细胞间粘附分子-1（ICAM-1）靶向超声分子成像评价大鼠移植肾急性排斥反应
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摘要
背景和目的：
移植肾急性排斥反应(Acute rejection, AR)是肾移植术后常见并发症,最易导致移植肾早期功能丧失,是移植肾存活和维持功能的主要障碍之一。研究表明,当AR发生时,首先表现为移植肾微血管内皮损伤,血管内皮细胞高表达或分泌一些与排斥反应有关的炎性分子标记物如细胞间粘附分子-1 (Intercellular adhesion molecule, ICAM-1)、血管细胞间粘附分子-1 (Vascular cell adhesion molecule, VCAM-1)等,这些炎性分子标记物能够促进T细胞、血小板或其它炎性细胞沿血管内皮滚动、粘附、聚集和外渗,后者可介导血管内皮炎症反应并释放多种炎症介质或促炎细胞因子进入到肾间质组织,如肿瘤坏死因子-α(TNF-α),白细胞介素-1(IL-1),血栓素A2(TXA2)等,进而导致移植肾脏组织及微血管损伤。因此,一定程度上讲,血管内皮损伤是移植肾AR的最早阶段,检测血管内皮所表达的特殊分子标记物如ICAM-1有可能早期评估移植肾AR的存在。
事实上,长期以来,学者们对移植肾排斥反应的诊断,特别是急性排斥反应的诊断进行了多种多样的研究,以求在组织学发生不可逆转改变之前达到早期、准确地诊断并开始治疗。众多研究者从免疫学监测、移植肾影像学和病理学检测等多方面进行了大量有益的探索与研究。但不同的检测手段和方法均有各自的局限性,如肾闪烁扫描术可以评价缺血性损伤,但在肾移植术后早期并不能鉴别AR、急性肾小管坏死和环孢素中毒,仅对何时恢复及移植肾短期或长期存活有一定价值。而MRI、PET等其它非靶向性放射性成像方法虽对评估肾移植后形态学和代谢变化能够提供有价值的参考信息,但PET和MRI等方法存在着一些无法克服的缺点：例如,仪器要求高,不能床边检查,放射示踪剂缺乏稳定性及具有放射污染等,使其临床应用受到一定的限制。目前,移植肾穿刺取组织进行病理学检查仍是诊断AR的最准确方法。但由于肾穿刺损伤大,易发生出血及其它严重并发症,故不能作为常规监测手段。因此,寻找一种安全、无创、敏感的检测手段对AR做出早期诊断具有重要的应用价值。
近年来,随着靶向超声微泡造影剂的出现,靶向超声分子显像(Targeted ultrasound molecular imaging)逐渐成为现实,极大拓展了传统超声微泡的应用范围,其作为血管内示踪剂能特异地粘附于血管内皮表面从而在行超声检查时被探测到,这使得应用超声技术从分子水平对各种疾病进行早期诊断成为可能。目前,靶向超声分子成像技术已经成功地对肾脏、下肢、肿瘤等部位的血管内皮炎症/血管新生进行了评价,对心脏移植排斥反应的监测也进行了初步探索。但移植肾AR由于小动物肾移植模型构建复杂、繁琐等原因,关于其靶向成像的研究尚属于空白阶段。我们实验室经过多年的探索和技术改进,靶向超声微泡构建研究也取得了相当大的成果,构建的靶向超声微泡大小和浓度、稳定性、抗体携带率等均已达到国际同等或更高水平,并且依靠血管内皮炎症相关因子(ICAM-1)靶向超声微泡成功实现了对肾脏、心脏等的血管内皮炎症反应评价。在前期工作基础之上,本研究尝试利用构建的靶向肾脏微血管内皮表面细胞间粘附分子-1(ICAM-1)靶向超声微泡和对比超声相结合,并采用先进的超声成像技术(将对微泡的破坏性降到最低)对移植肾AR进行早期评价和诊断,这无疑具有重要临床意义。
因此,本研究在普通脂质微泡的基础上,通过“抗生物素蛋白/生物素复合体”的化学桥接作用将抗大鼠ICAM-1单克隆抗体连接于脂质微泡外壳构建了携带抗ICAM-1的靶向超声微泡(MBI),并在大鼠移植肾AR模型上,应用MBI和对比超声(CEU)检查相结合,目的在于探讨MBI结合CEU评价大鼠移植肾AR的可行性。
材料和方法：
1、超声微泡的制备与评价
1.1、超声微泡的制备各种相关脂质材料按一定质量比例75℃水浴溶解于适量蒸馏水中,同时通全氟丙烷(C3F8)气体,超声振荡至形成乳白色液体制备生物素(biotin)化脂质微泡(MB)。MB经静置弃下清液并加入等量蒸馏水去除未结合脂质纯化1次后,按一定比例加入抗生蛋白链菌素(streptavidin)。继续静置弃下清液并加入等量蒸馏水去除未结合抗生蛋白链菌素纯化微泡1次后,按一定比例加入生物素化的抗大鼠细胞间粘附分子-1 (ICAM-1)和同型对照抗体(isotype control antibody),分别得到携带抗ICAM-1单抗靶向超声微泡(MBI)和同型对照微泡(MB),最后静置弃下清液并加入等量蒸馏水去除未结合抗体纯化微泡1次,冰箱中4℃保存备用。
1.2、超声微泡体外评价：
1.2.1、库尔特计数仪测量超声微泡的平均粒径及浓度。采用绿色荧光标记的二抗与抗大鼠ICAM-1单克隆抗体结合荧光显微镜下观察ICAM-1单抗与微泡外壳的连接情况。
1.2.2、平行板流动腔评价超声微泡粘附性能应用包被有大鼠ICAM-1抗原的聚苯乙烯培养皿作为平行板流动腔体外检测微泡粘附稳定性的平台。以普通脂质微泡非特异性粘附效果为对照,采用大鼠ICAM-1 (1000ng/μl抗原浓度)包被检测MBI粘附特异性及效能,所有分组均按平行板流动腔实验说明书要求检测3个样本(n=3)。MBI及MB(1×108/m1)经微量注射泵以0.5dyn/cm2剪切应力分别通过“大鼠ICAM-1抗原”包被的平行板流动腔,自显微镜下观察到有微泡出现后开始连续录像6min,观察每分钟微泡的靶向结合情况。
1.2.3大鼠肾脏对比超声(Contrast enhanced ultrasound, CEU)评价超声微泡显影效果
SD大鼠给予腹腔注射3%乌拉坦-水合氯醛麻醉后,背部备皮,尾静脉插管,使用sequoia512超声机,固定17L5探头在肾脏长轴,固定探头频率为7MHz,深度25mm,机械指数0.18,动态范围80分贝,经尾静脉注射1mlMB或MBI后以0.2ml生理盐水冲管,CPS实时成像,所有图像存储于MO盘备用。
2、大鼠移植肾AR模型的构建
2.1、移植前准备：供、受体均在移植前禁食12h,自由饮水,以3%戊巴比妥钠30mg/kg腹腔注射麻醉。
2.2、供体移植：麻醉成功后备皮,常规消毒铺巾。取腹部正中切口,由剑突至耻骨联合。以自制拉钩将腹壁向左右拉开,将全部肠管、胃、脾脏翻置腹腔外,湿纱布覆盖,显露左肾、左输尿管和膀胱。游离出腹主动脉、下腔静脉,用5/0丝线结扎左精索静脉及左肾上腺静脉并剪断,去除肾脂肪囊,分离左输尿管至膀胱,该过程中注意尽量保留输尿管周围脂肪组织,以免影响输尿管血供。显微血管钳阻断下腔静脉和腹主动脉上、下端,由腹主动脉下端插入灌注管,在下腔静脉下端剪一开口,4℃有肝素林格氏液(25U/m1)经腹主动脉插管灌注,灌洗至左肾和所属血管完全苍白,下腔静脉流出液变清澈为止,灌注量5-10ml。拔出腹主动脉插管,切断肾动、静脉,分离出左肾后放入盛有0℃-4℃乳酸林格氏液的小盘里。
2.3、受体移植：步骤同上,受体SD大鼠作腹部正中切口。由剑突至耻骨联合,同样将肠管推向右侧,以温盐水纱布包裹。近肾门处结扎左肾动、静脉及输尿管,并切除受体左肾。用10-0无损伤缝线将供体肾和受体肾的同名肾动、静脉做端端吻合。血流恢复后,移植肾颜色逐渐转为红润,明亮,肾静脉充盈,肾动脉搏动明显,5-10min左右可见输尿管蠕动,表明模型构建成功。
2.4、移植后处理：予肌注抗生素,视情况予以补液,注意保温。
3、靶向超声分子成像
实验分为两组,分别为接受肾移植术72h后的SD大鼠移植肾脏(肾移植组,n=10)与未经过任何处理SD大鼠右侧肾脏(对照组,n=10)。对肾移植组及对照组SD大鼠行二维、彩色多普勒及对比超声检查。超声造影时两组分别注入靶向超声微泡(MBI)和对照超声微泡(MB)。SD大鼠俯卧位,用支架固定Sequoia512超声机(Siemens,美国)的高频超声探头(17L5)于大鼠移植肾脏上方,调整探头位置获得良好肾脏显像后保持在整个实验过程中不变,仪器的各项参数在整个实验过程中不变。CEU检查均采用相干脉冲序列成像技术(Coherent Pulse Sequence, CPS)实时观察,探头发射频率为7.0MHZ,机械指数为0.2。每只大鼠采用微量进样器经尾静脉随机(间隔30min)先后注射MB和MBI的数量约为1×109个。在注入微泡3min后行CEU检查,获取第一帧图像后继续存储图像2-3帧后给予高MI的连续超声发射2-3s以破坏微泡,继续存储本底图像2-3帧,全部声学造影视频存于CD盘,以备脱机分析。微泡破坏前存储图像的平均声强度(Video Intensity, VI)可代表靶向超声微泡在组织中的总浓度,包括粘附和循环微泡。微泡破坏后存储图像上的平均VI代表的是在血池中循环微泡的浓度。前者减去后者得到的是粘附微泡在组织中的浓度。
