抗内皮细胞黏附分子-1靶向微泡介导hAng-1基因转染治疗兔心肌缺血的实验研究

目的 探讨应用抗心肌内皮细胞粘附分子-1（ICAM-1）靶向微泡能否提高血管生成素-1基因在缺血心肌中的转染效率.方法 体外实验检测抗ICAM-1靶向微泡与经人白细胞介素-1β刺激后的ECV304细胞结合的程度.体内实验分三组：①对照组：hAng-1基因＋超声照射;②非靶向微泡组：携hAng-1基因微泡＋超声照射;③靶向微泡组：携hAng-1基因的抗ICAM-1靶向微泡＋超声照射.制作兔急性心梗模型,分别用上述三种方法从静脉导入hAng-1基因.2周后心肌造影检测梗死心肌灌注状况聚合酶链反应（RT-PCR）检测hAng-1基因的mRNA表达以评价转染疗效.结果 携抗ICAM-1抗体的微泡在体外能大量靶向结合到产生炎性反应的ECV304细胞周边.应用靶向微泡转染2周后,左室内径减小,射血分数升高,但与非靶向微泡比较,差异无统计学意义（P〉0.05）,而心肌造影显示心肌内造影剂填充量较非靶向微泡组增多,且 RT-PCR定量灰度分析hAng-1mRNA（1.04±0.08）高于非靶向微泡组（0.53±0.04）,差异有统计学意义（P〈0.01）.结论 微泡与抗ICAM-1抗体连接后形成靶向微泡载体,可明显提高hAng-1基因在体内的转染效率,增加心肌梗死后局部的血管新生效应.     

抗VEGF抗体介导靶向超声造影剂的体外实验研究

目的以抗血管内皮因子（VEGF）抗体为配体研制能与血管内皮细胞特异性结合的靶向脂质体超声造影剂并检测其体外寻靶能力。 方法以静电吸附法将抗VEGF抗体连接到脂质体造影剂微泡的表面；体外培养ECV304人血管内皮细胞，用免疫荧光法检测靶向造影剂与其体外结合能力，以普通造影剂为对照组。 结果所制备的靶向超声造影剂与普通微泡无显著差异；免疫荧光实验结果显示靶向造影剂能在体外与血管内皮细胞特异性结合。 结论携抗VEGF抗体的脂质体靶向造影剂能通过静电吸附法成功制备，且在体外能与血管内皮细胞特异性结合。     

携抗ICAM-1靶向微泡定向转染Ang-1基因治疗急性心肌梗死

研究背景 基因治疗为难治性心肌缺血提供了新方法,但通过静脉途径导入体循环的外源性基因缺乏组织特异性,难以在靶组织局部停留,疗效较低。目的 利用缺血时心肌内皮细胞黏附分子-1(ICAM-1)表达增高的特点,探讨心肌梗死时利用抗ICAM-1靶向微泡能否提高血管生成素-1基因在体内的转染效率。方法 体外实验检测抗ICAM-1靶向微泡与经人白细胞介素-1β刺激后的ECV304细胞结合的程度。体内实验分三组:①对照组:hAng-1基因+超声照射;②非靶向微泡组:携hAng-1基因微泡+超声照射;③靶向微泡组:携hAng-1基因的抗ICAM-1靶向微泡+超声照射。制作兔急性心肌梗死模型,分别用上述三种方法从静脉导入hAng-1基因,各组动物均于梗死模型建立后当天和基因转染2周后进行常规超声检查和心肌造影;2周后心肌造影检测转染区心肌灌注状况,RT-PCR检测hAng-1基因的mRNA 表达以评价转染疗效。结果 携抗ICAM-1抗体的微泡在体外能大量靶向结合到产生炎性反应的ECV304内皮细胞周边。应用靶向微泡转染后缺血心肌2周后,左心室内径减小,左心室射血分数升高,但与非靶向微泡组无显著性差异,而心肌造影显示心肌内造影剂填充量较非靶向微泡组增多,RT-PCR定量灰度分析hAng-1mRNA高于非靶向微泡组(1.04±0.08vs0.53±0.04,P<0.01)。结论 微泡与抗ICAM-1抗体连接后形成靶向微泡载体,可明显提高hAng-1基因在体内的转染效率,增加心肌梗死后局部的血管新生效应。     

