本文選題：氧化錳納米粒子 + 分子影像�。� 參考：《第二軍醫(yī)大學》2014年博士論文

【摘要】：【研究背景】 腫瘤是目前全球最常見疾病之一，其發(fā)病率和死亡率都非常高。據(jù)世界衛(wèi)生組織報道，每年全球有超過1000萬新發(fā)病例，約700萬人死亡。美國2013年的新發(fā)癌癥病例為166萬，其中58萬癌癥死亡病例。在中國，到2012年底，新的癌癥病例每年為350萬，因癌癥死亡的人數(shù)是250萬�！霸缙诎l(fā)現(xiàn)”，“及時診斷”和“早期正確治療”是提高患者生存率的關(guān)鍵。如何在（�。┠[瘤的啟動或早期階段，及時發(fā)現(xiàn)和確診，是實現(xiàn)“三早”的重要途徑。腫瘤生長進展需要依靠新生血管供應(yīng)細胞代謝所必需的營養(yǎng)和氧氣，因此腫瘤的發(fā)生、發(fā)展及轉(zhuǎn)移與腫瘤血管生成密切相關(guān)。因此如能夠定量化、可視化地檢測并研究腫瘤新生血管，將有助于癌癥的早期診斷。而且，對于臨床腫瘤抗新生血管治療，能夠進行全程監(jiān)測，觀察新生血管的狀態(tài)，評估治療效果。腫瘤分子生物學研究表明，在腫瘤的血管生成和轉(zhuǎn)移等生物學事件中，腫瘤新生血管表面或某些腫瘤細胞表面存在的一種粘附分子超家族成員—整合素有著重要作用。目前，在腫瘤早期診斷的腫瘤新生血管靶點研究中，研究較多的是αvβ3整合素受體。整合素αvβ3受體在增殖的腫瘤新生血管內(nèi)皮細胞高度表達，而在靜息血管內(nèi)皮細胞上無表達，是腫瘤新生血管的標志分子之一。αvβ3受體能夠結(jié)合特異性識別并結(jié)合到細胞外基質(zhì)中的甘-精-天冬氨酸序列（RGD序列），，為在探針的設(shè)計和修飾方面，使其向腫瘤新生血管內(nèi)皮細胞靶向性聚合，提供了重要依據(jù)。目前在評價腫瘤血管的成像模式中，核磁共振MR成像是一種理想的、不斷發(fā)展的成像方式。它不僅可以多個參數(shù)、多種序列、軸冠矢狀多方位成像，而且無電離輻射，還可以較高的空間分辨率采集組織結(jié)構(gòu)解剖、生理病理及相關(guān)理化信息。MRI對比劑，尤其是靶向?qū)Ρ葎┑难邪l(fā)和應(yīng)用，在提高MRI的疾病診斷準確率的同時，賦予其反映病灶代謝特征、評估治療效果的能力，具有廣闊的臨床應(yīng)用前景。磁共振分子成像是分子影像學研究熱點。 第一部分磁共振靶向?qū)Ρ葎≧GD-mPEG-DA-MnO）的制備構(gòu)建和表征 【目的】制備整合素αvβ3介導(dǎo)的磁共振靶向?qū)Ρ葎┡悸?lián)RGD包被聚乙二醇并連接多巴胺的氧化錳納米粒子(RGD-mPEG-DA-MnO)并進行體外表征。 【材料和方法】以四水合氯化錳和油酸為原料，采用熱裂解法合成超小MnO納米粒子，對其進行mPEG和DA的表面修飾和RGD連接。使用透射電子顯微鏡鏡（TEM）、動態(tài)光散射（DLS）、氫質(zhì)子磁共振波譜（1HNMR）、傅里葉變換紅外光譜儀（FTIR）、電感耦合等離子體原子發(fā)射光譜儀（ICP-AES）及核磁共振分別對制備的靶向?qū)Ρ葎┻M行形態(tài)、粒徑、結(jié)構(gòu)、穩(wěn)定性、密度、樣品錳元素含量及弛豫效能進行體外表征。 【結(jié)果】新合成的mPEG-DA-MnO磁性納米粒子水合粒徑在150nm左右，粒徑分布比較均一，穩(wěn)定性高，其錳元素含量為2.48×10-3mg/MG（每毫克對比劑里面有2.48×10-3mg的錳）。五種不同樣品的表面修飾物mPEG-DA的密度范圍為6.51-17.2mmol/m2，其相應(yīng)r1弛豫效能的范圍為16.14mM-1S-1到5.98mM-1S-1，r1最大者約為醫(yī)用釓對比劑r1弛豫效能的3倍。成功偶聯(lián)RGD的RGD-mPEG-DA-MnO靶向磁性納米粒子對比劑具備高純度。 【結(jié)論】以四水合氯化錳和油酸為原料的熱裂解法可成功合成超小MnO納米粒子，經(jīng)mPEG和DA的表面修飾能夠制備納米化、單分散相、高純度、穩(wěn)定性、弛豫效能的納米粒子mPEG-DA-MnO，并偶聯(lián)RGD肽成功制備靶向磁性納米粒子RGD-mPEG-DA-MnO。 第二部分對比劑的毒性和造影效果的體外研究 【目的】探討新合成對比劑（RGD-mPEG-DA-MnO）的細胞毒性和造影效果。 【材料和方法】MTT法檢測新合成對比劑（RGD-mPEG-DA-MnO）在體外對A549細胞生存率的影響。采用PhillipAchieva3.0T磁共振對新合成對比劑進行T1WI及T1Mapping成像，T1加權(quán)成像信號強度、T1map值分別評價所制備不同粒徑和修飾物密度的對比劑的成像對比度和弛豫效能。 【結(jié)果】對比劑濃度在500ug/ml時，其細胞存活率仍較高，其IC50（半抑制濃度）為818.9ug/ml。隨著錳離子濃度的增大，粒徑5nmMnO核心的RGD-mPEG-DA-MnO造影圖像逐漸變亮。粒徑5nmMnO核心的RGD-mPEG-DA-MnO的r1弛豫效能為11.6s-1mM-1。 【結(jié)論】RGD-mPEG-DA-MnO磁性納米對比劑在一定濃度范圍內(nèi)對細胞無明顯毒性。其成像對比度較高，r1弛豫效能約為醫(yī)用Gd-DTPA弛豫效能的2倍。 