携Sialyl Lewisx和抗ICAM-1单抗双配体微泡靶向黏附行为方式的体外研究

目的 体外对比评价携Sialyl Lewisx和抗ICAM-1单抗双配体和同型对照单配体微泡的靶向黏附行为方式.方法 构建携Sialyl Lewisx与抗ICAM-1单抗双配体(MB-D)、携Sialyl Lewisx(MB-S)和抗ICAM-1单抗(MB-I)三种靶向微泡,利用平行板流动腔和Image-Pro-Plus图像分析软件,分别在0.6、2.0和4.0 dyn/cm2三种剪切应力下分析每种微泡流动过程的速度变化,测定滚动和牢固结合的微泡数目,并检测三种靶向微泡的半数解离应力.结果 0.6 dyn/cm2时单个视野内微泡的滚动数目,MB-D和MB-S明显高于MB-I(P＜0.05),而在2.0和4.0 dyn/cm2时MB-S分别约是MB-D和MB-I的1.47～1.70倍和4.52～10.65倍(P＜0.05).三种剪切应力下微泡的全程(6 min)结合数目MB-D分别为MB-S和MB-I的1.40～1.44倍和2.18～3.78倍(P＜0.05).MB-S、MB-D和MB-I的半数解离应力依次递增.结论 三种微泡具有不同的靶向黏附行为方式,MB-I表现为低效滚动后的牢固结合,MB-S呈高效滚动后的不稳定黏附,而MB-D能够实现高效滚动后相对牢固的结合,其靶向黏附效能明显高于同型对照微泡,有望在体内动脉系统高剪切应力状态下实现分子影像评价.

Abstract：

Objective To assess the adhesive behavior of dual-targeted microbubbles carrying both Sialyl Lewisx and anti-ICAM-1 monoclonal antibodies in vitro. Methods Selectin-targeted (with Sialyl Lewisx) microbubbles (MB-S),ICAM-1-targeted (with anti-ICAM-1 monoclonal antibodies) microbubbles (MB-Ⅰ),and dual-targeted (with both ligands) microbubbles(MB-D) were prepared by attaching the ligands to the biotinylated lipid-microbubbles via multi-step avidin biotin bridging chemistry. A parallel plate flow chamber combined with a novel automated tracking algorithm,were used to analyze the transient velocities,rolling and firmly adherent numbers of microbubbles at various shear stress (0. 6,2.0 and 4.0 dyn/cm2)over 6 min. Microbubbles detachments were tested by ramping up the shear stress at 30 s intervals. Results At 0.6 dyn/cm2 shear stress, the rolling numbers of MB-S and MB-D were remarkably more than that of MB-I( P＜0.05), while at 2.0 and 4.0 dyn/cm2 MB-S performed higher rolling efficiency as compared with either MB-I and MB-D ( P＜ 0.05). In all flow conditions, the adhesive numbers of MB-D to the targets were obviously greater than those of MB-S and MB-I ( P＜ 0.05). Half-maximal detachment decreased gradually in MB-I, MB-D and MB-S by turns ( P＜ 0.05). Conclusions MB-I, MB-S and MB-D have different adhesive behaviors. MB-I exhibites primarily firm adhesion with low rolling efficiency, while MB-S reveales unstable or transient adhesion with high rolling efficiency,and MB-D exhibites firm adhesion with high rolling efficiency. MB-D may be suitable for molecular imaging in high-flow vessels.     

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。     

体外评价携Sialyl Lewisx和抗ICAM-1单抗双配体超声微泡的靶向黏附性能

目的 构建携Sialyl Lewisx和抗ICAM-1单抗双配体和同型对照单配体靶向超声微泡,体外对比评价其靶向黏附性能.方法 采用"亲和素-生物素"桥接法构建携Sialyl Lewisx与抗ICAM-1单抗双配体(MB-D)、携Sialyl Lewisx(MB-S)和携抗ICAM-1单抗(MB-I)三种靶向超声微泡,以流式细胞术定量分析其配体结合率,并利用平行板流动腔分别在0.5、2.0和4.0 dyn/cm2三种剪切应力下的不同时间点观察微泡的结合及解离情况.结果 MB-D、MB-S和MB-I的配体结合率均达85%以上,各组间无统计学差异(P>0.05).在三种剪切应力下,MB-D和MB-S集中于前3～5 min高效结合,在5 min后两者的结合率均呈平台状态,而MB-I全程结合率(1～6 min)均呈低水平状态;MB-D的平均结合率和全程结合数目均明显高于MB-S和MB-I(P<0.05),而半数解离剪切应力则按MB-I、MB-D和MB-S顺序依次递减(P<0.05).结论 相同条件下,MB-S表现为早期快速、不稳定的黏附,MB-I为缓慢、牢固的结合,而MB-D呈早期高效、相对牢固的结合.MB-D有可能用于高流速的动脉超声分子成像.     

