VEGF-C靶向USPIO分子探针在肝癌特异性磁共振成像中的应用价值

发布时间：2018-03-22 10:42

本文选题：靶向成像　切入点：血管内皮生长因子C　出处：《山东大学》2014年硕士论文　论文类型：学位论文

【摘要】：研究背景肝细胞癌(hepatocellular carcinoma, HCC)是最常见的恶性肿瘤之一,超过50%的新发病例发生在中国,其病死率和复发率居高不下。HCC的侵袭、转移以及极高的术后复发率是影响患者生存和预后的主要障碍。近年来研究表明,血管内皮生长因子C (VEGF-C)通过与其配体R3(VEGFR-3)的结合介导肿瘤淋巴管生成,是形成肿瘤淋巴道转移的最重要因素,因而VEGF-C也成为反映肿瘤淋巴管生成、提示肿瘤侵袭转移的一个较为特异的标记物。目前HCC的诊断主要依赖影像学技术,但影像学对小病灶及不典型病灶诊断困难,同时对于肿瘤的转移倾向也难以准确评估。本研究构建靶向VEGF-C的超小型超顺磁性氧化铁颗粒(ultrasmall superparamagnetic iron oxide nanoparticles, USPIO)分子探针应用诱导性肝癌动物模型进行MR成像,旨在探讨该探针在HCC特异性成像中的价值,同时基于此反映肿瘤的转移倾向。目的探讨血管内皮生长因子C (VEGF-C)靶向超小型超顺磁性氧化铁(USPIO)分子探针在肝细胞肝癌中特异性MR成像中的价值。方法胺基修饰的USPIO连接VEGF-C抗体后构建VEGF-C-USPIO靶向分子探针。CCK-8法检测VEGF-C-USPIO探针对人脐静脉内皮细胞株(HUVEC)及HepG2细胞活性的影响。实验分为VEGF-C-USPIO组和USPIO组。将VEGF-C-USPIO和USPIO(含铁10μg/ml)按照20μl、40μl、80μl的剂量加入到HUVEC中孵育4h后,进行MR成像,测量其T2WI, T2*, R2*信号强度(SI)。构建大鼠原位肝细胞肝癌模型后随机分为两组(每组3只),分别于尾静脉注射VEGF-C-USPIO或USPIO,注射剂量为20μmol/Kg,于注射前、注射后0.5h、1h及1.5h进行磁共振成像,测量肿瘤组织T2WI及T2*WI的信号强度,并分析两组增强前后各时间点上述测量值的差异。细胞及肿瘤切片行普鲁士蓝染色以验证两组标本的铁含量,免疫荧光验证HUVEC中VEGF-C的表达情况,免疫组化染色验证肝癌组织中VEGF-C的表达情况。结果细胞毒性实验结果显示,不同浓度梯度、不同孵育时间VEGF-C-USPIO对HUVEC及HepG2细胞的细胞活力影响均较小。细胞悬液磁共振成像显示靶向组及非靶向组T2WI, T2*信号强度均随VEGF-C-USPIO和USPIO的剂量增加而下降,R2*信号强度随剂量的增加而上升,在同一剂量(40μl,80μl)时两组之间差异有统计学意义(P0.05)。细胞普鲁士蓝染色显示两组细胞内铁颗粒染色均随剂量的增加而增多,而靶向组细胞内染色铁颗粒明显多于非靶向组。动物实验结果显示,VEGF-C-USPIO注射后较注射前肝脏肿瘤T2WI及T2*WI的信号强度均明显下降,信号强度差异有统计学意义(P0.05),其中增强后1h下降程度最低；而注射USPIO后肝脏肿瘤信号强度下降不明显,差异无统计学意义(P0.05)；靶向组与非靶向组之间肿瘤强化后的T2WI及T2*WI信号强度差异也具有统计学意义(P0.05)。普鲁士蓝染色结果显示,靶向组肿瘤组织内见较多铁染色颗粒,非靶向组肿瘤组织内铁染色颗粒较少。结论 VEGF-C-USPIO分子探针无明显细胞毒性,对HUVEC及大鼠原位肝癌具有较好的主动靶向作用,实现了肝癌的特异性成像,同时也基于此对肿瘤的转移能力进行无创评估。
[Abstract]:Research background
Hepatocellular carcinoma (hepatocellular, carcinoma, HCC) is one of the most common malignant tumors, more than 50% new cases occurred in China, mortality rate and recurrence rate of high.HCC invasion, metastasis and high recurrence rate is the main obstacle to the survival and prognosis of patients. Recent studies indicate that vascular endothelial growth factor C (VEGF-C) by R3 (VEGFR-3) and its ligand binding mediated lymphangiogenesis, which is the most important factor of tumor lymphatic metastasis, so VEGF-C has become the reflection of tumor lymphangiogenesis, suggesting a more specific marker for tumor invasion and metastasis. The diagnosis of HCC mainly depends on imaging technology but, the imaging difficulties of small lesions and atypical lesions, while the tumor metastasis is also difficult to accurately assess. This study constructs targeting VEGF-C ultra small superparamagnetic iron oxide particles (ultrasmall superparamagnetic iron oxide nanoparticles, USPIO) molecular probe was applied to MR imaging of induced liver cancer animal models, aiming to explore the value of the probe in HCC specific imaging, and at the same time, to reflect the metastasis tendency of tumor.
objective
To explore the value of vascular endothelial growth factor C (VEGF-C) targeting super small superparamagnetic iron oxide (USPIO) molecular probe in the specific MR imaging of hepatocellular carcinoma.
Method