取图结束后,应用CEU图像分析软件(Virginia大学,美国)对图像进行分析,测量大鼠移植肾组及对照组肾脏造影VI值,单位为灰阶强度(U)；并用彩色编码技术制作肾脏显影的彩色编码图像,采用红色、橙色和白色依次代表显影强度由弱到强。
4、肾脏病理学检查：CEU图像采集后。取出大鼠肾脏,10%甲醛固定。常规脱水、石蜡包埋制片。切片作苏木素一伊红(HE)染色和免疫组化检查。
5、统计学处理：采用SPSS13.0软件包进行数据分析,计数资料用X±S表示,各组不同时间点结合微泡个数比较用重复测量的方差分析,各时间点内两组间单独效应比较采用单因素方差分析(One-way ANOVA)进行分析；动物实验组间比较采用两样本t检验,组内比较采用二阶段交叉设计方差分析,P<0.05为差异有显著性意义。
结果：
1、MBI构建及制备效果的体外评价
1.1、MBI制备及理化性质鉴定
采用声振法制备MB及MBI在显微镜下观察均为透亮微气泡,库尔特计数仪检测MB平均直径为2.86μm,浓度为1.7×109个/ml, MBI平均直径为2.71μm,浓度为1.5×109个/ml。
1.2、MBI制备效果体外评价：
1.2.1、荧光标记法评价抗体连接情况
采用绿色荧光标记的二抗与抗大鼠ICAM-1单抗结合,荧光显微镜下显示MBI外壳显明显绿色荧光,表明抗ICAM-1单抗与微泡外壳连接良好
1.2.2、平行板流动腔体外评价MBI靶向粘附效能
平行板流动腔体外评价结果显示,在模拟微血管生理血流条件的剪切应力(0.5dyn/cm2)下MBI可与包被于平行板流动腔的ICAM-1抗原有效结合,MBI结合数量随着时间延长而不断增加,显示了良好的主动性靶向黏附效能。普通对照微泡结合数量同样随着时间延长而增加但同靶向超声微泡相比其结合数量显著降低,两者差异显著(F=420.116,P=0.000)。
1.2.3、经外周静脉注射超声微泡MBI或MB,在大鼠肾脏中均可获得满意的声学造影效果。
2、靶向超声分子成像
2.1、二维超声及CEU检查结果：二维超声提示移植肾稍增大,形态稍饱满,肾皮质彩色血流信号略减少,对照组肾脏形态、大小正常,血流信号正常；移植肾及对照组肾在注入靶向超声微泡后均可见肾脏区域明显灌注显影,在延迟3min显象后第一帧CEU图像显示移植肾-MBI组可见显著的超声显影增强。而移植肾-MB组仅见轻度的超声显影增强,其显影强度较前者明显减弱；而对照-MBI组及对照-MB组延迟3min显象后,右侧肾脏均未见明显显影增强。
2.2、对比超声V1分析：移植肾-MBI组和移植肾-MB组VI值分别为(27.0±7.4)U、(10.2±2.4)U,前者同后者相比VI值增大,两者之间具有显著性差异(F=64.744,P=0.000)。对照-MBI组及对照-MB组VI值分别为(7.7±2.2)U、(6.5±1.0)U,两者之间差异无统计学意义(F＝2.868,P=0.129)。移植肾-MBI组VI值分别为对照-MBI组及对照-MB组VI值的(3.7±1.3)倍(t＝7.907,P=0.000)、(4.2±1.2)倍(t＝8.661,P=0.000),两者之间具有显著差异。而移植肾-MB组VI值同对照-MBI组及对照-MB组相比,两者之间具有显著差异(t=2.423,P=0.026；t=4.417,P=0.001),但仅分别为后两者(1.4±0.4)倍、(1.6±0.6)倍。
3、肾脏病理检查结果：移植肾组织中可见肾小管上皮细胞肿胀、变性,内可见管型和坏死脱落细胞,伴肾间质水肿及中性粒细胞增多；对照组肾脏组织中肾小管排列整齐,形态正常,间质无充血、水肿。
4、免疫组化检查：移植肾组织肾小球微血管内皮表面、肾小管上皮ICAM-1(棕黄色阳性反应物,箭头处)表达明显增加；而对照组肾组织血管内皮表面未见明显的ICAM-1表达。
结论：
1、平行板流动腔实验证实采用生物素-亲和素桥连方式制备出的携带ICAM-1靶向超声微泡随着时间的推移粘附数量不断地增加,提示其靶向粘附能力良好,可以用来评价病变组织的血管内皮炎症。
2、采用供肾血管与受体同名血管作端端吻合,大大减少了对受体循环系统的影响,保证了良好的术后生存期,为实验的进一步进行建立坚实基础。
3、应用携带抗ICAM-1单抗靶向超声微泡行对比超声检查可有效评价大鼠移植肾AR,提示将有望为临床提供一种早期、敏感、无创的检测移植肾AR的手段。
Background and Objective
Renal allograft acute rejection, a common complication after kidney transplantation, most likely leads to the lost of the early function of the transplanted kidney, and thus it becomes one of the main obstacles to the survival and function-maintenance of the transplanted renal. Studies have shown that the first sign of renal allograft acute rejection (AR) is the renal micro-vascular endothelial injury. The injury is characterized by the inflammatory and rejection-related molecular markers such as intercellular adhesion molecule, vascular cell adhesion molecule, which are expressed or secreted by the endothelial cells. These markers can promote the adhesion, aggregation and leakage of the platelets and neutrophilic granulocytes along the endothelium. What's worse, the neutrophilic granulocytes can result in inflammatory reaction and release several inflammatory mediators or pro-inflammatory cytokines into the renal interstitial tissues, such as lymphocytes, tumor necrosis factor-a, interleukin-1, thromboxane A2 and so on, which then lead to the injury of renal capillary vessel and cells. Therefore, to the extent that vascular endothelial injury is an important feature at the early stage of renal transplant rejection, we can presume the presence of AR considering the specific molecular markers such as ICAM-1 expressed by vascular endothelial.
