SPIO标记浓度对BMSCs生物学特性的影响及体外MRI表现

目的：观察不同浓度超顺磁性氧化铁（SPIO）标记对干细胞生物学特性的影响及SPIO标记后BMSCs体外的MRI特征。材料与方法分离、培养SD大鼠的BMSCs,取第四代的BMSCs,通过流式细胞仪检测表面抗原。不同浓度的SPIO标记液标记BMSCs,通过普鲁士兰染色检测标记效率,台盼兰活细胞计数检测细胞活力,MTT法检测细胞增殖活性。通过MRI检查,观察体外不同浓度SPIO标记细胞的信号变化情况。结果 SPIO在25～50μg/ml的浓度范围可以安全、高效地标记BMSCs,不会影响BMSCs的表型、活力和增殖等生物学特性;当SPIO浓度在75μg/ml以上时,BMSCs活性及增殖能力逐渐降低。随着SPIO标记浓度的增加,细胞内摄入的铁逐渐增多,而T2信号强度则逐渐降低;当SPIO浓度≥50μg/ml,标记细胞的T2信号强度与普通BMSCs相比较均有明显差异（P〈0.05）。结论适当浓度的SPIO标记BMSCs对其生物学活性没有明显影响,且MRI扫描能够有效检测其信号变化。     

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。     

SPIO标记BMSCs移植治疗局灶性脑梗死的MRI示踪研究

目的通过采用磁共振技术示踪在体超顺磁性氧化铁(super paramagnetic iron oxide,SPIO)标记骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs),观察SPIO标记的BMSCs在局灶性脑梗死模型中的迁移及治疗情况。材料与方法 28只雄性SD大鼠随机分为正常对照组(CON,n=4)、假手术组(SHAM,n=4)、局灶性脑梗死对照组(MCAO+SPIO,n=10)和SPIO标记BMSCs治疗组(MCAO+SPIO-BMSC,n=10)。通过普鲁士蓝染色法及CCK8法选取SPIO标记BMSCs的最佳浓度,并使用立体定向注射方法进行最佳浓度SPIO及SPIOBMSC的移植,术后第1、9、20、30、43天行1.5 T MRI扫描,检测两者在局灶性脑梗死中的生物学行为,普鲁士蓝染色观察细胞迁移分布情况。结果当SPIO标记浓度为50μg/ml时,BMSCs普鲁士蓝染色效率为100%,CCK8检测增殖活性较对照组及75μg/ml组均有明显差异(P0.05)。使用T2WI和SWI序列均能明确检测到MCAO+SPIO组和MCAO+SPIO-BMSC组中移植区域信号减低。与T2WI序列相比,SWI序列显示低信号范围更广,示踪时间更长。与MCAO+SPIO组比较,MCAO+SPIO-BMSC组可见SPIO标记的BMSCs向缺血梗死区迁移。普鲁士蓝染色检查发现:普鲁士蓝染色阳性的细胞聚集在梗死区域周围。结论MRI技术能够有效示踪50μg/ml SPIO标记的BMSCs,显示BMSCs在局灶性脑梗死模型中随时间变化发生的生物学行为,并对BMSCs在局灶性脑梗死中的迁移治疗情况进行动态观测。     

体外磁标记骨髓基质细胞移植治疗大鼠创伤性脑损伤的在体MRI观察

目的 采用高场MR在体监测超顺磁性氧化铁颗粒(SPIO)标记大鼠骨髓基质细胞(BMSCs)在脑损伤模型大鼠脑内的分布与迁移.方法 首先进行BMSCs体外培养,然后采用SPIO标记BMSC;采用Feeney法制作创伤性脑损伤模型(TBI).脑损伤24 h后于损伤区周围立体定向移植BMSCs,并于移植后1、3天及1、3周行MR检查.结果 倒置相差显微镜下观察,标记BMSCs的细胞内含有棕黄色铁颗粒,普鲁士蓝染色呈阳性;电镜下胞浆内可见散在分布的铁颗粒.细胞移植后MRI可见移植部位在MR各序列上均呈点状低信号,尤以磁敏感加权成像(SWI)上显示明确.结论 高场MRI能够在活体内连续示踪观察SPIO标记的BMSCs的分布与迁移,且SWI序列最为敏感.     

