靶向glypican-3荧光探针在肝细胞癌诊断中的应用研究

发布时间：2018-03-29 06:29

本文选题：肝细胞癌　切入点：glypican-3　出处：《中国人民解放军医学院》2017年博士论文

【摘要】：目的:基于glypican-3在人肝癌组织中的特异表达,构建靶向glypican-3荧光探针,在细胞、动物及人体水平探索其在肝细胞癌诊断中的应用。方法:(1)构建靶向glypican-3荧光探针并进行探针表征、安全性及稳定性验证;(2)利用Western blot及RT-PCR验证glypican-3在肝癌细胞系中的表达水平;(3)利用流式细胞术及细胞免疫荧光实验证实glypican-3在HepG2细胞膜表面表达;(4)设置不同时间梯度,利用细胞免疫荧光实验验证HepG2细胞及永生化正常肝细胞系L02对靶向glypican-3荧光探针的摄取水平;(5)建立裸鼠HepG2皮下瘤模型及U87皮下瘤模型,尾静脉注射靶向glypican-3荧光探针并利用小动物活体成像设备观察其在体分布及代谢,设置抗体提前封闭组及作为对照;(6)利用体式荧光显微镜验证该探针在近红外光下手术导航切除肝癌的应用;(7)对肝脏恶性肿瘤患者超声引导下组织活检,活检组织取出后即刻喷洒靶向glypican-3荧光探针,利用近红外显像系统观察肿瘤组织及周围正常组织的探针荧光蓄积情况。与患者术后病理活检诊断进行对比,探索肝细胞癌组织近红外荧光显像与病理诊断及glypican-3表达的相关性。结果:(1)成功构建靶向glypican-3荧光探针且该探针在血清中稳定性较高,体外及在体实验均证实该探针安全无毒;(2) Western blot 及 RT-PCR 证实 glypican-3 蛋白在 HepG2 细胞蛋白及 mRNA水平特异性高表达;(3)流式细胞术及激光共聚焦实验证实glypican-3在HepG2细胞膜表面高表达;(4)细胞免疫荧光实验证实与L02细胞系相比,HepG2细胞系与探针共孵育30分钟后即可特异摄取靶向glypican-3荧光探针,glypican-3抗体提前封闭HepG2细胞后可显著降低HepG2细胞对的特异探针摄取。HepG2细胞对靶探针的摄取能力随着孵育时间的延长而增加;(5)尾静脉注射靶向glypican-3荧光探针后,探针可在裸鼠HepG2肿瘤内特异性蓄积,尾静脉注射探针6小时后肿瘤与周边组织荧光对比度最强;(6)裸鼠模型证实该探针可应用于近红外光下手术导航切除肝癌;(7)对人肝脏恶性肿瘤组织的近红外成像显示,肝细胞癌喷洒荧光探针后荧光强度较肝转移癌及周边正常肝脏显著升高(P0.01)。当肿瘤组织与周边肝组织近红外荧光强度比值2.32时对HCC诊断效能最高,敏感度为83.3%、特异度为100%。肿瘤组织应用探针后荧光强度与组织glypican-3表达水平密切相关,且有无肝硬化对于肿瘤组织荧光强度无显著影响。结论:本研究中构建的靶向glypican-3近红外荧光探针可应用于用于肝细胞癌的临床诊断。
[Abstract]:Objective: the specific expression of Glypican-3 in human hepatocellular carcinoma based on the construction of the probe target, to explore the Glypican-3 fluorescence in the cell, animal and human level and its application in diagnosis of hepatocellular carcinoma. Methods: (1) construction of targeted Glypican-3 fluorescent probe and probe characterization, verification of safety and stability; (2) the expression level of blot using Western and RT-PCR verification of Glypican-3 in HCC cell lines; (3) using flow cytometry and immunofluorescence experiments confirmed that the expression of Glypican-3 on HepG2 cell membrane; (4) setting different time gradient of targeted Glypican-3 fluorescent probe uptake levels by immunofluorescence experiments and HepG2 cells immortalization of normal liver cell line L02; (5) establish HepG2 nude mice subcutaneous tumor model and U87 subcutaneous tumor model, intravenous injection of Glypican-3 targeted fluorescent probes and with small animal imaging equipment to observe the in vivo Distribution and metabolism, set in advance as control group and antibody closed; (6) to verify the application of navigation resection to the probe surgery in the near-infrared light with stereo fluorescence microscope; (7) guided biopsy of liver biopsy in patients with malignant tumor ultrasound, removed immediately after spraying the probe target to Glypican-3 fluorescence probe, fluorescence observation tumor tissue and surrounding normal tissue using near infrared imaging system. The accumulation were compared with the pathological diagnosis of biopsy patients, to investigate the relationship between hepatocellular carcinoma and the expression of near infrared fluorescence imaging and pathological diagnosis of Glypican-3. Results: (1) targeting Glypican-3 is successfully constructed and the fluorescence probe probe in the serum stability higher. In vitro and in vivo experiments have confirmed that the probe is safe and non-toxic; (2) Western blot and RT-PCR confirmed that Glypican-3 protein in HepG2 cells and mRNA protein level of specificity High expression; (3) flow cytometry and confocal laser experiments confirmed that the high expression of Glypican-3 in HepG2 cell membrane; (4) immunofluorescence experiments confirmed that compared with L02 cells, HepG2 cells and probe were incubated for 30 minutes specific uptake targeting Glypican-3 fluorescence probe, Glypican-3 antibody HepG2 closed in advance cells can significantly reduce the uptake ability of HepG2 cells to the specific probes of the probe target uptake in.HepG2 cells increased with prolonged incubation incubation time; (5) intravenous injection of Glypican-3 targeted fluorescent probe, the probe can accumulate in mice HepG2 tumor specific probe, intravenous injection 6 hours after the tumor and surrounding tissue fluorescence the strongest contrast; (6) nude mice confirmed that the probe can be applied to near infrared surgical navigation under the resection of hepatocellular carcinoma; (7) the near infrared imaging of tissues of liver malignant tumors showed hepatocellular carcinoma spray. The optical probe fluorescence intensity than liver metastatic carcinoma and surrounding normal liver was significantly increased (P0.01). When the tissue and surrounding liver tissues near infrared fluorescence intensity ratio of 2.32 HCC to the highest diagnostic efficiency, the sensitivity was 83.3%, specificity of 100%. in tumor tissue after application of probe fluorescence intensity and Glypican-3 levels are closely related, and there is no cirrhosis for tumor tissue fluorescence intensity had no significant effect. Conclusion: the constructed targeting Glypican-3 near infrared fluorescent probe can be applied for clinical diagnosis of hepatocellular carcinoma.

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7

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