In fact, scholars have made long researches and conducted a variety of studies on the diagnosis of renal transplant rejection, especially on the diagnosis of acute rejection, in order to make an accurate diagnosis and treatment before irreversible changes occur in the organization. Many researchers have carried out large numbers of explorations and studies from the aspect of the immunological monitoring、renal imaging and pathology testing and so on. However, different means and methods of examination have their own limitations.
For example, renal scintigraphy can evaluate the ischemic injury, but it can not identify the acute rejection, acute tubular interstitial nephritis and cyclosporine toxicity at the early stage after the renal transplantation, and only has certain value for the recovery and short-term or long-term of graft survival. Other non-targeted radiologic imaging methods, including MRI and PET, can provide valuable information for the assessment of morphological and metabolic changes respectively associated with rejection. Despite the advantages, these methods have some disadvantages which can not be solved. For example, instruments with high requirements, not the bedside examination, the lack of stability of the radioactive tracer, and the radioactive contamination have made certain restrictions in the clinical applications.
Nowadays, histopathological examination is still the most accurate method for the diagnosis of AR. However, due to its injury to the kidney, bleeding and other serious complications, it can not be used as routine monitoring tools. Thus, it is in great clinical significance to search for a safe, noninvasive, sensitive monitoring method for early diagnosis of AR.
In recent year, with the appearance of targeted ultrasound contrast agents, targeted ultrasound molecular imaging gradually becomes a reality, which has greatly expanded the scope of application of ultrasound. Its role as an intravascular tracer in being detected while adhering to endothelial surface makes it possible to apply the molecular lever ultrasound technology in the early diagnosis of various diseases. Currently, targeted ultrasound molecular imaging has successfully evaluated in the areas of vascular endothelial inflammation/angiogenesis of kidneys, limbs, tumor, and it has also conducted a preliminary exploration to the heart transplant rejection. But its targeted imaging research makes no progress, because renal transplantation models are difficult and fussy to build. After many years of exploration and technical improvements, our laboratory has made considerable achievements in building targeted ultrasound micro bubbles, the size, concentration, stability and antibody rate of which have reached the international equivalent or higher level. And we have successfully assessed the endothelial inflammatory response in the kidney/heart and other parts by targeting intercellular adhesion molecule (ICAM-1). On the basis of the preliminary study, this study tries to combine targeted micro bubbles and Ultrasound contrast to evaluate and diagnose of AR early, which will undoubtedly have great clinical significance.
In conclusion, on the basis of common lipid micro bubbles, we have constructed the micro bubbles targeted to ICAM-1 by combining the anti-rat-ICAM-lmonoclone antibodies to the shell of general lipid microbubbles via "avidin-biotin" bridging chemistry. By using this micro bubbles targeted to ICAM-1 (MBI) and the control microbubbles (MB), the VI (video intensity) of renal were measured separately by CEU. We hypothesized that the AR could be accurately evaluated with microbubbles targeted to ICAM-1 by using CEU.