磁探针对兔脂肪干细胞的生长影响及T2~*mapping成像

目的探讨磁探针(superparamagnetic iron oxide particles with poly-l-lysine,SPIO-PLL)标记兔脂肪干细胞(ADSCs)的安全浓度并体外T2*mapping定量成像。材料与方法经25、50、75μg/ml磁探针标记细胞后普鲁士蓝染色和透射电镜鉴定;流式法检测磁标记ADSCs的细胞周期及凋亡;分别对1×10~6个细胞(未标记),25μg/ml磁探针标记1×10~6个(1 d)、1×10~6个(3 d)和5×10~5个(1 d)行体外3.0 T MR GRE T2*WI及T2*mapping序列成像,测量各组弛豫时间。结果普鲁士蓝染色和透射电镜示铁颗粒位于胞浆内囊泡,标记率达100%;25μg/ml组细胞周期阻滞率及细胞凋亡率与对照组比较无明显差异(P0.05),25μg/ml磁探针为安全标记浓度;1×10~6个(1 d)信号强度最低,各组磁标记细胞T2*弛豫时间与对照组比较有明显统计学差异(F=169.837,P0.01)。结论 25μg/ml磁探针能安全高效标记ADSCs并可行体外MR成像,T2*mapping技术能定量监测磁标记细胞的弛豫时间。     

超顺磁性氧化铁标记对大鼠骨髓间充质干细胞生物学特性的影响

目的:探讨超顺磁性氧化铁(SPIO)标记骨髓间充质干细胞(MSCs)的方法、效果及其对MSCs生物学特性的影响.方法:用密度梯度离心加贴壁培养法分离、扩增大鼠MSCs.取第4代MSCs,用终浓度为25μg Fe/mL的SPIO-多聚赖氨酸(PLL)复合物进行标记.标记后动态观察MSCs的形态改变,用台盼蓝染色检测细胞的活力,MTT比色法检测细胞增殖能力,普鲁士蓝铁染色证实细胞内铁,分光光度法测定细胞铁含量.结果:MSCs的SPIO-PLL标记阳性率达100%,标记后的MSCs细胞内铁含量为(18.38±2.80)pg/cell,是未标记细胞的76倍.标记细胞的形态、活力、生长增殖能力与未标记细胞相比无明显改变.结论:PLL介导SPIO标记MSCs方法简便、效率高,不影响MSCs的生物学特性,可用于MSCs的标记和示踪.     