第三部分磁共振對比劑的動物在體成像研究 【目的】研究MR靶向?qū)Ρ葎㏑GD-mPEG-DA-MnO靶向荷人肺癌裸鼠、實現(xiàn)腫瘤血管及細胞靶向顯影的可行性，探討該磁性納米對比劑的成像特點。 【材料和方法】 1、非靶向磁共振對比劑(mPEG-DA-MnO)對荷人肺腺癌裸鼠的MRI研究BALB/C昆明白鼠經(jīng)尾靜脈注入靶向磁共振對比劑（mPEG-DA-MnO），觀察注射對比劑前后不同時間點肝臟、腎臟和肌肉的T1WI信號的變化。 2、磁共振靶向?qū)Ρ葎?RGD-mPEG-DA-MnO)對荷人肺腺癌裸鼠的MRI研究人肺腺癌A549細胞株傳代并對裸鼠皮下接種，制成荷人肺腺癌的裸鼠模型。通過尾靜脈注入（RGD-mPEG-DA-MnO）靶向磁共振對比劑，觀察注射前后不同時間點荷瘤鼠的瘤體、肝臟及頸背部肌肉T1WI信號值變化。 3、磁共振靶向?qū)Ρ葎?RGD-mPEG-DA-MnO)與非靶向?qū)Ρ葎?mPEG-DA-MnO)荷人肺癌裸鼠MRI成像對照研究按照隨機對照原則，將8只荷瘤裸鼠平均分為兩組，實驗組4只和對照組4只，實驗組通過尾靜脈注入靶向?qū)Ρ葎㏑GD-mPEG-DA-MnO，對照組注射mPEG-DA-MnO，觀察并比較在注射對比劑前后不同時間點兩組動物的腫瘤和肌肉T1WI信號強度值變化。 【結(jié)果】注射mPEG-DA-MnO對比劑15min后，其肝、腎臟區(qū)域有明顯增強效果，1h后增強效果減弱。2h后對比劑代謝進入膀胱。在靶向荷A549肺腺癌細胞腫瘤裸鼠MRI研究中，對比劑RGD-mPEG-DA-MnO有向瘤體局部聚集的趨勢，強化峰值出現(xiàn)在注射對比劑后約2小時左右，最大強化率達109.9％；肝臟也表現(xiàn)出明顯強化，強化峰值出現(xiàn)在注射對比劑后約1小時左右，之后呈現(xiàn)出比較緩慢的回落趨勢；和非靶向的mPEG-DA-MnO進行對照，(1)所制備的靶向?qū)Ρ葎㏑GD-mPEG-DA-MnO具有明顯的靶向瘤體效果，實驗組腫瘤各掃描時間點的信號強度值差異的統(tǒng)計學意義顯著(F=166.83，P0．0001)，強化峰值出現(xiàn)時間約注入對比劑后2小時；無靶向?qū)Ρ葎﹎PEG-DA-MnO組的瘤體各掃描時間點信號值差異亦有顯著統(tǒng)計學意義，強化高峰出現(xiàn)在注射后約1小時；靶向組（RGD-mPEG-DA-MnO）及無靶向組（mPEG-DA-MnO）的肌肉各掃描時間點信號強度值總體差異不大。 【結(jié)論】靶向?qū)Ρ葎㏑GD-mPEG-DA-MnO能與整合素αvβ3受體特異性結(jié)合，對荷瘤裸鼠的腫瘤新生血管MR成像的強化效果具持續(xù)性和特異性，使MR靶向顯像及靶向診斷具有可行性。 第四部分聚乙二醇包被的磁性氧化錳納米粒子的體外生物學效應(yīng)探討 【目的】探討聚乙二醇包被的磁性氧化錳納米粒子對比劑對人臍靜脈內(nèi)皮細胞（HUVECs）和人肺腺癌A549細胞的生物學效應(yīng)及相關(guān)機制。 【方法】采用多種細胞生物學研究方法探討聚乙二醇包被的磁性氧化錳納米粒子標記臍靜脈血管內(nèi)皮細胞(HUVECs)和A549人肺腺癌細胞后，對其增殖、遷移、侵襲、分化、細胞骨架及細胞亞器官等生物學行為的影響�！窘Y(jié)果】濃度超過500ug/L的包被聚乙二醇的氧化錳磁性納米粒子可抑制內(nèi)皮細胞HUVECs增殖、遷移和侵襲，此抑制能力表現(xiàn)出濃度依賴性；氧化錳納米粒子可抑制內(nèi)皮細胞的管腔分化形成能力；可引起細胞骨架發(fā)生重構(gòu)，粘著斑數(shù)目降低，細胞粘附能力呈濃度依賴性降低；去粘附可進一步引起細胞增殖、遷移、侵襲、分化能力的損害及抑制。高濃度的氧化錳納米粒子降低細胞線粒體膜電位，并誘導(dǎo)活性氧產(chǎn)生氧化應(yīng)激反應(yīng)�！窘Y(jié)論】高濃度的聚乙二醇包被的磁性氧化錳納米粒子（mPEG-DA-MnO）可通過誘導(dǎo)氧化應(yīng)激造成細胞毒作用。
[Abstract]:[research background]
Cancer is one of the most common diseases in the world and its incidence and mortality are very high. According to the WHO, there are more than 1000 million new cases in the world every year, about 7 million people die. In the United States, 1 million 660 thousand of new cases of cancer in 2013, of which 580 thousand are cancer deaths. In China, by the end of 2012, new cancer cases are 3 million 500 thousand a year of 3 million 500 thousand. The number of deaths due to cancer is 2 million 500 thousand. "Early detection", "timely diagnosis" and "early correct treatment" are the key to improve the survival rate of patients. How to find and diagnose in the start or early stage of (small) tumor is an important way to realize the "three early". The growth of tumor needs to rely on the supply of cell metabolism of the neovascularization. Essential nutrients and oxygen are necessary, so the occurrence, development and metastasis of tumor are closely related to the angiogenesis of tumor. Therefore, it will be helpful to the early diagnosis of cancer if it can be quantified and visualized and studied the neovascularization of tumor. Moreover, the whole course monitoring and observation of the neovascularization for the anti neovascularization of the clinical tumor The molecular biological study of tumor molecular biology shows that in the biological events such as angiogenesis and metastasis of tumor, a kind of adhesion molecule superfamily member of the tumor cell surface or some tumor cells has an important role. In the study, most of the research is the alpha v beta 3 integrin receptor. The integrin alpha v beta 3 receptor is highly expressed in the proliferating neovascular endothelial cells, but is not expressed on the resting vascular endothelial cells. It is one of the biomarkers of the neovascularization of the tumor. The alpha v beta 3 receptor can be combined with the specific recognition and combined with the glycinine aspartate in the extracellular matrix. The acid sequence (RGD sequence) provides an important basis for the design and modification of the probe into the targeting polymerization of neovascular endothelial cells in tumor cells. In the evaluation of tumor angiogenesis, magnetic resonance MR imaging is an ideal and constantly developing imaging mode. It can not only have multiple parameters, multiple sequences, and axial crown sagittal. Multidimensional imaging, without ionizing radiation, can also acquire tissue anatomy, physiological and pathological and related physical and chemical.MRI contrast agents, especially target contrast agents, to improve the diagnostic accuracy of MRI, and give them the ability to reflect the metabolic characteristics of the lesions and evaluate the therapeutic effect. Magnetic resonance molecular imaging (MRI) is a hot research area in molecular imaging.