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。     

携抗P-选择素单抗靶向微泡在炎症模型微循环中黏附机制及行为方式的研究

目的 荧光显微镜直视下对比评价携抗P-选择素单抗靶向微泡与同型对照微泡在微循环中的黏附机制及行为方式.方法 构建携荧光FITC的抗P-选择素单抗靶向微泡(MBp)和同型对照微泡(MBiso),并随机经静脉注入小鼠提睾肌炎症模型.20倍荧光显微镜直视下观察并记录5min内两种微泡在提睾肌微循环中的黏附情况,并对不同黏附方式的微泡进行计数.应用image-pro-plus分析软件对微泡进行定点追踪,并对其黏附过程中速度的变化进行定量测定.结果 荧光显微镜下观察可见MBp组与内皮黏附数量高达(8.4±2.1)个/视野,MBiso组仅为(0.8±0.8)个/视野,两者间差异有统计学意义(P＜0.01);MBp和MBiso组白细胞黏附数量分别为(3.6±0.6)个/视野、(2.2±0.8)个/视野,两者间差异无统计学意义(P＞0.05).微泡黏附过程速度变化曲线显示MBp和MBiso分别以流动速度逐渐和迅速降低两种方式实现对靶组织的黏附.结论 MBp和MBiso具有不同的黏附方式,MBp能更高效、特异地黏附于炎症组织血管内皮上,为评价血管内皮炎症反应或其他组织损伤的应用提供了理论基础.     

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。     

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IL-8单抗靶向超声微泡对损伤心肌细胞的体外寻靶实验研究

目的制备携IL-8单克隆抗体(以下简称单抗)靶向超声微泡,检测其基本特性,并探讨其对损伤心肌细胞的体外黏附能力。方法采用共价偶联法制备携IL-8单抗靶向超声微泡,利用缺氧、缺糖方法制备损伤心肌细胞,通过靶向超声微泡与损伤心肌细胞的体外结合实验检测其寻靶能力,并与Sono Vue微泡进行比较。结果携IL-8单抗靶向微泡与轻度及重度损伤心肌细胞的黏附比分别为(61.9±18.9)%和(86.6±5.1)%,明显高于Sono Vue微泡与轻度损伤及重度损伤心肌细胞的黏附比[(12.0±0.6)%和(11.8±1.0)%],差异均有统计学意义(均P0.01);且随着损伤程度的加重,携IL-8单抗靶向超声微泡与心肌细胞的黏附作用逐渐加强(r=0.945,P0.01)。结论携IL-8单抗靶向超声微泡对损伤心肌细胞具有较强的靶向性。     

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.     

携抗ICAM-1单抗和抗CD34单抗双靶向微泡的制备及体外寻靶研究

目的 制备同时携抗ICAM-1单抗和抗CD34单抗的双靶向微泡,鉴定其基本性质,并观察体外寻靶能力.方法 采用“生物素-亲和素”桥连法分别构建携抗ICAM-1单抗和抗CD34单抗双配体(MBD)、携抗ICAM-1单抗(MBICAM-1)和携抗CD34单抗(MBCD34)3种靶向微泡,光学显微镜下观察靶向微泡的形状,并用马尔文激光粒度分析仪、DFY超声图像定量分析仪、激光共聚焦显微镜、流式细胞仪检测靶向微泡的基本特性和抗体结合率.倒置显微镜下观察双靶向微泡与内皮祖细胞(EPCs)和损伤人脐静脉内皮细胞(HUVECs)的结合情况.结果 3种靶向微泡的形状、粒径、表面电位及抗体结合率差异均无统计学意义(P＞0.05),MBD的回声强度高于MBICAM-1、MBCD34和普通生物素化微泡(MBBiotin)(P＜0.01).MBD与EPCs和损伤HUVECs的结合率明显高于MBBiotin(P＜0.01),MBD和MBCD34与EPCs的结合率、MBD和MBICAM-1与损伤HUVECs的结合率差异均无统计学意义(P＞0.05).结论 成功制备了携抗ICAM-1单抗和抗CD34单抗的双靶向微泡,体外实验证实此双靶向微泡与内皮祖细胞和损伤血管内皮细胞均能特异性结合.     

Deformable gas-filled microbubbles targeted to P-selectin.