超声新技术预测心脏再同步化治疗疗效的动物实验和临床研究

目的 采用高场MR在体监测超顺磁性氧化铁颗粒(SPIO)标记大鼠骨髓基质细胞(BMSCs)在脑损伤模型大鼠脑内的分布与迁移。方法 采用Feeney法制作大鼠脑外伤(TBI)模型,然后平均分为4组:A 组:TBI后移植SPIO 标记BMSCs;B组:TBI后移植单纯SPIO;C组:TBI后移植未标记BMSCs;D组:TBI后移植培养液。其中,A组为实验组,其余均为对照组。①SPIO 标记:将1μl的SPIO(Resovist)加入2ml培养基混匀,使其终浓度为35μg/ml;然后将P6代细胞消化离心后加入上述加有SPIO 的培养基混匀;37℃、5% CO₂ 条件下共同孵育。②细胞内铁颗粒的鉴定:采用细胞普鲁士蓝染色法,将贴壁干细胞去除培养液后以磷酸盐缓冲液(PBS)冲洗,4%戊二醛固定30min,蒸馏水冲洗3次,2%亚铁氰化钾-6%盐酸水溶液孵育30min,1%核固红复染3min,蒸馏水洗去多余的核固红,显微镜下观察。③细胞移植:TBI模型鼠建立后24h进行立体定向移植手术。模型鼠用10%水合氯醛0.32ml/100g体质量腹腔注射再次麻醉后,俯卧位保定于立体定向架上,损伤顶叶皮层接受细胞移植。移植位点位于左顶叶TBI周围皮质(前囟后3mm,旁开2mm,深5mm),垂直距离以硬脑膜为标准。A组移植SPIO 磁化标记的BMSCs;B组仅移植等量等浓度的SPIO;C组移植等量等浓度的未标记BMSCs;D组仅移植等量的培养液。取10μl细胞(约0.5×10⁶ 个)悬液或对照液5min内注射,留针5min,然后缓慢拨出注射针头。移植完毕缝合头皮,腹腔注射庆大霉素2000U 抗菌治疗,回笼饲养。于移植后第1、3天及1、3周对各组实验动物行MR 扫描。结果 SPIO 标记后的BMSCs在倒置显微镜下可见细胞质内有黄色铁颗粒。行普鲁士蓝染色后,标记BMSCs胞质内可见阳性蓝色颗粒。透射电镜下胞浆内可见许多散在的铁颗粒。A组在各个时间点均可见位于左侧半球的移植部位在T1WI、T2WI、T2^* WI及磁敏感加权成像(SWI)序列上呈类圆形点状低信号,以T2^* WI及SWI序列的敏感度、对比度明显,SWI最明显(图3B);B组仅在移植后1天接受MR检查,呈低信号,3天及以后MR检查可见低信号消失;C、D两组在各个时间点均未见显示。A组动物左侧顶叶皮质的类圆形点状低信号在3天、1周及3周逐渐向损伤区迁移,尤其在SWI序列上显示更清楚。结论 高场MRI能够在活体内连续示踪观察SPIO 标记的BMSCs的分布与迁移,且以SWI序列最为敏感。     

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.     

SPIO标记猪脂肪干细胞的生物学特性与多向分化潜能及体外3.0TMR成像

背景:不同种类细胞的最佳化标记方案需要大量实验证明,而每种细胞对应标记策略的安全性检测至关重要.目的:应用超顺磁性氧化铁联合多聚左旋赖氨酸标记猪脂肪干细胞,探讨磁标记对细胞生物学特性和多向分化潜能的影响以及标记细胞体外3.0T MR成像特性.方法:五指山小型猪皮下脂肪分离培养脂肪干细胞;超顺磁性氧化铁-多聚左旋赖氨酸复合物标记液标记脂肪干细胞;应用3.0T MR对不同浓度标记细胞进行T1WI、T2WI及T2*WI序列体外成像.结果与结论:普鲁士蓝染色显示标记细胞胞质内含有多少不等的蓝染铁颗粒,细胞标记率近100%;标记细胞向心肌、骨、脂肪方向诱导分化成功;不同浓度标记细胞MR扫描显示,随细胞浓度升高,3种序列信号变化率均增加;相同浓度细胞T2*WI信号变化率最大,T1WI最小,同一浓度3种MR序列间信号变化率差异均有显著性意义(P < 0.01);3.0T MR成像能检测到至少1×106 L-1标记细胞.结果显示应用超顺磁性氧化铁联合多聚左旋赖氨酸标记方案可有效标记脂肪干细胞,不影响细胞活力、增殖及多向分化能力;T2*WI序列检测标记细胞最敏感.     