Methods
1.Microbubbles preparation
General lipid microbubbles (MB) and lipid microbubbles with biotin were prepared by sonication of perfluorocarbon gas (C3H8) with aqueous dispersion of several lipids in determinate ratio. After being washed (1×) to remove excess free unincorporated lipid, streptavidin in determinate ratio were added to the lipid microbubbles with biotin, then washed (1×) to removed excess free unincorporated streptavidin and the biotin conjugated mouse-anti rat ICAM-1 monoclone antibodies and isotype control antibodies in determinate ratio were added to complete the preparation of MBI and MB. At last, the MBI and MB were washed (1×) to remove excess free unincorporated antibodies. Both MB and MBI were storaged in refrigerator at 4℃.
2. Evaluation of microbubble in vitro:
2.1 The mean diameter and density in both MB and MBI were measured by coulter counter. Using green fluorescent-labeled antibody for anti rat ICAM-1 monoclone antibody to identify the linking antibodies on the MBI.
2.2 Assessment of microbubbles targeted to ICAM-1 with Parallel plate flow chamber
The binding and retention of targeted microbubbles to ICAM-1 immobilized on a culture dish were assessed in a parallel-plate flow chamber. Targeted microbubbles drawn through the flow chamber coated with ICAM-1 (1000ng/ml) at a shear stress of 0.5dyn/cm2. All groups use 3 samples according to the recommended protocol. The adhesion abilities of control microbubbles with isotype antibodies were tested as control groups. Quantitative analysis of microbubble accumulation was performed by counting the number of microbubbles adhered in the observed area and a graph of microbubble accumulation with time was plotted.
3. Rat model for acute transplant rejection
The 10 male Wistar rats and SD rats respectively(250-300g) used in this study were fasted 24 hours before surgery, but free of water. Wistar rats were anesthetized by intraperitoneal injection of Pentobarbital (1g/kg). The skin was incised by a longitudinal cut in the middle of abdomen. Left ureter, left renal artery and vein、vena cava and abdominal aorta were separated,4-0 silks were placed in the upper and lower of inferior vena cava and abdominal aorta. A small hole was cut between the traction line of the inferior vena cava to do as a perfusate outflow tract.
At the same time, Cut a hole in the abdominal aorta anterior to prime heparin Ringer's solution(4C,25u/ml), Until the color of kidneys paled and outflow of liquid of inferior vena cava turned clear, The amount of perfusate was 5-10ml. After removing the left renal artery and vein, ureter,the left kidney was placed in the small cap filled with Ringer's lactate(0-4℃).
After abdominal incision, SD rats were ligated renal artery and vein and ureter near the left renal hilum, and left kidney was removed. the same name of renal arterial and venous of the kidney of donor and recipient were anastomosed in turn. The kidney perfused well, turned pink very quickly, and functioned immediately. Rat model established successfully.
4.CEU imaging:Ten SD rats with renal allograftt acute rejection (AR) and ten normal SD rats were performed with CEU respectively by using MBI and MB, the intravenous injection of 1×109 microbubbles were made in random order with 30 minutes interval. After Three minutes of intravenous injection, microbubbles in the circulation were eliminated, the ultrasound signal (video intensity, VI) from MB and MBI were measured by second harmonic CEU imaging with pulsing interval time (PI) of ten seconds and a mechanical index (MI) of 0.2, transmission frequency of 7.0 MHz. After the first picture of CEU imaging being taken, the microbubbles were destroyed by two to three seconds of continuous imaging with a high MI of 1.9 and the background subtracted VI of Renal were measured.
5.Examination of pathology and immunohistochemisty:After CEU imaging, all kidney of experimental rats were cutted for the examination of pathology and immunohistochemisty.
6.Statistical analysis:Data are expressed as mean±SD. Experiment one: Comparisons between different groups were made with repeated measures; comparison between the two groups within each time point using One-way ANOVA to analyze; Experiment two:Comparisons between different groups were made with independent t-test, Interval comparisons were made with two stages cross-over design. Differences were considered significant at a value of P<0.05(2-sided).
Results
1.Results for microbubble preparation:The density of MBI and MB is about1.5×109/ml and 1.7×109/ml separately, the mean sizes for MBI and MB were about 2.71μm and 2.86μm respectively.
2.Results for evaluation of microbubble in vitro:The anti-Rat ICAM-1 monoclone antibodies linked well to the surface of microbubbles, which were observed with fluorescence microscopy, shell of ultrasound microbubbles showed obvious green fluorescence, That shows targeted microbubbles constructed successfully.
3.Results for evaluation of MBI using parallel plate flow chamber:Keeping the shear stress and microbubble concentration constant at 0.5dyn/cm2 and 2×108/ml, respectively, the microbubble accumulation over a 6-min interval was assessed. Numbers of microbubbles increased gradually with the increase of time. But there was only minimal and no significant adhesion in the control group.there was obvious differences(P=0.000).
4.Results for CEU imaging:A significant enhancement in ultrasound was observed in transplant kidney of MBI-group. Increase in VI value of transplant kidney region in MBI-group was great and it amounted to 27.0±7.4. However, increase in VI value of transplant kidney in MB-group was minor, it was just 10.2±2.4. Difference was evident in transplant kidney between of the two groups (P=0.000). The VI in transplant kidney of MBI-group is 3.7±1.3 times to the MBI-group of normal kidney (7.7±2.2) and 4.2±1.2times to the MB-group of normal kidney (6.5±1.0). There was obvious difference between the two groups (P=0.000) The VI in transplant kidney of MB-group is 1.4±0.4 times to the MBI-group of normal kidney and 1.6±0.6times to the MB-group of normal kidney. There was obvious difference between the two groups (P<0.05)
5. Results for examination of pathology and immunohistochemisty:Pathological examination showed that renal tubules arranged well and there was no congestion, interstitial edema in the normal kidney; We observed tubular epithelial cell becomed swelling, degeneration, and necrosis. Renal interstitial edema and neutrophil increased was also observed in transplant kidney. It was indicated by immunohistochemisty that the expression of endothelial ICAM-1 increased in transplant kidney compared to normal kidney.
Conclusions
1. Parallel plate flow chamber experiments show that targeted microbubbles can be successfully constructed via "avidin-biotin" bridging chemistry. The adhesion performance of targeted microbubble was very well, which indicated targeted microbubbles can be Possiblely used to evaluate the severity of lesions.
2. Anastomosis the same name of renal arterial and venous of donor and recipient, which reduced the impact on the circulatory system, ensuring a good postoperative survival, mading a solid foundation for further the experiments.
3.Microbubbles targeted to ICAM-1 (MBI) and CEU that create "active targeted CEU imaging" can effectively evaluate the acute renal allograft rejection injury in rat, and may be used to evaluate the microvascular inflammation and other endothelial responses.