Ultrasound contrast microbubbles have been successfully targeted to a number of intravascular disease markers. We hypothesized that targeted delivery could be improved further, by making the microbubbles deformable, leading to increased microbubble-endothelium adhesion contact area and stabilized adhesion. Activated leukocytes utilize such strategy; they deform after binding to inflamed endothelium in the vasculature. Lipid-shell microbubbles were targeted to the endothelial inflammatory protein P-selectin with a monoclonal anti-P-selectin antibody attached to the microbubble shell. Deformable microbubbles were created by controlled pressurization with partial gas loss, which generated an average excess shell surface area of approximately 30% and the formation of outward-projected wrinkles and folds. Targeted microbubble adhesion and deformability were assessed in the parallel plate flow chamber under shear flow. Sustained adhesion of deformable microbubbles at wall shear stresses between 0.4 and 1.35 dyn/cm(2) was consistently better than adhesion of wrinkle-free microbubbles. Over this shear range, targeted wrinkled microbubbles were deformed by shear flow, unlike wrinkle-free microbubbles. In a murine cremaster inflammation model, a significant improvement of deformable microbubble targeting was observed by intravital microscopy. Overall, the mechanical aspects of adhesion, such as particle shape, deformability and surface microstructure, are important in engineering efficient site-targeted particle-based agents for medical imaging and therapy.     

超声微泡连接特异性抗体的制备方法及靶向化处理因素对超声微泡物理性质的影响

目的 探讨超声微泡连接特异性抗体的制备方法及靶向化处理因素对超声微泡物理性质的影响.方法 应用生物素-链霉亲和素连接系统,使注射用六氟化硫微泡(SonoVue)与特异性抗血管细胞黏附分子-1(VCAM-1)抗体相连接,用间接免疫荧光法检测抗体与微泡的连接,用马尔文激光粒径分析仪分别测定生物素化处理前后微泡的粒径,采用超声诊断仪评价微泡靶向化构建前后的显像效果.结果 SonoVue与特异性抗体成功连接,间接免疫荧光检测呈阳性.生物素修饰后的微泡粒径分布变窄,与普通微泡的超声显像效果略有差异.结论 通过生物素-链霉亲和素系统可以成功靶向化构建超声微泡,靶向化处理因素对微泡的粒径有一定影响,对微泡的超声显影效果略有影响.     

人白血病抑制因子受体单抗靶向脂质体微泡的制备及其体外荧光鉴定

目的 制备人白血病抑制因子受体( LIFR)单抗靶向脂质体微泡,并对其微泡微囊的连接可靠性进行体外免疫荧光鉴定.方法 利用生物素-亲和素桥连技术,构建LIFR单抗脂质体微泡(MB-BSB-LIFR-AB).以FITC荧光亲和素标记普通脂质体微泡(MB)、生物素化脂质体微泡(MB-B),以两个不同浓度梯度(1:4和1:16)的DTAF荧光二抗分别标记MB、MB-B、生物素-亲和素脂质体微泡(MB-BS)、MB-BSB-LIFR-AB;荧光显微镜下观察微泡荧光强度并分为0级、1级、2级、3级.结果 加入FITC荧光亲和素后,MB-B呈现3级明亮的绿色荧光,而MB无荧光显示(0级);以1:4和1:16浓度梯度的DTAF荧光二抗孵育后,MB-BSB-LIFR-AB均发出3级明亮的绿色荧光;而MB-BS、MB-B仅在1:4时显示1级微弱的绿色荧光;MB在两个浓度梯度下均无荧光显示(0级).结论 携LIFR单抗通过生物素-亲和素桥连技术有效连接在MB-B微囊;体外荧光法是鉴定靶向脂质体微泡配体连接可靠性的简便方法.     

靶向性声学造影剂与血管内皮细胞相互作用的实验研究

目的 制备携抗人VCAM-1单克隆抗体的白蛋白声学造影剂，观察其与损伤血管内皮细胞的相互作用，探讨评价血管内皮功能的新方法。 方法 采用交联法将抗人VCAM-1单克隆抗体共价偶联到自制氟碳气体为核心的白蛋白微气泡表面，制备靶向性声学微气泡；倒置显微镜下分别观察普通白蛋白微气泡、靶向性微气泡与正常内皮细胞、损伤内皮细胞的结合作用，高倍视野下计数内皮细胞及黏附的微气泡的数目，通过计算微气泡与内皮细胞的比值对两者之间的结合作用进行定量分析。 结果 无论是正常内皮细胞，或损伤内皮细胞，仅见少量的对照组微气泡的黏附作用；而镜下可见大量携VCAM-1单抗的白蛋白微气泡黏附在损伤内皮细胞表面，黏附数目显著高于黏附于正常内皮细胞表面的数目。 结论 携VCAM-1单抗的靶向性声学造影剂能够特异性结合在损伤内皮细胞表面，开拓了超声成像技术检测血管内皮损伤、评价血管内皮功能新的研究领域。     