Part 1 Preparation and characterization of magnetic resonance targeted contrast agent (RGD-mPEG-DA-MnO).
[Objective] to prepare an integrin - alpha v beta 3 - mediated magnetic resonance targeting contrast agent coupled with RGD - coated peg and dopamine - based manganese oxide nanoparticles (RGD-mPEG-DA-MnO) and to characterize in vitro.
[materials and methods] super small MnO nanoparticles were synthesized with four hydrated manganese chloride and oleic acid by thermal splitting. The surface modification of mPEG and DA and RGD connection were used. The transmission electron microscope (TEM), dynamic light scattering (DLS), hydrogen proton magnetic resonance spectroscopy (1HNMR), Fourier transform infrared spectrometer (FTIR), inductance coupling, etc. were used. The morphology, particle size, structure, stability, density, manganese content and relaxation efficiency of the target contrast agent prepared by atomic emission spectrometer (ICP-AES) and nuclear magnetic resonance (NMR) were characterized respectively.
[results] the diameter of the newly synthesized mPEG-DA-MnO magnetic nanoparticles is about 150nm, the particle size distribution is uniform, the stability is high, the manganese content is 2.48 x 10-3mg/MG (2.48 10-3mg Mn in the contrast agent per milligram). The density range of the surface modifier of five different samples is 6.51-17.2mmol/m2, and its corresponding R1 relaxation The range of efficiency is from 16.14mM-1S-1 to 5.98mM-1S-1, and the largest of R1 is about 3 times the relaxation efficiency of the medical gadolinium contrast agent R1. The successful coupling of RGD's RGD-mPEG-DA-MnO target to the magnetic nanoparticle contrast agent has high purity.
[Conclusion] ultra small MnO nanoparticles can be successfully synthesized with four hydrated manganese chloride and oleic acid as raw materials. By surface modification of mPEG and DA, nano, monodisperse, high purity, stable and relaxor nanoparticles mPEG-DA-MnO are prepared, and the target magnetic nanoparticles RGD-mPEG-DA-MnO. can be prepared by coupling RGD peptide.
The second part: the toxicity and contrast effect of contrast agents in vitro.
[Objective] to explore the cytotoxicity and contrast effect of the new synthetic contrast agent (RGD-mPEG-DA-MnO).
[materials and methods] MTT method was used to detect the effect of new synthetic contrast agent (RGD-mPEG-DA-MnO) on the survival rate of A549 cells in vitro. T1WI and T1Mapping imaging of new synthetic contrast agents were performed by PhillipAchieva3.0T magnetic resonance (MRI), T1 weighted imaging signal intensity, and T1map value to evaluate the imaging of contrast agents with different particle sizes and modifier densities respectively. Contrast and relaxation efficiency.
[results] the cell survival rate of the contrast agent at 500ug/ml was still high, and its IC50 (semi inhibitory concentration) was 818.9ug/ml. with the increase of the concentration of manganese ions, the RGD-mPEG-DA-MnO contrast image of the kernel of the particle size 5nmMnO gradually brightened. The R1 relaxation efficiency of the RGD-mPEG-DA-MnO of the particle size 5nmMnO core was 11.6s-1mM-1.
[Conclusion] RGD-mPEG-DA-MnO magnetic nano contrast agent has no obvious toxicity to cells in a certain concentration range. Its imaging contrast is high, and the relaxation efficiency of R1 is about 2 times of the efficacy of medical Gd-DTPA relaxation.
The third part is animal imaging in vivo.
[Objective] to study the feasibility of MR targeted contrast agent RGD-mPEG-DA-MnO targeting human lung cancer in nude mice, to explore the feasibility of tumor angiogenesis and cell targeting, and to explore the imaging characteristics of the magnetic nano contrast agent.
