USPIO标记LIVIN反义寡脱氧核苷酸靶胰腺癌的磁共振分子成像研究

The early diagnosis and targeting treatment of pancreatic cancer is still the hot and hard question in the field of the tumor study.Livin is recently found one member in inhibitor of apoptosis proteins.It can be highly expressed in various tumors such as pancreatic cancer,but not or lower expressed in normal pancreas.This subject will use the antisense technique of molecular biology in the field of imaging medcine and construct molecular probe.It prepares the antisense oligonucleotide targeting the mRNA of Livin gene and is labeled by ultrasmall superparamagnetic iron oxide.The qualities and functions of this probe were tested by superconductive magnetic resonance unit and vibration magnetometer.Secondly, a series of experiments in vitro were performed with molecular probe and pancreatic cancer cells PANC1.Thirdly,targeted molecular probe were injected into animal models of pancreatic cancer.Targeted MR molecular imaging was done in vivo.Finally the tissue specimens were tested by routine pathology and immunohistochemistry We aimed to make early and specific diagnosis of pancreatic cancer by magnetic resonance molecular imaging.And we will make clear the silencing effect of the probe on Livin gene and Caspase-3 protein expression to reveal the mechanism of effect of the probe on the pancreatic cancer cell apoptosis and to provide a new approach to early and specific diagnosis and targeting gene therapy of pancreatic cancer.

胰腺癌的早期诊断及靶向治疗一直是目前肿瘤研究领域的热点及难点之一。Livin是近年发现的一个凋亡抑制蛋白家族中的成员，可在胰腺癌等多种实体肿瘤细胞中高度表达，而癌旁胰腺组织或正常组织低表达或不表达，我们以Livin基因的mRNA片段作为靶点，制备与该序列互补的反义寡脱氧核苷酸，利用超顺磁性氧化铁标记，制成反义探针，采用超导型磁共振仪及振动磁场计等方法对该探针进行性能检测，然后将该探针和胰腺癌细胞PANC1进行系列体外实验，并将靶向探针注入胰腺癌小鼠动物模型体内，进行靶向活体MR分子水平显像，最后进行常规病理和免疫组织化学检测。其研究目的在于通过磁共振分子成像，早期特异性诊断胰腺癌，并明确该探针对Livin基因的沉默效应及对Caspase-3蛋白表达的影响，进而揭示其对胰腺癌细胞凋亡的影响机制，为胰腺癌的早期诊断及靶向治疗提供新思路。

胰腺导管细胞腺癌是最具侵略性的恶性肿瘤之一，尽管对胰腺导管细胞腺癌的生物学特性和遗传变异的研究已经比较深入，但胰腺导管细胞腺癌的治疗手段仍然是暂时的、无效的。对治疗无效的一个合理解释就是抗癌药物的非特异性递送和到达肿瘤部位的量不足。Livin是与肿瘤发生发展密切相关的凋亡抑制蛋白家族成员之一，可在胰腺癌等多种实体肿瘤细胞中高度表达，而癌旁胰腺组织或正常组织低表达或不表达。我们设计并体外化学合成针对Livin基因的小干扰RNA序列，通过超顺磁性氧化铁纳米粒子（SPIONs）标记，制备靶向反义探针（siLivin-SPIONs），旨在达到靶向特异性递送药物及非侵入性评估运输的效率、活体监测肿瘤反应的目的。siLivin-SPIONs设计合成通过一种驱动内涵体逃逸的膜转位肽（肉豆蔻酰化聚精氨酸肽）作为中介运输到细胞质以及肿瘤选择性肽（EPPT1）以增加细胞内传递和肿瘤特异性。我们已经成功制备反义探针（siLivin-SPIONs），并应用于胰腺癌细胞PANC1进行体内及体外实验。研究结果显示，siLivin-SPIONs聚集于细胞内及肿瘤内，能有效沉默PANC1细胞中Livin基因的表达。肿瘤特异性沉默Livin基因的表达能有效抑肿瘤细胞增殖并增加细胞凋亡，Livin基因通过Caspase蛋白酶水解途径调控PANC1细胞生长。我们的数据表明，siLivin-SPIONs具有肿瘤靶向性及非侵入性评估运输效率的双重特异性探针，为胰腺导管细胞腺癌的靶向治疗提供机会和路径。