本文選題：靶向成像　切入點：血管內皮生長因子C　出處：《山東大學》2014年碩士論文　論文類型：學位論文

【摘要】：研究背景 肝細胞癌(hepatocellular carcinoma, HCC)是最常見的惡性腫瘤之一,超過50%的新發(fā)病例發(fā)生在中國,其病死率和復發(fā)率居高不下。HCC的侵襲、轉移以及極高的術后復發(fā)率是影響患者生存和預后的主要障礙。近年來研究表明,血管內皮生長因子C (VEGF-C)通過與其配體R3(VEGFR-3)的結合介導腫瘤淋巴管生成,是形成腫瘤淋巴道轉移的最重要因素,因而VEGF-C也成為反映腫瘤淋巴管生成、提示腫瘤侵襲轉移的一個較為特異的標記物。目前HCC的診斷主要依賴影像學技術,但影像學對小病灶及不典型病灶診斷困難,同時對于腫瘤的轉移傾向也難以準確評估。本研究構建靶向VEGF-C的超小型超順磁性氧化鐵顆粒(ultrasmall superparamagnetic iron oxide nanoparticles, USPIO)分子探針應用誘導性肝癌動物模型進行MR成像,旨在探討該探針在HCC特異性成像中的價值,同時基于此反映腫瘤的轉移傾向。 目的 探討血管內皮生長因子C (VEGF-C)靶向超小型超順磁性氧化鐵(USPIO)分子探針在肝細胞肝癌中特異性MR成像中的價值。 方法 胺基修飾的USPIO連接VEGF-C抗體后構建VEGF-C-USPIO靶向分子探針。CCK-8法檢測VEGF-C-USPIO探針對人臍靜脈內皮細胞株(HUVEC)及HepG2細胞活性的影響。實驗分為VEGF-C-USPIO組和USPIO組。將VEGF-C-USPIO和USPIO(含鐵10μg/ml)按照20μl、40μl、80μl的劑量加入到HUVEC中孵育4h后,進行MR成像,測量其T2WI, T2*, R2*信號強度(SI)。構建大鼠原位肝細胞肝癌模型后隨機分為兩組(每組3只),分別于尾靜脈注射VEGF-C-USPIO或USPIO,注射劑量為20μmol/Kg,于注射前、注射后0.5h、1h及1.5h進行磁共振成像,測量腫瘤組織T2WI及T2*WI的信號強度,并分析兩組增強前后各時間點上述測量值的差異。細胞及腫瘤切片行普魯士藍染色以驗證兩組標本的鐵含量,免疫熒光驗證HUVEC中VEGF-C的表達情況,免疫組化染色驗證肝癌組織中VEGF-C的表達情況。 結果 細胞毒性實驗結果顯示,不同濃度梯度、不同孵育時間VEGF-C-USPIO對HUVEC及HepG2細胞的細胞活力影響均較小。細胞懸液磁共振成像顯示靶向組及非靶向組T2WI, T2*信號強度均隨VEGF-C-USPIO和USPIO的劑量增加而下降,R2*信號強度隨劑量的增加而上升,在同一劑量(40μl,80μl)時兩組之間差異有統(tǒng)計學意義(P0.05)。細胞普魯士藍染色顯示兩組細胞內鐵顆粒染色均隨劑量的增加而增多,而靶向組細胞內染色鐵顆粒明顯多于非靶向組。動物實驗結果顯示,VEGF-C-USPIO注射后較注射前肝臟腫瘤T2WI及T2*WI的信號強度均明顯下降,信號強度差異有統(tǒng)計學意義(P0.05),其中增強后1h下降程度最低；而注射USPIO后肝臟腫瘤信號強度下降不明顯,差異無統(tǒng)計學意義(P0.05)；靶向組與非靶向組之間腫瘤強化后的T2WI及T2*WI信號強度差異也具有統(tǒng)計學意義(P0.05)。普魯士藍染色結果顯示,靶向組腫瘤組織內見較多鐵染色顆粒,非靶向組腫瘤組織內鐵染色顆粒較少。 結論 VEGF-C-USPIO分子探針無明顯細胞毒性,對HUVEC及大鼠原位肝癌具有較好的主動靶向作用,實現(xiàn)了肝癌的特異性成像,同時也基于此對腫瘤的轉移能力進行無創(chuàng)評估。
[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.

【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R735.7;R445.2

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本文編號：1648304