超声靶向造影及弹性成像对移植肝胆道并发症评价体系的建立及其机制研究

Liver transplantation remained to be the most effective therapeutic option for patients with end-stage liver diseases. However, biliary complications, once considered as the technical 'Achilles heel' of orthotopic liver transplantation, still remained a common source of morbidity and mortality. The diagnosis of early stage of the desease is extremely important..Biliary complications appeared attributable to various factors, including hepatic artery thrombosis or stenosis, technical reasons, as well as ischemia-reperfusion injury and immunological injury, the typical characteristic of which was the pathological deposit of collagen, which leaded to cholangiohepatitis eventually..The mechenism of the development of biliary complication after liver transplant remained to be unclear. However, it seemed that epithelial mesenchymal transfer might play an important role in it. EMT describeed the process by which cells gradually lose typical epithelial characteristics and acquire mesenchymal traits, such as disruption of cell-cell adherence, loss of apico-basal polarity, matrix remodeling, migration and so on..Evidence had been shown that the biliary epithelial cells had the potential to undergo EMT, and various mesenchymal proteins were demonstrated during the process of biliary hepatopathy. However, assessment of EMT in adult liver had generally been addressed by immunohistochemistry, which technically could be demonstrated in an given time. Thus, a method which could assess the expression of the markers was strongly needed..Targeted contrast-enhanced ultrasound imaging had emerged as a promising new non-invasive imaging strategy for imaging biological processes at the molecular level recent years. The basis of targeted contrast-enhanced ultrasound imaging is the combination of microbubbles and specific antibodies, of which the effect could be amplified by the biotin-avidin linkage. This linkage had been used in the targeted imaging of neoplastic angiogenesis and was high predictable to be effective in the imaging of epithilial markers..Ultrasound elastography had emerged as a noninvasive method to evaluate the tissue elastic properties, which used the transient vibration or acoustic radiation force to induce a difference between the scattered signals of soft and hard tissures. It had been proved to be helpful in the diagnosis in many diseases, including cirrhosis, throbosis, neoplasma and so on. The mechanism was demonstrated to be correlated to collagen deposit. However, its application in the predict of molecular expression had not been practiced..Therefore, we decided to design a microbubble which can be specifically combined with the biotin-antibody, which can be used as a marker imaging methods as well as the ultrasound elastography, to effectively diagnosing the early stage biliary complications after liver transplant, and hopefully, demonstrate the role of EMT in the disease process eventually.

肝移植是目前治疗终末期肝胆疾病最有效的方法，但高发生率和死亡率的胆道并发症成为其致命缺陷。该并发症的主要病理表现为胆管上皮细胞缺失及功能改建，产生病理性修复，最终引起胆道狭窄及胆源性纤维化。早期发现和评价该病在临床工作中具有重要意义。本课题组前期研究表明该病早期胆管上皮细胞可能发生间叶组织标记物的上调，产生胶原沉积，从而最终引起胆源性肝纤维化和胆源性肝硬化。超声靶向造影将纳米级微气泡和特异性抗体相连，可用于特定分子的靶向显影，而超声弹性成像则利用剪切波对组织硬度和粘弹性进行实时测量，与胶原含量相关密切。因此，在分子学检测技术的基础上，本课题组拟构建特异性造影剂，与胆管上皮细胞表面的间质标记物进行特异性结合和显影，同时测定胆管壁和肝实质弹性指数，研究随时间和空间分布动态变化的特征，以进一步阐明该并发症发生的分子机制，并建立对移植肝早期胆道并发症的无创评价体系，以提高该病的早期诊断率和治疗率。

缺血再灌注损伤（IRI）是目前肝移植术后并发症的重要因素之一，临床上对于这种并发症的处理相当棘手，主要是由于缺乏早期、特异性的诊断方法。为了解决这一问题，本研究应用亲和素-生物素法，构建一种新型的靶向超声分子显像剂，探讨LSEC表达ICAM-1分子与肝移植后肝功能损伤的关系，从细胞和分子水平，检测肝内血流灌注，评估肝移植后肝脏缺血再灌注损伤的病理生理变化及显影特征，并考察靶向脂质纳泡体内显影的可重复性及造影参数的稳定性，探讨显影强度与病理及分子标记物的相关性，提示疾病的发展过程。.研究表明，自制靶向脂质纳泡粒径约148.15±39.75nm，Zeta电位-28.4 ± 7.9 mV，浓度（3.6~7.4）×109个/ml，激光共聚焦显微镜下观察靶向脂质纳泡的绿色荧光较好地粘附在H/R组人肝窦内皮细胞表面。肝脏IRI后，以细胞凋亡为主。肝窦内皮细胞凋亡比例17.53±1.19%，肝细胞凋亡比例5.61±0.85%。IRI后，肝窦内皮细胞ICAM-1显著表达，且与ALT、LDH呈线性相关，相关系数分别为r=0.812，r=0.854。靶向脂质纳泡体内显影效果稳定，组内相关系数ICC=0.938，可重复性好。超声造影测量参数选取肝包膜下3cm深度作为ROI区域，变异性最小，CV 3.9%。两名观测者在相同条件下测量结果一致性好，ICC=0.948，信度较高。动物体内超声造影显示，I/R组靶向脂质纳泡PI及增强持续时间明显高于普通纳泡，达峰时间明显提前；靶向脂质纳泡的PI及增强持续时间在I/R组明显高于SO组。I/R组靶向脂质纳泡PI与肝脏IRI病理Suzuki’s评分呈正相关，秩相关系数rs =0.648；与IRI后肝内ICAM-1表达呈线性相关，相关系数r=0.706。.综上所述，利用超声靶向造影对分子标记物进行显影，是一种可用于活体、动态评价组织内细胞因子变化的分子显像方法，早于形态学的改变，具有较好的创新性和应用价值；使超声从传统的解剖、形态学成像发展为功能与分子水平的成像方法，从分子水平，无创、定量评估肝移植后肝脏缺血再灌注损伤，提高了超声分子成像技术的诊断能力，具有可行性，为临床基于疾病分子表型的诊断与个性化精准治疗提供重要的依据。