携带抗P-选择素单抗靶向微泡和对比超声评价肾缺血再灌注损伤

目的探讨携带抗小鼠P-选择素单抗的靶向超声微泡（MBp）和对比超声（CEU）评价肾缺血再灌注损伤的可行性。方法12只实验小鼠随机均分为2组：缺血再灌注（IR组）和假手术组（SH组）,应用＂亲和素-生物素＂桥连法构建MBp。所有小鼠分别随机（间隔30min）经静脉弹丸注射给予普通脂质微泡（MB）和MBp,10min后行肾CEU检查,测量肾显影的声强度（VI）,最后进行肾组织免疫组化检测。结果第一帧CEU图像显示MBp及MB在IR组缺血再灌注肾分别可见显著及轻度的超声显影,而两者在SH组假手术肾均无明显的超声显影。VI值在IR组MBp较IR组MB及SH组MBp均明显增大（P〈0.05）;而在IR组MB较SH组MB轻度增大（P〈0.05）。免疫组化检测显示缺血再灌注肾血管内皮P-选择素表达较假手术肾明显增加。结论应用MBp行CEU检查可有效评价小鼠肾缺血再灌注损伤,将可用于评价微血管炎症或相关的血管内皮反应。     

携Sialyl Lewis~X和抗小鼠P-选择素单抗靶向超声微泡评价心肌缺血再灌注损伤对比研究

目的探讨携Sialyl Lewis~X靶向超声微泡结合对比超声分子成像评价心肌缺血再灌注损伤可行性并与携抗小鼠P-选择素单抗靶向超声微泡对比分析。方法采用"亲和素-生物素"桥接法构建携Sialyl Lewis~X和抗小鼠P选择素单抗靶向超声微泡(MB_(slex)、MBp),并应用平行板流动腔技术在体外模拟的生理血流条件下评价MBp和MB_(slex)与小鼠P-选择素Fc段的靶向黏附效能。然后,20只心肌缺血再灌注(IR)小鼠随机经静脉注入MB_(slex)和MBp,分别于注入5min后行心肌对比超声心动图(MCE)检查,测量心肌缺血区和非缺血区的声强度(VI)。结果平行板流动腔实验显示:在第6分钟时,MB_(slex)与小鼠P-选择素Fc段结合数目为MBp的1.7倍。对比超声图像显示MB_(slex)组和MBp组缺血区心肌均见显著造影增强,声强度(VI)值分别为(23.52±1.08)U、(25.98±6.23)U,两者相比无显著差异(P0.05)。但无论是MBp组还是MB_(slex)组的缺血区心肌VI值均明显高于非缺血区心肌VI值[(6.53±0.95)U,(7.13±0.91)U,(P0.05)]。结论 MB_(slex)对炎症组织靶向检出能力与MBp相似,它和对比超声结合可有效评价心肌缺血再灌注损伤。     

流式细胞术定量评价携抗P-选择素单抗靶向超声微泡配体结合效率

目的 流式细胞术定量评价应用生物素(B)-亲和素(S)-生物素(B)桥连技术构建的携抗P-选择素靶向微泡(MB-BSBp)的配体结合效率.方法 以未标记荧光的MB-B作对照,用不同荧光物质分别标记MB-BSBp不同部位, 应用流式细胞仪分析各组微泡的荧光强度并定量配体结合率.C57小鼠8只建立肾脏缺血再灌注模型载体评价靶向微泡成像质量.结果 MB-B与FITC-streptavidin的结合率达99%;DTAF-二抗与MB-BSBp的结合率为(59.66±2.30)%,明显高出荧光二抗与MB-BS和MB-B的结合,比率分别为(29.87±1.68)%和(26.50±3.86)%(P<0.05).结论 抗P-选择素单抗通过生物素桥连法有效装配在MB-B表面,流式细胞术可能是定量评价靶向微泡配体结合效率的有效方法.     