移植肾急性排斥反应(Acute rejection, AR)是肾移植术后常见并发症,最易导致移植肾早期功能丧失,是移植肾存活和维持功能的主要障碍之一。研究表明,当AR发生时,首先表现为移植肾微血管内皮损伤,血管内皮细胞高表达或分泌一些与排斥反应有关的炎性分子标记物如细胞间粘附分子-1 (Intercellular adhesion molecule, ICAM-1)、血管细胞间粘附分子-1 (Vascular cell adhesion molecule, VCAM-1)等,这些炎性分子标记物能够促进T细胞、血小板或其它炎性细胞沿血管内皮滚动、粘附、聚集和外渗,后者可介导血管内皮炎症反应并释放多种炎症介质或促炎细胞因子进入到肾间质组织,如肿瘤坏死因子-α(TNF-α),白细胞介素-1(IL-1),血栓素A2(TXA2)等,进而导致移植肾脏组织及微血管损伤。因此,一定程度上讲,血管内皮损伤是移植肾AR的最早阶段,检测血管内皮所表达的特殊分子标记物如ICAM-1有可能早期评估移植肾AR的存在。
事实上,长期以来,学者们对移植肾排斥反应的诊断,特别是急性排斥反应的诊断进行了多种多样的研究,以求在组织学发生不可逆转改变之前达到早期、准确地诊断并开始治疗。众多研究者从免疫学监测、移植肾影像学和病理学检测等多方面进行了大量有益的探索与研究。但不同的检测手段和方法均有各自的局限性,如肾闪烁扫描术可以评价缺血性损伤,但在肾移植术后早期并不能鉴别AR、急性肾小管坏死和环孢素中毒,仅对何时恢复及移植肾短期或长期存活有一定价值。而MRI、PET等其它非靶向性放射性成像方法虽对评估肾移植后形态学和代谢变化能够提供有价值的参考信息,但PET和MRI等方法存在着一些无法克服的缺点：例如,仪器要求高,不能床边检查,放射示踪剂缺乏稳定性及具有放射污染等,使其临床应用受到一定的限制。目前,移植肾穿刺取组织进行病理学检查仍是诊断AR的最准确方法。但由于肾穿刺损伤大,易发生出血及其它严重并发症,故不能作为常规监测手段。因此,寻找一种安全、无创、敏感的检测手段对AR做出早期诊断具有重要的应用价值。
近年来,随着靶向超声微泡造影剂的出现,靶向超声分子显像(Targeted ultrasound molecular imaging)逐渐成为现实,极大拓展了传统超声微泡的应用范围,其作为血管内示踪剂能特异地粘附于血管内皮表面从而在行超声检查时被探测到,这使得应用超声技术从分子水平对各种疾病进行早期诊断成为可能。目前,靶向超声分子成像技术已经成功地对肾脏、下肢、肿瘤等部位的血管内皮炎症/血管新生进行了评价,对心脏移植排斥反应的监测也进行了初步探索。但移植肾AR由于小动物肾移植模型构建复杂、繁琐等原因,关于其靶向成像的研究尚属于空白阶段。我们实验室经过多年的探索和技术改进,靶向超声微泡构建研究也取得了相当大的成果,构建的靶向超声微泡大小和浓度、稳定性、抗体携带率等均已达到国际同等或更高水平,并且依靠血管内皮炎症相关因子(ICAM-1)靶向超声微泡成功实现了对肾脏、心脏等的血管内皮炎症反应评价。在前期工作基础之上,本研究尝试利用构建的靶向肾脏微血管内皮表面细胞间粘附分子-1(ICAM-1)靶向超声微泡和对比超声相结合,并采用先进的超声成像技术(将对微泡的破坏性降到最低)对移植肾AR进行早期评价和诊断,这无疑具有重要临床意义。
因此,本研究在普通脂质微泡的基础上,通过“抗生物素蛋白/生物素复合体”的化学桥接作用将抗大鼠ICAM-1单克隆抗体连接于脂质微泡外壳构建了携带抗ICAM-1的靶向超声微泡(MBI),并在大鼠移植肾AR模型上,应用MBI和对比超声(CEU)检查相结合,目的在于探讨MBI结合CEU评价大鼠移植肾AR的可行性。
材料和方法：
1、超声微泡的制备与评价
1.1、超声微泡的制备各种相关脂质材料按一定质量比例75℃水浴溶解于适量蒸馏水中,同时通全氟丙烷(C3F8)气体,超声振荡至形成乳白色液体制备生物素(biotin)化脂质微泡(MB)。MB经静置弃下清液并加入等量蒸馏水去除未结合脂质纯化1次后,按一定比例加入抗生蛋白链菌素(streptavidin)。继续静置弃下清液并加入等量蒸馏水去除未结合抗生蛋白链菌素纯化微泡1次后,按一定比例加入生物素化的抗大鼠细胞间粘附分子-1 (ICAM-1)和同型对照抗体(isotype control antibody),分别得到携带抗ICAM-1单抗靶向超声微泡(MBI)和同型对照微泡(MB),最后静置弃下清液并加入等量蒸馏水去除未结合抗体纯化微泡1次,冰箱中4℃保存备用。
1.2、超声微泡体外评价：
1.2.1、库尔特计数仪测量超声微泡的平均粒径及浓度。采用绿色荧光标记的二抗与抗大鼠ICAM-1单克隆抗体结合荧光显微镜下观察ICAM-1单抗与微泡外壳的连接情况。
1.2.2、平行板流动腔评价超声微泡粘附性能应用包被有大鼠ICAM-1抗原的聚苯乙烯培养皿作为平行板流动腔体外检测微泡粘附稳定性的平台。以普通脂质微泡非特异性粘附效果为对照,采用大鼠ICAM-1 (1000ng/μl抗原浓度)包被检测MBI粘附特异性及效能,所有分组均按平行板流动腔实验说明书要求检测3个样本(n=3)。MBI及MB(1×108/m1)经微量注射泵以0.5dyn/cm2剪切应力分别通过“大鼠ICAM-1抗原”包被的平行板流动腔,自显微镜下观察到有微泡出现后开始连续录像6min,观察每分钟微泡的靶向结合情况。
1.2.3大鼠肾脏对比超声(Contrast enhanced ultrasound, CEU)评价超声微泡显影效果
SD大鼠给予腹腔注射3%乌拉坦-水合氯醛麻醉后,背部备皮,尾静脉插管,使用sequoia512超声机,固定17L5探头在肾脏长轴,固定探头频率为7MHz,深度25mm,机械指数0.18,动态范围80分贝,经尾静脉注射1mlMB或MBI后以0.2ml生理盐水冲管,CPS实时成像,所有图像存储于MO盘备用。
2、大鼠移植肾AR模型的构建
2.1、移植前准备：供、受体均在移植前禁食12h,自由饮水,以3%戊巴比妥钠30mg/kg腹腔注射麻醉。
2.2、供体移植：麻醉成功后备皮,常规消毒铺巾。取腹部正中切口,由剑突至耻骨联合。以自制拉钩将腹壁向左右拉开,将全部肠管、胃、脾脏翻置腹腔外,湿纱布覆盖,显露左肾、左输尿管和膀胱。游离出腹主动脉、下腔静脉,用5/0丝线结扎左精索静脉及左肾上腺静脉并剪断,去除肾脂肪囊,分离左输尿管至膀胱,该过程中注意尽量保留输尿管周围脂肪组织,以免影响输尿管血供。显微血管钳阻断下腔静脉和腹主动脉上、下端,由腹主动脉下端插入灌注管,在下腔静脉下端剪一开口,4℃有肝素林格氏液(25U/m1)经腹主动脉插管灌注,灌洗至左肾和所属血管完全苍白,下腔静脉流出液变清澈为止,灌注量5-10ml。拔出腹主动脉插管,切断肾动、静脉,分离出左肾后放入盛有0℃-4℃乳酸林格氏液的小盘里。
2.3、受体移植：步骤同上,受体SD大鼠作腹部正中切口。由剑突至耻骨联合,同样将肠管推向右侧,以温盐水纱布包裹。近肾门处结扎左肾动、静脉及输尿管,并切除受体左肾。用10-0无损伤缝线将供体肾和受体肾的同名肾动、静脉做端端吻合。血流恢复后,移植肾颜色逐渐转为红润,明亮,肾静脉充盈,肾动脉搏动明显,5-10min左右可见输尿管蠕动,表明模型构建成功。
2.4、移植后处理：予肌注抗生素,视情况予以补液,注意保温。
3、靶向超声分子成像
实验分为两组,分别为接受肾移植术72h后的SD大鼠移植肾脏(肾移植组,n=10)与未经过任何处理SD大鼠右侧肾脏(对照组,n=10)。对肾移植组及对照组SD大鼠行二维、彩色多普勒及对比超声检查。超声造影时两组分别注入靶向超声微泡(MBI)和对照超声微泡(MB)。SD大鼠俯卧位,用支架固定Sequoia512超声机(Siemens,美国)的高频超声探头(17L5)于大鼠移植肾脏上方,调整探头位置获得良好肾脏显像后保持在整个实验过程中不变,仪器的各项参数在整个实验过程中不变。CEU检查均采用相干脉冲序列成像技术(Coherent Pulse Sequence, CPS)实时观察,探头发射频率为7.0MHZ,机械指数为0.2。每只大鼠采用微量进样器经尾静脉随机(间隔30min)先后注射MB和MBI的数量约为1×109个。在注入微泡3min后行CEU检查,获取第一帧图像后继续存储图像2-3帧后给予高MI的连续超声发射2-3s以破坏微泡,继续存储本底图像2-3帧,全部声学造影视频存于CD盘,以备脱机分析。微泡破坏前存储图像的平均声强度(Video Intensity, VI)可代表靶向超声微泡在组织中的总浓度,包括粘附和循环微泡。微泡破坏后存储图像上的平均VI代表的是在血池中循环微泡的浓度。前者减去后者得到的是粘附微泡在组织中的浓度。
