本文選題：活體熒光成像 + 二氧化硅熒光納米探針��； 參考：《中國人民解放軍醫(yī)學院》2016年博士論文

【摘要】：研究背景：分子影像學(Molecular imaging, MI)是近年來隨著現(xiàn)代化科學技術和生命醫(yī)學的飛速發(fā)展應用而生的新的學科,是將醫(yī)學影像技術和分子生物學技術相結合的成像方法。利用多種影像學方法和分子探針,能夠在活體狀態(tài)下對生物過程進行細胞層面和分子水平上,甚至基因水平的定性和定量研究。以研究生物體內生化進程和生理變化為目標的新興學科。MI最顯著特點是：借助處于前沿領域的各種分子探針,利用多種影像學方法,如MRI、CT、核素、PET、超聲和光學等影像學技術,選擇特異性介質,實現(xiàn)對特定甚至單個細胞、分子的非侵入性顯像。目的：本課題組利用自行研發(fā)構建的分子靶向探針進行光學成像,以考察其生物相容性及特異靶向性為目的,進行了一系列研究,包括探針的體內體外實驗、人三陰性裸鼠乳腺癌模型構建、腫瘤的活體成像診斷及治療為一體的研究。方法：文章的第一部分采用反相微乳液法制備了介孔二氧化硅(MSNs),以此為載體介質,選用近紅外熒光染料吲哚菁綠(ICG)為熒光材料,制備了ICG-MSNs熒光生物化學納米顆粒,通過共價偶聯(lián)的方法上載了特異性靶向腫瘤新生血管的靶向配體RGD,獲得熒光信號較強的納米探針RGD-ICG-MSNs 。利用熒光顯微鏡觀察探針對RGD(+)表達的MDA-MB-231三陰性乳腺癌細胞的靶向性,以RGD(-)表達的乳腺癌細胞系MCF-7為對照,考察其用于標記與靶向吞噬性的效能。采用慢病毒轉染了熒光素酶和綠色熒光蛋白的人類三陰性乳腺癌MDA-MB-231細胞系,傳代培養(yǎng),接種于BALB/C裸鼠的肩胛部皮下、乳房墊下及尾靜靜注射,建立三陰性乳腺癌裸鼠皮下、原位及靜脈轉移性腫瘤模型。文章第二部分利用熱層析成像儀和超聲儀在腫瘤生長早期進行檢測,并用小動物活體成像儀監(jiān)測腫瘤細胞在裸鼠體內的生長情況及探針的主動靶向性能。在癌細胞接種的第5天時,熱層析成像系統(tǒng)報告了該部位的異常表現(xiàn),而同一組裸鼠在第9天時超聲才可以探及結節(jié)樣結構,因此,熱層析成像儀對微小腫瘤的檢測要早于超聲。熒光靶向探針在小動物活體熒光儀的監(jiān)視下,不僅可以準確的靶向活體的皮下和原位表淺的乳腺腫瘤,而且可以靶向裸鼠腹腔內的多發(fā)轉移性淋巴結。文章的第三部分利用所構建的活體腫瘤模型進行超聲引導下微波消融治療,并且術前與術后均用超聲造影來評價腫瘤血管的灌注情況及毀損情況,為腫瘤治療提供了可視化原位滅活的新方法。結果：實驗結果表明,RGD(+)表達的MDA-MB-231細胞較RGD(-)表達的乳腺癌MCF-7細胞對熒光納米顆粒的吞噬效果明顯增強;RGD-ICG-MSNs具有生物相容性好、細胞毒性低及化學穩(wěn)定性好的優(yōu)點;成功建造了三陰性乳腺癌的裸鼠皮下瘤、原位瘤及靜脈轉移瘤模型,該動物模型用于后續(xù)的熱層析成像實驗及活體熒光成像實驗,用以考察腫瘤細胞的最早檢測時間及探針在體內的尋靶性能;熱層析成像儀和超聲儀在腫瘤生長早期進行檢測結果表明,在癌細胞接種的第5天時,熱層析成像系統(tǒng)報告了該部位的異常表現(xiàn),而同一組裸鼠在第9天時超聲才可以探及結節(jié)樣結構,因此,熱層析成像儀對微小腫瘤的檢測要早于超聲;熒光靶向探針可以準確的靶向活體的皮下和原位表淺的乳腺腫瘤,可以靶向裸鼠腹腔內的多發(fā)轉移性淋巴結;超聲引導下微波消融治療裸鼠皮下及原位腫瘤,并且術前與術后均用超聲造影來評價腫瘤血管的灌注情況及毀損情況,力求為腫瘤治療提供可視化原位滅活的新方法提供實驗依據。結論：RGD-ICG-MSNs具有良好的化學穩(wěn)定性及生物相容性,細胞毒性小,可以用于人三陰性乳腺癌裸鼠皮下及原位腫瘤的靶向診斷;利用熱層析及熒光成像系統(tǒng)為腫瘤早期、精準的定性診斷奠定實驗基礎;超聲可視化引導下微波消融可以徹底消滅腫瘤細胞。
[Abstract]:Background: Molecular imaging (MI) is a new discipline developed in recent years with the rapid development of modern science and technology and life medicine. It is an imaging method combining medical imaging technology with molecular biology technology. By using a variety of imaging methods and molecular probes, it can be used in living organisms. The process carries on the qualitative and quantitative study of the cell level and molecular level, and even the gene level. The most prominent feature of the new subject.MI, which aims at studying biochemical processes and physiological changes in the organism, is to use a variety of molecular probes in the frontiers and use a variety of imaging methods, such as MRI, CT, nuclide, PET, ultrasound and optical image. In order to investigate the biocompatibility and specific targeting, a series of studies have been carried out, including the in vitro and in vitro experiments of the probe. Three the construction of the breast cancer model in negative nude mice, the research of the living imaging diagnosis and treatment of the tumor. Method: the first part of the article uses the reverse microemulsion method to prepare the mesoporous silica (MSNs) as the carrier medium, and uses the near infrared fluorescent dye indocyanine green (ICG) as the fluorescent material to prepare the ICG-MSNs fluorescence biochemistry. Nanoparticles, by covalently coupled method, loaded the targeted ligand RGD for specific targeting tumor neovascularization, and obtained a strong nanoscale probe RGD-ICG-MSNs. The targeting of MDA-MB-231 three negative breast cancer cells expressed by RGD (+) was observed by fluorescence microscopy, and MCF-7 of the breast cancer cell line expressed by RGD (-) was the pair. The human three negative breast cancer MDA-MB-231 cell