携P选择素单抗靶向微泡在高剪切应力下体外血栓超声分子成像的效果

目的制备携P-选择素单抗的靶向超声微泡(MBp),通过体外流动腔模型评价其在高剪切应力下行血栓超声分子成像的效果。方法采用"亲和素-生物素"桥接法制备MBp和同型对照微泡(MB),应用自制琼脂糖流动腔模型模拟体内高剪切应力血流环境,将MBp和MB随机先后注入琼脂糖模型内,与小鼠血栓孵育30min后,PBS液15cm/s流速不间断冲洗血栓,分别在冲洗2、4、6、8和10min时行对比超声检查并测量声强度(VI)值。免疫组化评价小鼠血栓的P-选择素表达。结果冲洗前MBp组和MB组血栓VI值无明显差异(P0.05);冲洗后VI值在各时间点MBp组均较MB组大(P0.05)。冲洗10min后MBp组血栓仍有可视性对比增强,而MB组血栓在冲洗2min后已无可视性对比增强。免疫组化显示血栓表面有明显的P-选择素表达。结论以P-选择素为靶标的MBp在高血流剪切应力下与血栓的靶向结合稳定可靠,可用于动脉血栓的超声分子成像。     

双靶向超声微泡造影剂评价小鼠缺血再灌注心肌

目的 制备同时携载P选择素和ICAM-1抗体的靶向超声微泡造影剂,以评估小鼠缺血再灌注损伤心肌声学造影显像效果.方法 采用生物素-亲和素方法制备靶向微泡,于激光共聚焦显微镜下观察微泡形态,流式细胞仪检测连接效率.将24只健康昆明小鼠随机分成3组:结合双抗体选择素微泡组(MBd组)10只、结合P选择素单抗组(MBp组)10只,空白微泡组(MBc组)4只.结扎冠状动脉左前降支近左主干分支,制作心肌缺血再灌注模型,60 min后经尾静脉分别注射MBd、MBp及MBc,采集心肌对比造影图像.所有图像均采用Sonomath超声影像分析仪处理.结果 MBd组和MBp组对缺氧内皮细胞的黏附力以及对缺血再灌注区心肌显像增强程度显著高于MBc组(P均＜0.05),MBd组对缺血再灌注区心肌显像延迟时间高于MBp组(P＜0.05).结论 双靶向微泡联合超声造影是检测和评估小鼠缺血再灌注心肌的无创性手段.     

亲和素桥连构建携抗P选择素单抗靶向超声微泡及体外荧光鉴定

目的 体外荧光方法鉴定生物素-亲和素-生物素(BSB)桥连技术构建携带抗P-选择素单抗靶向超声微泡(MB-BSBp)的町靠性.方法 不同荧光标记物分别标记MB-BSBp的不同部位,观测微泡荧光强度(0、1、2和3级),以普通脂质超声微泡(MB)作对照.结果 加入FITC荧光亲和素孵育,生物紊化脂质微泡(MB-B)呈现明亮的绿色荧光(3级),而MB无荧光显示(0级).以两个浓度梯度(1:4和1:16)的DTAF荧光二抗(抗抗P-选择素单抗抗体)标记MB、MB-B、MB-BS和MB-BSBp,MB-BSBp在两个浓度梯度下均发出明亮的绿色荧光(3级);而MB-B、MB-BS仅在1:4时显示微弱的绿色荧光(1级),MB在两个浓度梯度下均无荧光显示(0级).结论 抗P-选择素单抗通过亲和素桥连有效装配在MB-B表面,体外荧光法是鉴定靶向微泡配体连接可靠性的简便方法.     

在生理血流条件下靶向超声微泡对P-选择素的靶向黏附效能

目的平行板流动腔评价在生理血流条件下携抗小鼠P-选择素单抗靶向超声微泡（MBp）的靶向黏附效能。方法采用＂亲和素-生物素＂桥接法构建MBp;在3种浓度（10、100和1000ng/ml）小鼠P-选择素Fc段（PSFc）包被的平行板流动腔和固定剪切应力下,以及最大包被浓度和不同剪切应力（0.2-1.7dyn/cm^2）下分别检测MBp的每分钟结合数量（结合率）,以抗小鼠P-选择素单抗封闭组和空白组为对照。在3种包被浓度下检测MBp达半数解离的剪切应力。所有分组样本数均为3。结果两对照组均未见有明显的MBp结合。实验组MBp结合率随包被浓度的增高而增加（P〈0.05）,然而与剪切应力呈现双向性（P〈0.05）。MBp达半数解离的剪切应力随包被浓度的增加而增大（P〈0.05）。结论MBp在生理条件下可与PSFc特异有效地结合,体外对靶向超声微泡的靶向黏附效能评价将有助于判定超声分子成像的效果和靶向微泡的在体应用环境条件。     