取图结束后,应用CEU图像分析软件(Virginia大学,美国)对图像进行分析,测量大鼠移植肾组及对照组肾脏造影VI值,单位为灰阶强度(U)；并用彩色编码技术制作肾脏显影的彩色编码图像,采用红色、橙色和白色依次代表显影强度由弱到强。
4、肾脏病理学检查：CEU图像采集后。取出大鼠肾脏,10%甲醛固定。常规脱水、石蜡包埋制片。切片作苏木素一伊红(HE)染色和免疫组化检查。
5、统计学处理：采用SPSS13.0软件包进行数据分析,计数资料用X±S表示,各组不同时间点结合微泡个数比较用重复测量的方差分析,各时间点内两组间单独效应比较采用单因素方差分析(One-way ANOVA)进行分析；动物实验组间比较采用两样本t检验,组内比较采用二阶段交叉设计方差分析,P<0.05为差异有显著性意义。
结果：
1、MBI构建及制备效果的体外评价
1.1、MBI制备及理化性质鉴定
采用声振法制备MB及MBI在显微镜下观察均为透亮微气泡,库尔特计数仪检测MB平均直径为2.86μm,浓度为1.7×109个/ml, MBI平均直径为2.71μm,浓度为1.5×109个/ml。
1.2、MBI制备效果体外评价：
1.2.1、荧光标记法评价抗体连接情况
采用绿色荧光标记的二抗与抗大鼠ICAM-1单抗结合,荧光显微镜下显示MBI外壳显明显绿色荧光,表明抗ICAM-1单抗与微泡外壳连接良好
1.2.2、平行板流动腔体外评价MBI靶向粘附效能
平行板流动腔体外评价结果显示,在模拟微血管生理血流条件的剪切应力(0.5dyn/cm2)下MBI可与包被于平行板流动腔的ICAM-1抗原有效结合,MBI结合数量随着时间延长而不断增加,显示了良好的主动性靶向黏附效能。普通对照微泡结合数量同样随着时间延长而增加但同靶向超声微泡相比其结合数量显著降低,两者差异显著(F=420.116,P=0.000)。
1.2.3、经外周静脉注射超声微泡MBI或MB,在大鼠肾脏中均可获得满意的声学造影效果。
2、靶向超声分子成像
2.1、二维超声及CEU检查结果：二维超声提示移植肾稍增大,形态稍饱满,肾皮质彩色血流信号略减少,对照组肾脏形态、大小正常,血流信号正常；移植肾及对照组肾在注入靶向超声微泡后均可见肾脏区域明显灌注显影,在延迟3min显象后第一帧CEU图像显示移植肾-MBI组可见显著的超声显影增强。而移植肾-MB组仅见轻度的超声显影增强,其显影强度较前者明显减弱；而对照-MBI组及对照-MB组延迟3min显象后,右侧肾脏均未见明显显影增强。
2.2、对比超声V1分析：移植肾-MBI组和移植肾-MB组VI值分别为(27.0±7.4)U、(10.2±2.4)U,前者同后者相比VI值增大,两者之间具有显著性差异(F=64.744,P=0.000)。对照-MBI组及对照-MB组VI值分别为(7.7±2.2)U、(6.5±1.0)U,两者之间差异无统计学意义(F＝2.868,P=0.129)。移植肾-MBI组VI值分别为对照-MBI组及对照-MB组VI值的(3.7±1.3)倍(t＝7.907,P=0.000)、(4.2±1.2)倍(t＝8.661,P=0.000),两者之间具有显著差异。而移植肾-MB组VI值同对照-MBI组及对照-MB组相比,两者之间具有显著差异(t=2.423,P=0.026；t=4.417,P=0.001),但仅分别为后两者(1.4±0.4)倍、(1.6±0.6)倍。
3、肾脏病理检查结果：移植肾组织中可见肾小管上皮细胞肿胀、变性,内可见管型和坏死脱落细胞,伴肾间质水肿及中性粒细胞增多；对照组肾脏组织中肾小管排列整齐,形态正常,间质无充血、水肿。
4、免疫组化检查：移植肾组织肾小球微血管内皮表面、肾小管上皮ICAM-1(棕黄色阳性反应物,箭头处)表达明显增加；而对照组肾组织血管内皮表面未见明显的ICAM-1表达。
结论：
1、平行板流动腔实验证实采用生物素-亲和素桥连方式制备出的携带ICAM-1靶向超声微泡随着时间的推移粘附数量不断地增加,提示其靶向粘附能力良好,可以用来评价病变组织的血管内皮炎症。
2、采用供肾血管与受体同名血管作端端吻合,大大减少了对受体循环系统的影响,保证了良好的术后生存期,为实验的进一步进行建立坚实基础。
3、应用携带抗ICAM-1单抗靶向超声微泡行对比超声检查可有效评价大鼠移植肾AR,提示将有望为临床提供一种早期、敏感、无创的检测移植肾AR的手段。
Background and Objective
Renal allograft acute rejection, a common complication after kidney transplantation, most likely leads to the lost of the early function of the transplanted kidney, and thus it becomes one of the main obstacles to the survival and function-maintenance of the transplanted renal. Studies have shown that the first sign of renal allograft acute rejection (AR) is the renal micro-vascular endothelial injury. The injury is characterized by the inflammatory and rejection-related molecular markers such as intercellular adhesion molecule, vascular cell adhesion molecule, which are expressed or secreted by the endothelial cells. These markers can promote the adhesion, aggregation and leakage of the platelets and neutrophilic granulocytes along the endothelium. What's worse, the neutrophilic granulocytes can result in inflammatory reaction and release several inflammatory mediators or pro-inflammatory cytokines into the renal interstitial tissues, such as lymphocytes, tumor necrosis factor-a, interleukin-1, thromboxane A2 and so on, which then lead to the injury of renal capillary vessel and cells. Therefore, to the extent that vascular endothelial injury is an important feature at the early stage of renal transplant rejection, we can presume the presence of AR considering the specific molecular markers such as ICAM-1 expressed by vascular endothelial.