Amine modified USPIO connection VEGF-C antibody to construct VEGF-C-USPIO molecular probes targeting.CCK-8 VEGF-C-USPIO probe detection method of human umbilical vein endothelial cells (HUVEC) and the activity of HepG2 cells. The experiment was divided into VEGF-C-USPIO group and USPIO group. VEGF-C-USPIO and USPIO (FE 10 g/ml) with 20 L, 40 L, 80 l dose added to HUVEC after 4H incubation, MR imaging, measuring the T2WI, T2*, R2* signal intensity (SI). The establishment of rat orthotopic hepatocellular carcinoma model were randomly divided into two groups (n = 3), respectively in the intravenous injection of VEGF-C-USPIO or USPIO, injection volume 20 mol/Kg before injection, after injection of 0.5h, 1H and 1.5h magnetic resonance imaging, the signal intensity of T2WI and T2*WI were measured in tumor tissue, and analyze the differences between the two groups increased at all time points before and after the measurements. Cells and tumor sections stained with Pu Lu Shilan verification of two groups of specimens The expression of VEGF-C in HUVEC was verified by iron content and immunofluorescence, and the expression of VEGF-C in liver cancer tissues was verified by immunohistochemical staining.
Result
The results of cytotoxicity test showed that different concentration gradient were smaller in different incubation time of VEGF-C-USPIO on HUVEC and HepG2 cell activity. The cell suspension with magnetic resonance imaging target group and non targeted group of T2WI, the signal intensity of T2* increased with VEGF-C-USPIO and USPIO dose increased and decreased, the signal intensity of R2* increased with the increase of dose, at the same dose (40 L, 80 l) was statistically significant when the differences between the two groups (P0.05). Prussian blue staining showed two groups of cells were stained iron particles were increased with the increase of dose, while the target group of intracellular staining was significantly more than that of non target iron particles to the group. Animal experiments showed that after VEGF-C-USPIO injection than before the injection signal intensity of liver tumor T2WI and T2*WI decreased significantly, statistically significant differences in signal intensity (P0.05), which enhanced the lowest degree of 1H decreased; and after the injection of USPIO of liver Dirty tumor signal intensity did not decrease obviously, the difference was not statistically significant (P0.05); T2WI and T2*WI signal strength difference of target group and non targeted groups after the tumor enhancement was also statistically significant (P0.05). Prussian blue staining showed that the target group was found in tumor tissues stained granules of more iron, non target to the group in tumor tissue iron staining particles smaller.
conclusion
VEGF-C-USPIO molecular probe has no obvious cytotoxicity. It has a good targeting effect on HUVEC and hepatocellular carcinoma in situ, and achieves the specific imaging of liver cancer. Meanwhile, it also carries out non-invasive evaluation of tumor metastasis ability.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R735.7;R445.2

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