携Sialyl Lewis~X与携抗P-选择素单抗靶向超声微泡粘附性的对比研究

目的对比评价携sialylLewis。与携抗P-选择素单抗靶向超声微泡粘附特性。方法采用“亲和素-生物素”桥接法构建携Sialyl Lewis、（MB-S）、携抗P-选择素单抗靶向超声微泡（MB-P）及携同型抗体微泡（MB-C）。MB-S、MB-P及MB-C以相同流速通过相应小鼠P-选择素Fc段包被培养皿时,利用平行板流动腔在不同时间点测定相应的MB-S、MB-P及MB-C（对照组）的结合数目、滚动数目以及解离时达到半数解离的剪切应力。结果MB-S结合数量前3min快速增加,其后随时间增加无明显变化,而MB-P结合数量与时间呈正相关（P〈0．05）,且MB-S结合数目是MB-P的2～4倍;对照组MB-C未见明显结合（P〈0．05）。MB-S滚动数目大于MB-P（P〈0．05）;MB-S半数解离时剪切力小于MB-P（P〈0．05）。结论靶向超声微泡MB-S表现为早期、快速、不稳定的结合及滚动,MB-P表现为缓慢牢固结合。     

定点追踪技术评价携Sialyl Lewisx和抗P-选择素单抗靶向微泡在高剪切应力下的黏附行为

目的构建携Sialyl Lewisx和抗P-选择素单抗靶向微泡,用定点追踪技术在体外高剪切应力下评价其黏附行为。方法构建携Sialyl Lewisx靶向微泡(MB-S)、携抗P-选择素单抗靶向微泡(MB-P)、携Sialyl Lewisx和抗P-选择素单抗双配体靶向微泡(MB-D)。利用平行板流动腔和Image-Pro-Plus软件绘制微泡滚动的时间-速度折线图。微泡第1帧的速度为V1,第2帧至黏附前倒数第3帧的平均速度为V2,黏附前倒数第2帧的速度为V3。结果 MB-P高速滚动,速度骤降为0;MB-S从高速平缓过渡到低速,再渐降低至0;MB-D从高速过渡到低速,再骤降为0。3种微泡V1差异无统计学意义(P>0.05);MB-P的V2明显高于MB-S和MB-D(P<0.01);MB-S和MB-D间V2差异无统计学意义(P>0.05)。V3在3种微泡中的顺序为MB-P>MB-D>MB-S(P<0.01)。结论Sialyl Lewisx介导高效滚动,抗P-选择素单抗介导微泡速度的骤降,两者在微泡靶向黏附时可发挥互补作用。     

P-选择素靶向超声微泡与普通超声微泡粘附途径和效能的对比研究

目的荧光显微镜直视下对比评价P-选择素靶向超声微泡与普通超声微泡在炎症内皮上的粘附途径和效能。方法构建携荧光FITC、抗P-选择素单抗的靶向超声微泡（MBp）和携荧光FITC普通超声微泡（MB）,随机经静脉弹丸式分别注入对照组（10只）和肿瘤坏死因子（TNF—α）处理组（10只）的小鼠提睾肌炎症模型。同时,20高倍荧光显微镜观测5min内两组不同微泡在提睾肌微血管中的粘附数量和粘附途径。结果TNF—α处理组MBp粘附数量为（12±2．6）个／视野,而MB粘附数量为（3±1．2）个／视野,两者差异有统计学意义（P〈0．01）;且TNF—α处理组MBp粘附数量分别为对照组MBp和MB的（6．4±1．7）倍和（9．9±2．1）倍（P〈0．01）。TNF-Ⅱ处理组和对照组分别有（69．6±2．6）％和（56．6±25．4％）的MBp通过直接与内皮细胞结合实现粘附,而MB内皮粘附率仅为（24．6±23．3）％和（20．0±27．4）％。结论与MB比较,MBp能高效、特异地粘附于炎症组织血管内皮上,应用其可有效评价血管内皮炎症反应或其他组织损伤。     