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。     

Guns and Knives in New Mexico: Patterns of Penetrating Trauma, 1978–1993

Objective: To identify patterns of nonfatal and fatal penetrating trauma among children and adults in New Mexico using ED and medical examiner data.
Methods: The authors retrospectively sampled in 5-year intervals all victims of penetrating trauma who presented to either the state Level-1 trauma center or the state medical examiner from a 16-year period (1978–1993). Rates of nonfatal and fatal firearm and stabbing injury were compared for children and adults.
Results: Rates of nonfatal injury were similar (firearm, 34.3 per 100,000 person-years; stabbing, 35.1). However, rates of fatal injury were significantly different (firearm, 21.9; stabbing, 2.7; relative risk: 8.2; 95% confidence interval: 5.4, 12.5). From 1978 to 1993, nonfatal injury rates increased for children (p = 0.0043) and adults (p < 0.0001), while fatal penetrating injury remained constant. The increase in nonfatal injury in children resulted from increased firearm injury rates. In adults, both stabbing and firearm nonfatal injury rates increased.
Conclusions: Nonfatal injury data suggest that nonfatal violence has increased; fatal injury data suggest that violent death rates have remained constant. Injury patterns vary by age, mechanism of trauma, and data source. These results suggest that ED and medical examiner data differ and that both are needed to guide injury prevention programs.     

Systematic Reviews Published in the January 2011 Issue of The Cochrane Library

ABSTRACT

The Cochrane Library of Systematic Reviews is published quarterly as a DVD and monthly online. The January 2011 issue (first quarterly DVD for 2011) contains 4515 complete reviews, 1985 protocols for reviews in production, and 13,521 one-page summaries of systematic reviews published in the general medical literature. In addition, there are citations of 641,000 randomized controlled trials, and 14,018 cited papers in the Cochrane methodology register. The health technology assessment database contains over 9300 citations. One hundred and seven new reviews have been published in the last 3 months, of which five have potential relevance for practitioners in pain and palliative medicine.     

Prise en charge des entorses externes de cheville : étude clinique préliminaire sur l’efficacité du dispositif Myolux™

Ankle sprains are the most common injury of the musculoskeletal system and are associated with significant societal and economic impacts. It has been proven that classical therapeutic strategies may not be effective in preventing recurrent injuries: the recurrence rates reported in the literature can reach 73%. In order to provide an effective rehabilitation solution, a destabilizing orthosis was developed. This device is equipped with a mechanical articulator reproducing the subtalar mechanics and placed under the heel. In this paper, we present the main results of a preliminary clinical study conducted between 2004 and 2007. All subjects included in this study were treated with the abovementioned orthosis during 10 rehabilitation sessions of 30 minutes each. Data show a relatively low recurrence rate of 12% for the overall population. Moreover, it's of primary importance to note that this satisfactory ratio is largely reduced (3% of recurrence rate) for the 29 patients who performed one training session per month after the 10th initial rehabilitation sessions. Hence, the destabilizing orthosis appears to be an effective solution to prevent recurrent ankle sprains. However, joint protection requires long-term and regular training sessions. This result has motivated the development of a similar device allowing patients to perform training sessions at home. Finally, data obtained in this study are promising awaiting the final results of the comparative, multicentric and independent clinical trials currently managed by the Hospices Civils de Lyon.     

Clinical Trial Updates and Hotline Sessions presented at the European Society of Cardiology Congress 2007

This article provides information and commentaries on trials which were presented at the Hotline and Clinical Trial Update Sessions at the European Society of Cardiology Congress 2007 in Vienna. The key presentations were performed by leading experts in the field with relevant positions in the trials or registries. It is important to note that unpublished reports should be considered as preliminary data, as the analysis may change in the final publications. The comprehensive summaries have been generated from the oral presentation and the webcasts of the European Society of Cardiology and should provide the readers with the most comprehensive information of relevant publications.     

Balance and Boundaries in Grief Counseling: An Intervention Framework for Volunteer Training

Volunteers or paraprofessional counselors are commonly used to provide supportive care to the bereaved. These counselors generally are trained in basic listening skills, providing a generic, nonspecific approach to intervention that remains to be proven effective. The present paper outlines a framework that provides paraprofessionals with a broader model for intervention with the bereaved. Attention to boundaries as a helper and balance in the grief recovery are central to the model. Interventions are described that provide the paraprofessional counselor with more options for tailoring their counseling strategy to the individual. These include techniques that are presumed to be more specific to the enhancement of grief recovery.     

