HGF into a Rat Model of Liver Fibrosis

Purpose

The aim of this study is to evaluate the effect of overexpressing human hepatocyte growth factor (HGF) for mesenchymal stem cells (MSCs) in liver fibrosis regeneration and magnetic resonance (MR) tracking of MSCs in rat liver.

Procedures

MSCs were transfected with ad-HGF/ad-green fluorescent protein (GFP) and labeled with superparamagnetic iron oxide (SPIO). The characteristics of SPIO-HGF/MSCs were investigated. Prussian blue staining for iron assessment was conducted in vitro and in vivo. SPIO-HGF/MSCs (group A) or SPIO-GFP/MSCs (group B) were transplanted into a rat model of liver fibrosis, and MR imaging of the rat liver was performed. The signal to noise ratio (SNR) and R2* (1/T2*) value were measured. Prussian blue staining was performed to detect the in vivo distribution of MSCs, and liver Ki67 immunohistochemistry (IHC) staining was studied. The serum levels of HGF, alanine aminotransferase (ALT) and hyaluronic acid (HA) were determined.

Results

The positive rate of HGF transfection was 93.17 % and the HGF/MSCs were labeled with SPIO successfully (97.80?±?1.06 %). Labeling of MSCs with SPIO did not alter cell proliferation in vitro. The signal intensity of liver T2* WI images decreased on day 1 after cell transplantation and recovered to pre-transplantation level on day 15 (group A) and day 13 (group B). The SNR of group A were significantly lower than that of group B (P?=?0.006), and the R2* values of group A were significantly higher than those of group B (P?<?0.001). The R2* value had a significantly negative correlation with SNR. There were more Prussian blue-positive cells in of group A were more than in group B in vivo. The positive rate of Ki67 was 16.11?±?2.13 %, and the serum level of ALT/HA was decreased in group A.

Conclusion

HGF transfection improved MSCs localization in the liver and aided liver repair. The R2* value might be a feasible index in addition to SNR to track the SPIO-MSC transplantation in the liver.

     

携带人肝细胞生长因子的腺病毒转染人骨髓间充质干细胞及超顺磁性氧化铁标记细胞体外MR成像

目的 评价携带人源化绿色荧光蛋白(hrGFP)-人肝细胞生长因子(hHGF)的腺病毒载体对人永生化骨髓间充质干细胞UE7T-13生物学特征的影响,并探讨对超顺磁性氧化铁(SPIO)标记细胞进行体外MR成像的可行性。 方法 构建和包装携带hrGFP-hHGF基因的腺病毒载体;以hrGFP-hHGF腺病毒转染UE7T-13细胞,检测细胞中hrGFP表达阳性率、hHGF mRNA及细胞上清液中hHGF水平;检测hrGFP-hHGF腺病毒对H₂O₂诱导细胞凋亡的影响。以SPIO标记UE7T-13细胞,检测标记后细胞内铁含量、细胞增殖及分化能力;对不同铁浓度SPIO标记的细胞行MRI。结果 成功构建hrGFP-hHGF腺病毒载体,将其转染UE7T-13细胞48 h后hrGFP阳性表达率达93.17%,hHGF mRNA表达提高3075.63倍,细胞上清液中hHGF水平显著升高,随后下降,于第14天仍高于hrGFP腺病毒转染细胞。hrGFP-hHGF腺病毒可抑制H₂O₂介导的细胞凋亡。SPIO标记后细胞铁染色阳性率达100%,细胞铁含量明显高于未标记细胞;SPIO标记不影响细胞增殖及分化能力;T2WI信号随标记铁浓度增高而降低。 结论 hrGFP-hHGF腺病毒载体可抑制H₂O₂介导的细胞凋亡;SPIO能高效标记细胞,不影响细胞增殖及分化能力,可用于细胞体外MR成像。     

Long-term engraftment of multipotent mesenchymal stromal cells that differentiate to form myogenic cells in dogs with Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an incurable genetic disease with early mortality. Multipotent mesenchymal stromal cells (MSCs) are of interest because of their ability to differentiate to form myogenic cells in situ. In the present study, methods were developed to expand cultures of MSCs and to promote the myogenic differentiation of these cells, which were then used in a new approach for the treatment of DMD. MSC cultures enriched in CD271(+) cells grew better than CD271-depleted cultures. The transduction of CD271(+) MSCs with MyoD caused myogenic differentiation in vitro and the formation of myotubes expressing late myogenic markers. CD271(+) MSCs in the myogenic cell lineage transplanted into dog leukocyte antigen (DLA)-identical dogs formed clusters of muscle-like tissue. Intra-arterial injection of the CD271(+) MSCs resulted in engraftment at the site of the cardiotoxin (CTX)-injured muscle. Dogs affected by X-linked muscular dystrophy in Japan (CXMD(J)) treated with an intramuscular injection of CD271(+) MSCs similarly developed muscle-like tissue within 8-12 weeks in the absence of immunosuppression. In the newly formed tissues, developmental myosin heavy chain (dMyHC) and dystrophin were upregulated. These findings demonstrate that a cell transplantation strategy using CD271(+) MSCs may offer a promising treatment approach for patients with DMD.     

LyP-1标记超顺磁性氧化铁的制备及体外成像

目的 探讨LyP-1标记超顺磁性氧化铁(SPIO)与乳腺癌细胞结合进行体外成像的可行性。方法 以共沉淀法制备SPIO,用LyP-1与3-氨丙基三甲氧基硅烷(APTMS)包被的SPIO耦联,以乳腺癌MDA-MB-231细胞株为研究对象,设立LyP-1-SPIO组、竞争组、SPIO组和对照组,普鲁士蓝染色,评价不同组中铁颗粒在细胞中的分布情况,四甲基偶氮唑盐(MTT)比色法检测细胞活性并观察其体外MR成像效果。结果 成功制备SPIO。 LyP-1-SPIO组有较多铁颗粒进入细胞内,竞争组和SPIO组仅有少量铁颗粒进入细胞中,对照组细胞中无铁颗粒。MTT比色法检测结果显示不同作用时间LyP-1-SPIO和SPIO对细胞的生长均无显著影响;体外MR成像提示LyP-1-SPIO能够显著增强阴性对比效果。结论 LyP-1-SPIO对MDA-MB-231细胞具有较好的靶向作用,可用于对肿瘤细胞的影像学诊断。