小鼠心肌缺血再灌注模型制备及其超声分子成像研究

目的以显微外科技术建立小鼠心肌缺血-再灌注模型,用其评价携抗P-选择素单抗靶向微泡(MBp)的"心肌炎症"超声分子成像效果。方法 30只昆明小鼠随机均分为缺血-再灌注(ischemia-reperfusion,IR)和假手术(sham surgery,SH)组,手术组结扎冠脉前降支15min,再灌注1h。心电图、M型超声、2,3,5-氯化三苯基四氮唑(TTC)和HE染色评价模型构建情况,分别将普通微泡和MBp经静脉注射到实验小鼠体内,评价MBp的超声分子成像效果。结果结扎前降支时前壁心肌呈明显充盈缺损,心电图ST段呈弓背向上抬高。再灌注后充盈缺损消失,ST段降至正常;M型超声检查显示,IR组左室短轴缩短率(LV-%FS)明显低于假手术组(P0.05);心脏TTC染色无坏死表现,而HE染色提示缺血区心肌存在炎症改变;MBp造影显示前壁心肌呈选择性显影增强,前壁视频强度(video intensity,Ⅵ)明显高于后壁(P0.01)。增强区域与结扎前降支时的充盈缺损区域高度相关(r=0.95)。结论 MBp可以有效评价心肌缺血再灌注"炎症"损伤,小鼠心肌缺血再灌注模型适宜于超声和其它显像方法的"炎症"分子成像研究。     

超声辐照载10-HCPT微泡在小鼠H22肝癌移植瘤的定位释放研究

目的制备载10-HCPT脂质超声微泡(HLM),结合超声定位破坏微泡的方法实现10-HCPT在H22移植瘤的定位释放。方法用机械振荡法制备HLM。用荧光显微镜观察荧光标记的HLM在肿瘤中的分布情况。将荷瘤小鼠分为5组:超声+10-HCPT注射液组、超声+空白脂质微泡+10-HCPT注射液组、单独HLM组、超声+HLM组及生理盐水对照组。于处理1h后处死小鼠剥取肿瘤及心、肝、脾、肺、肾,测定各组织内药物浓度。结果病理切片见荧光素在肿瘤的分布超声+HLM组明显多于单独HLM组。药物浓度检测显示:1 h后肿瘤内10-HCPT浓度最高的是超声+HLM组,其他组织中10-HCPT浓度使用HLM的两组显著高于使用10-HCPT注射液的两组(P0.05)。结论局部超声辐照破坏HLM的方法能定位释放10-HCPT,提高移植瘤局部的10-HCPT含量,同时脂质微泡作为10-HCPT的载体能延长其体内循环时间。     

Evaluation of Transfection Efficiency in Skeletal Muscle Using Nano/Microbubbles and Ultrasound

Recent studies have revealed that ultrasound contrast agents with low-intensity ultrasound, namely, sonoporation, can noninvasively deliver therapeutic molecules into target sites. However, the efficiency of molecular delivery is relatively low and the methodology requires optimization. Here, we investigated three types of nano/microbubbles (NMBs)—human albumin shell bubbles, lipid bubbles and acoustic liposomes—to evaluate the efficiency of gene expression in skeletal muscle as a function of their physicochemical properties and the number of bubbles in solution. We found that acoustic liposomes showed the highest transfection and gene expression efficiency among the three types of NMBs under ultrasound-optimized conditions. Liposome transfection efficiency increased with bubble volume concentration; however, neither bubble volume concentration nor their physicochemical properties were related to the tissue damage detected in the skeletal muscle, which was primarily caused by needle injection. (E-mail: kodama@bme.tohoku.ac.jp)     

Comparison of Magnetic Microbubbles and Dual-modified Microbubbles Targeted to P-selectin for Imaging of Acute Endothelial Inflammation in the Abdominal Aorta

Purpose

Ultrasound molecular imaging (UMI) has potential to evaluate an inflammatory profile of endothelium. However, it is less successful in large arteries. This study compared magnetic microbubbles (MBs) selectively targeted to endothelial P-selectin and dual-targeting MBs in vitro and in vivo.

Procedures

MBs were modified with P-selectin antibody (MB_PM) or isotype control antibody (MB_CM) via a magnetic streptavidin bridge, and MBs were conjugated to P-selectin antibody (MB_P) or both P-selectin antibody and PAA-sialyl Lewis^x (MB_D) via regular streptavidin linker. Adherence of MBs was determined by using a parallel plate flow chamber at variable shear stress (0.5–24 dyn/cm²). Adhesive and magnetic behaviors of MBs were analyzed at 4.0 dyn/cm² or at a flow rate of 50 mm/s. Attachment of MBs to P-selectin was determined with contrast-enhanced ultrasound (CEU) imaging of murine abdominal aorta inflammation. The expression of P-selectin was assessed by immunohistochemistry.