A new method for the simultaneous analysis of unmodified and modified urinary nucleosides and nucleobases by high performance liquid chromatography

Details are given of a new, rapid and simple pre-fractionation method and an isocratic high performance liquid chromatography system suitable for parallel analysis of nucleosides and nucleobases from urine and other biological fluids. The quantitative recovery and excellent reproducibility of the method is demonstrated by analysis of representative standard RNA catabolites. The advantage of this new method for application to biological samples is discussed.     

Adsorption of Charged Non-albumin Bound Drugs by Amorphous Silica

We investigated the in vitro drug adsorption of PQ 10150 sodium silicate gel (AIS, Santa Clara, CA) with particle size of 230 um and surface area of 400 nr/g. We observed 99% to 88% adsorption of gentamicin; a mean 91 % of disopyramide; a mean 89% of quinidine at low concentration, falling to 75% at higher concentration. Insulin was 88% adsorbed at low concentrations but less so (65%) at higher concentrations. We observed a mean 83 % adsorption of procainamide, a mean 84% of N-acetyl procainamide, 74% oflidocaine, 73% of amitriptyline; and 44% of desipramine. We found an average 14% reduction of total digoxin concentration when serum containing digoxin (2 to 33 ng/mL) was exposed to sodium silicate, while the reduction in free digoxin concentration was 16%. Five percent ethosuximide was also removed. The adsorption of theophylline, phenobarbital, acetaminophen, phenytoin, ethylene glycol, methotrexate, salicylate, thiocyanate and diazepam was minimal and not significant. We conclude that significant amounts of charged, non-albumin bound drugs can be removed by PQ 10150 sodium silicate gel.     

自动化酸碱平衡图在急诊社区获得性肺炎诊治中的应用

目的 探讨自动化酸碱平衡图在急诊科社区获得性肺炎(CAP)患者诊断中的价值.方法 根据病史、肺功能测定结果、慢性阻塞性肺疾病(COPD)诊断标准,将111例CAP患者分为单纯CAP组(56例)和COPD合并CAP组[即慢性阻塞性肺疾病急性加重(AECOPD)组,55例].询问患者病史后即刻抽取动脉血测血气并进行自动化酸碱平衡图分析.结果 血气分析结果显示,AECOPD组动脉血二氧化碳分压(PaCO2,kPa)、HCO3- (mmol/L)、剩余碱(BE,mmol/L)均显著高于CAP组(PaCO2:7.714±2.414比5.896±1.308,HCO3-:30.767±7.185比25.014±3.043,BE:4.345±5.371比-0.354±3.180,均P＜0.01).自动化酸碱平衡图分析结果显示,AECOPD组患者酸碱平衡紊乱高达89.1％,CAP组为66.1％.将AECOPD组和CAP组患者中正常(10.9％、33.9％)、急性呼吸性酸中毒(急性呼酸,12.7％、14.3％)、慢性呼吸性酸中毒(慢性呼酸,49.1％、10.7％)、呼吸性碱中毒(呼碱,7.3％、14.3％)、代谢性酸中毒(代酸,12.7％、17.9％)、代谢性碱中毒(代碱,12.7％、8.9％)综合进行x2分析,差异有统计学意义(x2=24.421,P=0.001),而将正常、急性呼酸、呼碱、代酸及代碱进行x2分析,差异无统计学意义(x2=5.280,P=0.260),提示AECOPD患者慢性呼酸的发生率较单纯CAP患者显著增加.结论 自动化酸碱平衡图能帮助急诊科医师快速识别CAP患者是否存在多重酸碱平衡紊乱,并可快速识别急、慢性呼吸系统疾病.