携抗ICAM-1超声造影剂对兔腹主动脉损伤内膜靶向显影的实验研究

目的探讨携抗ICAM-1的超声造影剂微泡靶向显影对损伤血管内膜的诊断价值.方法使用自制含氟碳声振白蛋白造影剂,制备FITC标记的携抗ICAM-1的靶向超声造影剂.荧光素标记普通造影剂微泡以示对照.新西兰大白兔12只,随机分2组,通过高脂饮食建立腹主动脉内膜损伤模型.模型建立前后分别经兔耳缘静脉推注普通(第1组)、靶向(第2组)2种造影剂微泡,超声监测不同时期、2类微泡在内膜显影情况的差异并做比较分析.造影后取腹主动脉标本做病理对照.结果模型建立前,2组造影结果无明显差异.内膜损伤后,靶向微泡持续显影时间明显延长,有延迟排空现象,与普通微泡造影结果相差显著.光镜见第2组内皮黏附微泡明显多于第1组,内膜面有强绿色荧光带形成.结论携抗黏附分子-1造影剂微泡可靶向黏附于损伤血管内膜,超声造影可据此判定早期血管内膜损伤.     

携载PSMA单抗靶向前列腺癌的纳米级脂质微泡的制备及体外寻靶能力

目的制备携载PSMA单抗靶向人前列腺癌的纳米级脂质微泡,并观察其体外寻靶能力。方法利用静电吸附法制备携载PSMA单抗靶向前列腺癌的脂质纳米级微泡,检测其一般特性;免疫荧光法检测抗体与纳米级微泡的结合情况;用细胞免疫荧光分析鉴定PSMA的表达及其在细胞膜上的定位;普通光镜下观察靶向微泡对前列腺癌细胞的寻靶能力,同时以胃癌细胞作对照。结果携载PSMA单抗的靶向纳米级微泡分布均匀,平均粒径为(623.70±66.05)nm,免疫荧光法显示微泡表面可见绿色荧光,细胞免疫荧光检测前列腺癌细胞膜高表达PSMA,体外寻靶实验显示该靶向微泡可与人前列腺癌LNCAP及C4-2细胞牢固结合,而与胃癌MKN45细胞不结合。结论本实验成功制备的携载PSMA单抗靶向人前列腺癌的纳米级脂质微泡具有较强的体外寻靶能力,能特异性地与前列腺癌细胞结合。     

携GL-7靶向微泡造影剂对高脂饮食兔腹主动脉内膜的靶向作用

目的 探讨携GL-7靶向微泡造影剂对高脂饮食兔腹主动脉内膜体外特异性结合及体内增强显影效果.方法 14只新西兰兔用高脂饮食法建立腹主动脉内膜损伤模型,并随机分为两组,4周后分别使用对照微泡和靶向微泡造影剂进行腹主动脉超声造影,以视频密度法评价两种造影剂对动脉内膜的增强效果,荧光显微镜观察两种造影剂体内结合情况及与动脉内膜荧光染色的结合情况及荧光强度统计分析.结果 对照微泡、靶向微泡造影后血管内膜回声均较造影前增强,靶向微泡造影与对照微泡造影比较,血管内膜峰值视频密度差异有统计学意义(P<0.05).荧光显微镜观察,靶向微泡血管腔内呈现绿色荧光,而对照微泡血管腔内仅有微弱的绿色荧光.动脉血管冰冻切片结果显示,靶向微泡造影组动脉内膜有绿色荧光表达,而对照微泡造影组绿色荧光表达较弱,两组荧光强度差异有统计学意义(P<0.05).结论 GL-7靶向微泡造影剂与兔动脉血管内膜体内、体外特异性结合,可显著增强兔腹主动脉内膜靶向显影.     