Results

The adhesive efficacy of MB_D was greater than MB_P and MB_CM, but lower than MB_PM under all shear stress conditions (P?<?0.05). The behaviors of fast-binding and rolling slow down were noted in MB_D and MB_PM; meanwhile, magnetic shifting of MBs centerline was presented in MB_PM. Contrast video intensity (VI) from adhered MB_PM to P-selectin of the inflammatory aorta was significantly higher than those from MB_D and MB_P (P?<?0.05).

Conclusions

MB_PM may be a better molecular probe than MB_D for detection of P-selectin on aorta with CEU, likely due to the shifting of axial distribution. Thus, it may improve the detection of the inflammatory profile on large arteries by UMI.

     

自制靶向脂质微泡与普通脂质微泡超声显影的对比实验研究

目的探讨自制的靶向脂质微泡超声造影剂在正常兔肝脏与VX2兔肿瘤模型的超声成像特点,对比研究其与普通脂质做泡超声造影剂超声成像的异同。方法分别从正常兔及VX2肿瘤兔耳缘静脉团注普通脂质微泡超声造影剂与自制靶向脂质微泡超声造影剂,使用飞利浦iU22超声诊断仪的造影模式,实时监控2种不同造影剂各自的超声显影特点。DFY定量分析诊断仪分析图像的达峰时间、峰值回声强度、清除时间并对各组参数进行比较。结果在正常兔肝实质,普通脂质微泡超声造影剂(MB)达峰时间明显早于自制的靶向脂质微泡超声造影剂(MB’)达峰时间,峰值回声强度前者高于后者,清除时间前者显著短于后者。在VX2肿瘤区,MB达峰时间明显早于MB’,峰值回声强度前者明显高于后者,清除时间前者显著短于后者。结论自制靶向脂质微泡超声造影剂有其自身独特的超声显像过程和特点,呈"负向显影"模式。     

Real-Time Contrast-Enhanced Ultrasound for the Assessment of Perfusion Dynamics in Skeletal Muscle

We developed a real-time low-MI contrast-enhanced ultrasound method (CEUS), compared it with venous occlusion plethysmography (VOP) and evaluated its robustness in the quantification of skeletal muscle perfusion during exercise. Contrast pulse sequencing (7 MHz) during continuous intravenous infusion of SonoVue (4.8 mL/300s) was used repeatedly in eight healthy volunteers to monitor changes of the muscle perfusion before, during and after isometric exercises (10 to 50% of individual maximum strength for 20 to 30 s) of the gastrocnemius muscle in real time. CEUS was correlated with VOP at different time points, and the exactness of several CEUS parameters obtained from ultrasound-signal-intensity-time curves was evaluated. Real-time CEUS depicted a large variability of the skeletal muscle blood volume at rest (mean, 3.48; range, 0.60 to 9.92 [mL]), with a significant reproducibility (r = 0.72, p < 0.05) and correlation with VOP (r = 0.59, p < 0.001). Mean blood volume during exercise was 1.58(mL), increased to a mean maximum after exercise of 8.88(mL), the mean change of the local blood volume during and directly after the exercise was –0.10 and +1.57(mL/s). The average CEUS signal during exercise decreased (mean area under the curve, –50.4[mL·s]) and subsequently increased post exercise (mean 118.6[mL·s]). CEUS parameters could be calculated with mean relative errors between 6 and 36%. Continuous assessment of local muscle microcirculation during exercise is possible with real-time CEUS with an acceptable robustness. Its application may be of particular interest in a better understanding of the role of perfusion during muscle training, and the monitoring of pathological vascular response, such as in diabetic microvessel diseases. (E-mail: martin.krix@kabelbw.de)     

Determination of Skeletal Muscle Microvascular Flowmotion with Contrast-Enhanced Ultrasound

Most methods of assessing flowmotion (rhythmic oscillation of blood flow through tissue) are limited to small sections of tissue and are invasive in tissues other than skin. To overcome these limitations, we adapted the contrast-enhanced ultrasound (CEUS) technique to assess microvascular flowmotion throughout a large region of tissue, in a non-invasive manner and in real time. Skeletal muscle flowmotion was assessed in anaesthetised Sprague Dawley rats, using CEUS and laser Doppler flowmetry (LDF) for comparison. Wavelet transformation of CEUS and LDF data was used to quantify flowmotion. The α-adrenoceptor antagonist phentolamine was infused to predictably blunt the neurogenic component of flowmotion. Both techniques identified similar flowmotion patterns, validating the use of CEUS to assess flowmotion. This study demonstrates for the first time that the novel technique of CEUS can be adapted for determination of skeletal muscle flowmotion in large regions of skeletal muscle.