Annexin V微泡对凋亡组织寻靶的试验研究

目的制备Annexin V靶向微泡,评价其理化性质,并进行初步体外寻靶试验探究。方法生物素-亲和素桥接法制备Annexin V靶向微泡,粒径分析仪、Malvern电位仪评价其理化性质,免疫荧光染色法评估靶向配体结合状况,用荧光显微镜观察其形态、大小及分布状态。建立人甲状腺未分化癌裸鼠模型,对其进行微波消融,诱导肿瘤组织凋亡。随机分组,A组为普通微泡,B组为Annexin V抗体预饱和+靶向微泡,C组为Annexin V靶向微泡,比较3组微泡与消融后凋亡肿瘤组织的结合情况。结果成功制备Annexin V靶向微泡,微泡形态大小一致,理化性质稳定;A组和B组,基本未见微泡与肿瘤组织切片稳定结合,而C组见Annexin V微泡与组织稳定结合。结论采用生物素-亲和素桥接法制成Annexin V靶向微泡;且在体外能与凋亡肿瘤组织稳定结合,即成功寻靶,从而实现识别检测凋亡组织的功能。     

Platelet membrane glycoproteins and microvesicles in blood from postoperative salvage: a study in cardiac bypass patients

BACKGROUND: Transfusion of blood collected by intraoperative and postoperative salvage systems has been linked to the development of thrombocytopenia and disseminated intravascular coagulation. Although functional defects have been reported in platelets from unwashed salvaged blood, platelet membrane glycoprotein (GP) composition, a potentially important determinant of function and survival, has not been studied. STUDY DESIGN AND METHODS: Platelets from 22 patients whose blood was salvaged at the completion of surgery were analyzed and compared to platelets obtained from the venous blood from the same patient. Platelet membranes were stained with fluorescein isothiocyanate-conjugated CD41a monoclonal antibody (anti-GPIIb/IIIa) to identify platelets, a phycoerythrin-conjugated monoclonal antibody, CD62 (anti-P-selectin) to identify activated platelets, and CD42b (anti- GPIb) or anti-GPIb/IX to assess GPIb. Samples were analyzed with a flow cytometer using software. RESULTS: Platelets obtained from salvaged blood demonstrated lower GPIb expression (CD42b and GPIb/IX monoclonal antibody binding), higher P-selectin expression, and greater numbers of platelet-derived microvesicles. CONCLUSION: The clinical significance of transfusing blood containing activated platelets and microvesicles merits investigation.     

Sialyl salivary-type amylase associated with ovarian cancer

BACKGROUND: There have been many reports describing hyperamylasemia, with a salivary-type amylase phenotype, in patients with malignant tumors and/or multiple myelomas. In contrast, we have discovered and characterized a sialyl salivary-type amylase from multiple myeloma and/or lung cancer cells. This paper reports the first association of sialyl salivary-type amylase with ovarian cancer, discovered and characterized using sera from retrospective studies. METHODS: Based on strictly retrospective observation of amylase zymograms, three samples of patients' sera with abnormally fast-migrating isoamylases were detected. Sialyl salivary-type amylase was determined by neuraminidase treatment and reaction with anti-salivary monoclonal antibody, and the extra elution peak of amylase was detected by size-exclusion HPLC analysis. RESULTS: Sialyl salivary-type amylase was detected in the sera of three female patients with ovarian cancer. The ratio of S3 to S2 sub-band in isoamylase electrophoresis, was slightly over 1.00 in two cases and below 1.00 in the other. These cases were not recognized in routine isoamylase electrophoretic analyses, because the abnormal patterns were weak. CONCLUSION: Sialyl salivary-type amylase was characterized for the first time in the sera of patients with ovarian cancer.     

细胞间黏附分子-1靶向微泡超声造影成像评价肾移植后急性排异反应

目的 探讨靶向超声分子成像评价肾移植后急性排异反应的可行性.方法 采用“亲和素-生物素”桥接法构建携抗细胞间黏附分子-1(ICAM-1)靶向微泡(MBI)和携同型抗体对照微泡(MB).10只SD大鼠行左侧肾异种移植术,术后72 h移植肾随机先后注入MBI和MB(间隔30 min),分别于注入3 min后行移植肾超声造影检查,并测量移植肾声强度(VI),最后进行肾组织病理及免疫组化检测.结果 移植肾在注入靶向超声微泡后可见肾区域明显灌注显影,延迟3 min显像MBI组在移植肾可见显著的超声显影增强.而MB组移植肾仅见轻度的超声显影增强,其显影强度较前者明显减弱.MBI组和MB组移植肾VI值分别为(27.0±7.4)U、(10.2±2.4)U,两者之间差异有统计学意义(F=64.744,P＜0.05).结论应用靶向ICAM-1超声微泡和超声造影结合能有效评价大鼠肾移植急性排异.