含氟烷人血白蛋白靶向超声造影剂的制备研究

目的为靶向超声造影剂的制备进行基础实验研究。方法将含氟烷人血白蛋白超声造影剂与ICAM-1抗体混合,加入0.1%戊二醛,4℃孵育2h,促使抗体与微泡外壳充分交联。普通光镜评估靶向微泡的大小、形态。免疫荧光法进行靶向微泡的鉴定。结果普通光镜下可见携ICAM-1抗体的靶向微泡大小、形态与普通白蛋白微泡对照无明显改变。荧光显微镜证实微泡与ICAM-1抗体整合成功。结论戊二醛交联法适用于白蛋白靶向超声造影剂的制备,方法简便,成本低,重复性好。     

携ICAM-1单抗超声造影剂体外评估兔腹主动脉内皮损伤实验研究

目的探讨携ICAM-1 单抗的超声造影剂评价血管内皮损伤的可行性及其价值.方法 FITC标记自制含氟碳声振白蛋白造影剂及携ICAM-1 单抗的靶向超声造影剂.新西兰大白兔8只,高脂饮食建立内皮损伤动物模型后,取兔腹主动脉做冰冻病理切片,随机分两组,分别滴加非靶向造影剂、靶向造影剂,比较两种微泡内皮分布情况及相应荧光染色度的差异.结果靶向造影剂组内皮黏附微泡明显增多,荧光染色较强.结论携ICAM-1 单抗的造影剂微泡可靶向黏附于损伤内皮,据此可评价早期内皮损伤.     

靶向超声微泡MB_(ICAM-1)的制备及靶向实验研究

目的制备载抗ICAM-1(细胞间黏附分子-1)的靶向超声微泡MBICAM-1,鉴定制备微泡的基本性质,研究其在体内及体外的靶向能力。方法通过"生物素-亲和素"桥接法将抗ICAM-1结合到生物素化脂质微泡,制成MBICAM-1;检测制备微泡的浓度、粒径,观察微泡的形态;检测抗ICAM-1与微泡的结合率;ICAM-1质粒转染Hela细胞,观察转染后的Hela细胞与MBICAM-1结合,验证MBICAM-1体外靶向功能;制备家兔左侧跟腱炎症模型,用制备的微泡对家兔双侧跟腱行造影检查,验证MBICAM-1体内靶向能力。结果三种微泡平均粒径1.00~2.4μm,浓度2.4×108~10/ml;微泡的形态完整、规则,分布较匀;微泡和抗ICAM-1单抗的平均结合率为(86.5±5.3)%。MBICAM-1环绕在转染后的Hela细胞周围。MBICAM-1对家兔左侧跟腱造影呈高增强。结论采用"生物素-亲和素"桥接法可制备MBICAM-1;MBICAM-1在体外、体内均具有靶向功能。     

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.     

携抗ICAM-1靶向微泡定向转染Ang-1基因治疗急性心肌梗死的实验研究

目的 探讨携抗ICAM-1的靶向微泡定向转染基因至心肌细胞的能力及其作为新型载体治疗急性心肌梗死的可行性.方法 37只家兔制备急性心肌梗死模型.3只经耳缘静脉注射携ICAM-1抗体靶向微泡,心肌组织冷冻切片后观察其对受损血管内皮的吸附.余34只兔随机分为3组:IM组(心肌注射Ang-1基因组)、ICAM-1组(注射携抗ICAM-1靶向微泡+Ang-1基因组)、对照组(空白对照),于基因转染前后分别行常规超声心动图检查,处死后取心肌组织及ICAM-1组家兔肝、肾组织行RT-PCR及Western-Blot分别检测目的 基因及蛋白表达;免疫组化Ⅷ因子染色检测其新生毛细血管密度.结果 IM组、ICAM-1组基因转染后2周左室射血分数(LVEF)均较转染前显著提高(P＜0.05),其中IM组及ICAM-1组之间差异无统计学意义;IM组及ICAM-1组行RT-PCR及Western-Blot均可检测出目的 基因及蛋白表达,且两组之间表达量差异无统计学意义;对照组及ICAM-1组肝肾组织未见明显Ang-1 mRNA及蛋白表达.心肌组织Ⅷ因子染色后高倍镜下显示IM组及ICAM-1组均可见大量新生毛细血管,其中相对于ICAM-1组,IM组新生毛细血管计数更高.结论 携抗ICAM-1的靶向微泡可定向转染Ang-1基因至心肌细胞,其转染效率与目前认为最高效的心肌内注射基因的转染效率近似,可作为新型靶向载体定向转染血管新生基因,对急性心肌梗死具有治疗作用.