In fact, scholars have made long researches and conducted a variety of studies on the diagnosis of renal transplant rejection, especially on the diagnosis of acute rejection, in order to make an accurate diagnosis and treatment before irreversible changes occur in the organization. Many researchers have carried out large numbers of explorations and studies from the aspect of the immunological monitoring、renal imaging and pathology testing and so on. However, different means and methods of examination have their own limitations.
For example, renal scintigraphy can evaluate the ischemic injury, but it can not identify the acute rejection, acute tubular interstitial nephritis and cyclosporine toxicity at the early stage after the renal transplantation, and only has certain value for the recovery and short-term or long-term of graft survival. Other non-targeted radiologic imaging methods, including MRI and PET, can provide valuable information for the assessment of morphological and metabolic changes respectively associated with rejection. Despite the advantages, these methods have some disadvantages which can not be solved. For example, instruments with high requirements, not the bedside examination, the lack of stability of the radioactive tracer, and the radioactive contamination have made certain restrictions in the clinical applications.
Nowadays, histopathological examination is still the most accurate method for the diagnosis of AR. However, due to its injury to the kidney, bleeding and other serious complications, it can not be used as routine monitoring tools. Thus, it is in great clinical significance to search for a safe, noninvasive, sensitive monitoring method for early diagnosis of AR.
In recent year, with the appearance of targeted ultrasound contrast agents, targeted ultrasound molecular imaging gradually becomes a reality, which has greatly expanded the scope of application of ultrasound. Its role as an intravascular tracer in being detected while adhering to endothelial surface makes it possible to apply the molecular lever ultrasound technology in the early diagnosis of various diseases. Currently, targeted ultrasound molecular imaging has successfully evaluated in the areas of vascular endothelial inflammation/angiogenesis of kidneys, limbs, tumor, and it has also conducted a preliminary exploration to the heart transplant rejection. But its targeted imaging research makes no progress, because renal transplantation models are difficult and fussy to build. After many years of exploration and technical improvements, our laboratory has made considerable achievements in building targeted ultrasound micro bubbles, the size, concentration, stability and antibody rate of which have reached the international equivalent or higher level. And we have successfully assessed the endothelial inflammatory response in the kidney/heart and other parts by targeting intercellular adhesion molecule (ICAM-1). On the basis of the preliminary study, this study tries to combine targeted micro bubbles and Ultrasound contrast to evaluate and diagnose of AR early, which will undoubtedly have great clinical significance.
In conclusion, on the basis of common lipid micro bubbles, we have constructed the micro bubbles targeted to ICAM-1 by combining the anti-rat-ICAM-lmonoclone antibodies to the shell of general lipid microbubbles via "avidin-biotin" bridging chemistry. By using this micro bubbles targeted to ICAM-1 (MBI) and the control microbubbles (MB), the VI (video intensity) of renal were measured separately by CEU. We hypothesized that the AR could be accurately evaluated with microbubbles targeted to ICAM-1 by using CEU.
