【摘要】：腦膠質(zhì)瘤是神經(jīng)外科中最常見的顱內(nèi)腫瘤,發(fā)病率約占中樞神經(jīng)系統(tǒng)腫瘤的40%,成人神經(jīng)惡性腫瘤的78%[1]。近30年來,雖然神經(jīng)影像學(xué)及膠質(zhì)瘤的治療都取得了很大進(jìn)展,但惡性膠質(zhì)瘤的預(yù)后仍然悲觀。成人中惡性膠質(zhì)瘤的1年及5年生存率分別約為30%和13%,其中GBM的中位生存時(shí)間僅12月左右[2]。而腫瘤的發(fā)生從根本上講是基因的病變,目前基因治療從理論到實(shí)踐為徹底地攻克腫瘤,改善患者預(yù)后帶來了極大的希望。但仍然有很多環(huán)節(jié)亟待研究和早日攻破,其中由于缺乏高效穩(wěn)定的載體系統(tǒng),加之中樞神經(jīng)系統(tǒng)血腦屏障結(jié)構(gòu)的特殊性,是導(dǎo)致其治療效率不高的重要原因之一。 本實(shí)驗(yàn)中,通過兩步溶膠-凝膠法,初步合成了明膠-硅氧烷納米粒子(GS NPs),并在此基礎(chǔ)上構(gòu)建了一種經(jīng)親水性聚乙二醇(PEG)、陽離子多肽Tat、核酸適配體TTAl和Fe304共修飾的納米粒子基因載體系統(tǒng)。它不僅能透過血腦屏障,特異靶向腦部腫瘤細(xì)胞,并具有MRI顯影效果。考察其攜帶質(zhì)粒DNA在細(xì)胞水平的轉(zhuǎn)染效率,為該納米粒子作為入腦轉(zhuǎn)運(yùn)載體的應(yīng)用提供理論和實(shí)踐支持。 第一部分通過傅里葉變換紅外光譜(FT-IR)表征GS NPs與Fe304的偶聯(lián)。通過對(duì)納米顆粒的表面電位分析(Zeta potential)、透射電子顯微鏡(TEM)、納米顆粒粒徑分析(DLS)、傅里葉變換紅外光譜(FT-IR)、熱失重分析(TGA)等理化分析檢測方法,研究該材料的理化特性。 第二部分通過激光共聚焦、MRI等成像技術(shù),驗(yàn)證GS NP-Fe3O4作為基因載體可被C6細(xì)胞有效攝取,并能攜帶GFP (green fluorescent protein)報(bào)告基因在腫瘤細(xì)胞中高效靶向表達(dá)。使用磁共振成像對(duì)經(jīng)過細(xì)胞共培養(yǎng)后一定濃度范圍內(nèi)的GS NP-Fe3O4和Fe304進(jìn)行T2加權(quán)序列掃描并分別測量其弛豫率r2值,驗(yàn)證GS NP-Fe3O4作為MRI對(duì)比劑的可能性。 第三部分通過在腦立體定位儀引導(dǎo)下注射C6膠質(zhì)瘤細(xì)胞的方法建立大鼠腦膠質(zhì)瘤模型,之后經(jīng)尾靜脈注射分別GS NPs-Fe3O4和Fe304,比較不同納米粒子之間的MRI成像效果,觀察信號(hào)變化情況。對(duì)腫瘤標(biāo)本切片進(jìn)行HE和普魯士藍(lán)染色,檢測其病理學(xué)改變。通過對(duì)大鼠C6腦膠質(zhì)瘤切片及MRI成像檢測,探討GS NPs-Fe3O4是否具有特異性靶向標(biāo)記成像的特性,進(jìn)而能夠在腫瘤診斷、藥物轉(zhuǎn)運(yùn)及靶向治療等方面發(fā)揮巨大優(yōu)勢。
[Abstract]:Glioma is the most common intracranial tumor in neurosurgery, accounting for 40% of central nervous system tumors and 78% of adult neuromalignant tumors. In recent 30 years, although neuroimaging and glioma treatment have made great progress, the prognosis of malignant gliomas is still pessimistic. The 1-year and 5-year survival rates of adult malignant gliomas were about 30% and 13%, respectively. The median survival time of GBM was only about 12 months [2]. At present, gene therapy from theory to practice has brought great hope to thoroughly conquer the tumor and improve the prognosis of patients. However, there are still many links to be studied and broken down as soon as possible, among which the lack of efficient and stable carrier system and the particularity of blood-brain barrier structure in central nervous system are one of the important reasons leading to the inefficiency of its treatment. In this experiment, a two-step sol-gel method was used. The gelatin-siloxane nanoparticles (GS NPs),) were synthesized and a novel carrier system was constructed on the basis of which an aptamer TTAl and Fe304 were co-modified by polyethylene glycol (PEG), cationic polypeptide Tat, aptamer. It can not only penetrate the blood-brain barrier and specifically target brain tumor cells, but also have the effect of MRI imaging. The transfection efficiency of the plasmid DNA was investigated at the cell level to provide theoretical and practical support for the application of the nanoparticles as brain transporter. In the first part, the coupling of GS NPs and Fe304 was characterized by Fourier transform infrared spectroscopy (FT-IR). The physicochemical properties of the nanoparticles were studied by means of surface potential analysis, (Zeta potential), transmission electron microscope, (TEM), particle size analysis, (DLS), Fourier transform infrared spectroscopy (FT-IR) and thermogravimetric analysis (TGA). In the second part, laser confocal MRI and other imaging techniques were used to verify that GS NP-Fe3O4 as a gene vector could be effectively ingested by C6 cells, and GFP (green fluorescent protein) reporter gene could be efficiently expressed in tumor cells. Magnetic resonance imaging (MRI) was used to scan GS NP-Fe3O4 and Fe304 in a certain concentration range after cell coculture and the relaxation rate R2 was measured respectively to verify the possibility of GS NP-Fe3O4 as a contrast agent for MRI. In the third part, C6 glioma model was established by injecting C6 glioma cells under the guidance of stereotactic locator, and then the MRI imaging effects between different nanoparticles were compared by injecting GS NPs-Fe3O4 and Fe304, respectively through tail vein. Observe the change of signal. HE and Prussian blue staining were used to detect the pathological changes. By detecting rat C6 glioma slices and MRI imaging, this paper discusses whether GS NPs-Fe3O4 has the characteristics of specific targeted imaging, and can play a great advantage in tumor diagnosis, drug transport and targeted therapy.
【學(xué)位授予單位】：廈門大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.41

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