[materials and methods]
1, MRI study of non targeted magnetic resonance contrast agent (mPEG-DA-MnO) in nude mice bearing human lung adenocarcinoma BALB/C mice were injected with target magnetic resonance contrast agent (mPEG-DA-MnO) into the tail vein of Kunming white mice, and the changes of T1WI signals in the liver, kidney and muscle were observed at different time points before and after the injection of contrast agent.
2, magnetic resonance targeting contrast agent (RGD-mPEG-DA-MnO) for human lung adenocarcinoma MRI research human lung adenocarcinoma A549 cell line and subcutaneous inoculation of nude mice into nude mice bearing lung adenocarcinoma. Through the tail vein injection (RGD-mPEG-DA-MnO) target magnetic resonance contrast agent, the tumor body, liver and neck were observed at different time points before and after injection. The T1WI signal value of the back muscles changes.
3, magnetic resonance targeting contrast agent (RGD-mPEG-DA-MnO) and non target contrast agent (mPEG-DA-MnO) nude mice with lung cancer MRI imaging control study, according to the random control principle, 8 tumor bearing nude mice were divided into two groups, 4 experimental group and 4 control group, the experimental group was injected into the target contrast agent RGD-mPEG-DA-MnO through the tail vein, and the control group was injected mPEG-DA-MnO, The T1WI and signal intensity of two groups of animals before and after injection of contrast agent were observed and compared.
[results] after the injection of mPEG-DA-MnO contrast agent 15min, the liver and kidney region had an obvious enhancement effect. After 1h, the enhancement effect weakened the.2h contrast agent metabolism into the bladder. In the MRI study on A549 lung adenocarcinoma cell tumor, the contrast agent RGD-mPEG-DA-MnO had the trend of local aggregation of the tumor to the tumor body, and the enhancement peak appeared after the injection contrast agent. About 2 hours or so, the maximum intensification rate was 109.9%, the liver also showed obvious enhancement, and the intensification peak appeared about 1 hours after the injection contrast agent, and then showed a slow downward trend, compared with the non target mPEG-DA-MnO, and (1) the target contrast agent RGD-mPEG-DA-MnO has obvious target tumor effect. The statistical significance of the difference of signal intensity of each time point of the tumor was statistically significant (F=166.83, P0.0001), and the peak time of the enhancement was about 2 hours after the injection of contrast agent; the difference of the signal value of each scanning time point of the tumor body without target contrast agent mPEG-DA-MnO group also had significant statistical significance, and the peak of the intensities appeared about 1 hours after the injection. The signal intensity values of target group (RGD-mPEG-DA-MnO) and non target group (mPEG-DA-MnO) at different scanning time points were not significantly different.
[Conclusion] target contrast agent RGD-mPEG-DA-MnO can be specifically combined with integrin alpha v beta 3 receptor. The enhancement effect of MR imaging in tumor neovascularization of tumor bearing nude mice is continuous and specific, which makes MR target imaging and target diagnosis feasible.
The fourth part of polyethylene glycol coated magnetic manganese oxide nanoparticles in vitro biological effects
[Objective] to investigate the biological effect and mechanism of the magnetic manganese oxide nanoparticle contrast agent coated with polyethylene glycol (PEG) on human umbilical vein endothelial cells (HUVECs) and human lung adenocarcinoma A549 cells.
[Methods] the effects of polyethylene glycol coated magnetic manganese oxide nanoparticles on the proliferation, migration, invasion, differentiation, cytoskeleton and cell suborgans of the umbilical vein endothelial cells (HUVECs) and A549 human lung adenocarcinoma cells were investigated by multiple cell biological methods. [results] the concentration was more than 500ug/L The manganese oxide magnetic nanoparticles coated with polyethylene glycol can inhibit the proliferation, migration and invasion of endothelial cell HUVECs, and the inhibition ability shows a concentration dependence. Manganese oxide nanoparticles can inhibit the formation of endothelium cells, cause the remodeling of the cytoskeleton, the number of adhesion spots and the concentration of cell adhesion. Dependence can be reduced; adhesion can further induce cell proliferation, migration, invasion, differentiation and inhibition. High concentrations of manganese oxide nanoparticles reduce the cell mitochondrial membrane potential and induce reactive oxygen species to produce oxidative stress reactions. [Conclusion] high concentration of polyethylene glycol coated magnetic manganese oxide nanoparticles (mPEG-DA-MnO) can be used. Cytotoxic effects are induced by inducing oxidative stress.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R943;R96

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