Abstract：

Objective To explore the capability of Ang-1 gene delivery to acute myocardial infarction using targeted microbubbles carrying ICAM-1 antibody.Methods Thirty-seven rabbits' left circumfles branch coronary arteries were ligated for models.Three rabbits were injected with microbubbles carrying ICAM-1 to detect the ability of targeting.Thirty-four rabbit models were divided into 3 groups randomly as follow:IM group (n=12,accept direct intramuscular injection),ICAM-1 group (n=12,accept intravenous injection of targeted microbubbles and Ang-1) and control group (n=10,without any treatment).Ultrasonography were executed on all animals before and 2 weeks after the treatment.All rabbits were killed after 2 weeks and examined for Ang-1 mRNA and protein by RT-PCR and Western-Blot respectively.Microvessel density (MVD) counting of infracted myocardium,observed by factor Ⅷ immunochemical staining,was performed to value the proangiogenesis effect of Ang-1 delivered by targeted microbubbles carrying ICAM-1 antibody.The liver and the kidney in ICAM-1 group were taken to assess the systemic delivery.Results IM and ICAM-1 group showed significantly improvement in the ejection fraction (P＜0.05) while control group did not.Ang-1 mRNA and protein could be detected in IM and ICAM-1 group;however,the expression between the two groups showed no siginificant difference.None of the control animals showed Ang-1 expression.compared with ICAM-1 group,the MVD was greater in IM group.Ang-1 was not detected either in liver or in kidney in ICAM-1 group.Conclusions Targeted microbubbles carrying ICAM-1 antibody can deliver Ang-1 gene to ischemic myocardium directly.Meanwhile,it's as effective as IM injections besides the greater angiogenesis effect.This strategy improves the perfusion of acute myocardial infarction and the function of heart.     

LHRHa靶向微泡造影剂的制备及体外寻靶实验

目的 制备人卵巢癌靶向超声造影剂,观察其体外寻靶能力。方法 采用生物素-链霉亲和素法制备促黄体生成素释放激素类似物(LHRHa)靶向脂质微泡,以免疫荧光染色验证LHRHa与脂质微泡的结合情况,并以普通脂质微泡作为对照,在倒置显微镜下观察LHRHa靶向脂质微泡对人卵巢癌A2780/DDP的体外寻靶能力。结果 LHRHa靶向脂质微泡免疫荧光阳性;体外寻靶实验显示LHRHa靶向脂质微泡能够与表面存在LHRH受体的A2780/DDP细胞特异性结合,而普通脂质微泡则不能结合。结论 利用生物素-链霉亲和素方法可成功制备LHRHa靶向脂质微泡造影剂,该靶向造影剂具有体外寻靶能力。     

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.     

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.     

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。     

Nitric Oxide Pretreatment Enhances Atheroma Component Highlighting in Vivo with Intercellular Adhesion Molecule-1-Targeted Echogenic Liposomes

We present an ultrasound technique for the detection of inflammatory changes in developing atheromas. We used contrast-enhanced ultrasound imaging with (i) microbubbles targeted to intercellular adhesion molecule-1 (ICAM-1), a molecule of adhesion involved in inflammatory processes in lesions of atheromas in New Zealand White rabbits, and (ii) pretreatment with nitric oxide-loaded microbubbles and ultrasound activation at the site of the endothelium to enhance the permeability of the arterial wall and the penetration of ICAM-1-targeted microbubbles. This procedure increases acoustic enhancement 1.2-fold. Pretreatment with nitric oxide-loaded echogenic liposomes and ultrasound activation can potentially facilitate the subsequent penetration of targeted echogenic liposomes into the arterial wall, thus allowing improved detection of inflammatory changes in developing atheromas.     

Design and characterization of targeted ultrasound microbubbles for diagnostic use

Targeted ultrasound (US) contrast agents represent, because of their size (1 to 5 μm), a unique class of diagnostic imaging agents enabling true vascular imaging of conditions like inflammation and tumor angiogenesis. The objective of this study was to develop technology for preparing targeted microbubbles with binding and acoustic properties compatible with diagnostic use. Phosphatidylcholine (PC) was shown to represent the most favorable wall material. Various thiolated peptide binders were effectively conjugated to PC-based microbubbles containing maleimide functionalized lipids (95:5) without the need for biotin-streptavidin or antibody technology. By optimizing the technology, specific targeting of the inflammatory target E-selectin and the angiogenic target VEGFR2 in the presence of 100% serum was achieved. Increased phospholipid chain length from 18 carbons to 22 carbons improved the stability of the microbubbles during US exposure, without compromising binding or acoustic properties. (E-mail: roald.skurtveit@ge.com)