Methods
1.Microbubbles preparation
General lipid microbubbles (MB) and lipid microbubbles with biotin were prepared by sonication of perfluorocarbon gas (C3H8) with aqueous dispersion of several lipids in determinate ratio. After being washed (1×) to remove excess free unincorporated lipid, streptavidin in determinate ratio were added to the lipid microbubbles with biotin, then washed (1×) to removed excess free unincorporated streptavidin and the biotin conjugated mouse-anti rat ICAM-1 monoclone antibodies and isotype control antibodies in determinate ratio were added to complete the preparation of MBI and MB. At last, the MBI and MB were washed (1×) to remove excess free unincorporated antibodies. Both MB and MBI were storaged in refrigerator at 4℃.
2. Evaluation of microbubble in vitro:
2.1 The mean diameter and density in both MB and MBI were measured by coulter counter. Using green fluorescent-labeled antibody for anti rat ICAM-1 monoclone antibody to identify the linking antibodies on the MBI.
2.2 Assessment of microbubbles targeted to ICAM-1 with Parallel plate flow chamber
The binding and retention of targeted microbubbles to ICAM-1 immobilized on a culture dish were assessed in a parallel-plate flow chamber. Targeted microbubbles drawn through the flow chamber coated with ICAM-1 (1000ng/ml) at a shear stress of 0.5dyn/cm2. All groups use 3 samples according to the recommended protocol. The adhesion abilities of control microbubbles with isotype antibodies were tested as control groups. Quantitative analysis of microbubble accumulation was performed by counting the number of microbubbles adhered in the observed area and a graph of microbubble accumulation with time was plotted.
3. Rat model for acute transplant rejection
The 10 male Wistar rats and SD rats respectively(250-300g) used in this study were fasted 24 hours before surgery, but free of water. Wistar rats were anesthetized by intraperitoneal injection of Pentobarbital (1g/kg). The skin was incised by a longitudinal cut in the middle of abdomen. Left ureter, left renal artery and vein、vena cava and abdominal aorta were separated,4-0 silks were placed in the upper and lower of inferior vena cava and abdominal aorta. A small hole was cut between the traction line of the inferior vena cava to do as a perfusate outflow tract.
At the same time, Cut a hole in the abdominal aorta anterior to prime heparin Ringer's solution(4C,25u/ml), Until the color of kidneys paled and outflow of liquid of inferior vena cava turned clear, The amount of perfusate was 5-10ml. After removing the left renal artery and vein, ureter,the left kidney was placed in the small cap filled with Ringer's lactate(0-4℃).
After abdominal incision, SD rats were ligated renal artery and vein and ureter near the left renal hilum, and left kidney was removed. the same name of renal arterial and venous of the kidney of donor and recipient were anastomosed in turn. The kidney perfused well, turned pink very quickly, and functioned immediately. Rat model established successfully.
4.CEU imaging:Ten SD rats with renal allograftt acute rejection (AR) and ten normal SD rats were performed with CEU respectively by using MBI and MB, the intravenous injection of 1×109 microbubbles were made in random order with 30 minutes interval. After Three minutes of intravenous injection, microbubbles in the circulation were eliminated, the ultrasound signal (video intensity, VI) from MB and MBI were measured by second harmonic CEU imaging with pulsing interval time (PI) of ten seconds and a mechanical index (MI) of 0.2, transmission frequency of 7.0 MHz. After the first picture of CEU imaging being taken, the microbubbles were destroyed by two to three seconds of continuous imaging with a high MI of 1.9 and the background subtracted VI of Renal were measured.
5.Examination of pathology and immunohistochemisty:After CEU imaging, all kidney of experimental rats were cutted for the examination of pathology and immunohistochemisty.
6.Statistical analysis:Data are expressed as mean±SD. Experiment one: Comparisons between different groups were made with repeated measures; comparison between the two groups within each time point using One-way ANOVA to analyze; Experiment two:Comparisons between different groups were made with independent t-test, Interval comparisons were made with two stages cross-over design. Differences were considered significant at a value of P<0.05(2-sided).
Results
1.Results for microbubble preparation:The density of MBI and MB is about1.5×109/ml and 1.7×109/ml separately, the mean sizes for MBI and MB were about 2.71μm and 2.86μm respectively.
2.Results for evaluation of microbubble in vitro:The anti-Rat ICAM-1 monoclone antibodies linked well to the surface of microbubbles, which were observed with fluorescence microscopy, shell of ultrasound microbubbles showed obvious green fluorescence, That shows targeted microbubbles constructed successfully.
3.Results for evaluation of MBI using parallel plate flow chamber:Keeping the shear stress and microbubble concentration constant at 0.5dyn/cm2 and 2×108/ml, respectively, the microbubble accumulation over a 6-min interval was assessed. Numbers of microbubbles increased gradually with the increase of time. But there was only minimal and no significant adhesion in the control group.there was obvious differences(P=0.000).
4.Results for CEU imaging:A significant enhancement in ultrasound was observed in transplant kidney of MBI-group. Increase in VI value of transplant kidney region in MBI-group was great and it amounted to 27.0±7.4. However, increase in VI value of transplant kidney in MB-group was minor, it was just 10.2±2.4. Difference was evident in transplant kidney between of the two groups (P=0.000). The VI in transplant kidney of MBI-group is 3.7±1.3 times to the MBI-group of normal kidney (7.7±2.2) and 4.2±1.2times to the MB-group of normal kidney (6.5±1.0). There was obvious difference between the two groups (P=0.000) The VI in transplant kidney of MB-group is 1.4±0.4 times to the MBI-group of normal kidney and 1.6±0.6times to the MB-group of normal kidney. There was obvious difference between the two groups (P<0.05)
5. Results for examination of pathology and immunohistochemisty:Pathological examination showed that renal tubules arranged well and there was no congestion, interstitial edema in the normal kidney; We observed tubular epithelial cell becomed swelling, degeneration, and necrosis. Renal interstitial edema and neutrophil increased was also observed in transplant kidney. It was indicated by immunohistochemisty that the expression of endothelial ICAM-1 increased in transplant kidney compared to normal kidney.
Conclusions
1. Parallel plate flow chamber experiments show that targeted microbubbles can be successfully constructed via "avidin-biotin" bridging chemistry. The adhesion performance of targeted microbubble was very well, which indicated targeted microbubbles can be Possiblely used to evaluate the severity of lesions.
2. Anastomosis the same name of renal arterial and venous of donor and recipient, which reduced the impact on the circulatory system, ensuring a good postoperative survival, mading a solid foundation for further the experiments.
3.Microbubbles targeted to ICAM-1 (MBI) and CEU that create "active targeted CEU imaging" can effectively evaluate the acute renal allograft rejection injury in rat, and may be used to evaluate the microvascular inflammation and other endothelial responses.
引文
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