lines, which were transfected with luciferase and green fluorescent protein, were cultured, inoculated in the subcutaneous tissue of the scapula of BALB/C nude mice, under the breast padding and tail injection to build the subcutaneous, in situ and venous transfer of three negative breast cancer in nude mice. The second part of this article is to detect the growth of tumor cells in nude mice and the active target performance of the probe in nude mice by using the thermal tomography instrument and the ultrasound apparatus in the early stage of tumor growth. The thermal layer analysis system reports the abnormal manifestation of the tumor at the fifth day of the cancer cell inoculation. In the same group of nude mice, the nodular structure can be detected by ultrasound at ninth days. Therefore, the detection of microtumors by the thermal tomography instrument should be earlier than that of the ultrasound. Under the monitoring of the small animal living fluorograph, the fluorescent target probe can not only target the subcutaneous and the superficial breast tumors of the living body accurately, but also target the nude mice intraperitoneally. The third part of the article uses the constructed living tumor model for ultrasound guided microwave ablation, and both preoperative and postoperative ultrasound contrast is used to evaluate the perfusion and damage of tumor vessels. A new method of visualizing the in-situ inactivation of tumor is provided for the treatment of tumor. The MDA-MB-231 cells expressed by RGD (+) are more effective than RGD (-) expressed in RGD (-) breast cancer MCF-7 cells, and RGD-ICG-MSNs has the advantages of good biocompatibility, low cytotoxicity and good chemical stability. The subcutaneous tumor of nude mice with three negative breast cancer, in situ tumor and vein metastatic tumor model have been successfully constructed. The object model was used for subsequent thermal tomography and in vivo fluorescence imaging experiments to investigate the earliest detection time of the tumor cells and the searching performance of the probe in the body. The thermal tomography imaging instrument and the ultrasound apparatus were used to detect the early growth of the tumor. The thermal tomography system reported the site at the fifth day of the cancer cell inoculation. The same group of nude mice can detect the nodular structure at ninth days. Therefore, the detection of microtumors by the thermal tomography instrument should be earlier than that of ultrasound; the fluorescent target probe can target the subcutaneous and shallow breast tumor in situ, and can target the multiple metastatic lymph nodes in the abdominal cavity of nude mice; Microwave ablation in the treatment of subcutaneous and orthotopic tumors in nude mice, and the preoperative and postoperative ultrasonography were used to evaluate the perfusion and damage of tumor vessels, and to provide experimental basis for the new method of visualizing in situ inactivation of tumor. Conclusion: RGD-ICG-MSNs has good chemical stability and biocompatibility. Small toxicity, can be used in the target diagnosis of subcutaneous and in situ tumors in nude mice with three negative breast cancer; using thermal tomography and fluorescence imaging system for early tumor, accurate qualitative diagnosis lay the experimental basis; ultrasound visualization guided microwave ablation can completely eliminate tumor cells.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R737.9;R445

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