本文關(guān)鍵詞：構(gòu)建透明質(zhì)酸功能化三氧化二鉍納米顆粒用于腫瘤靶向成像及其放療增敏研究　出處：《江蘇大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 鉍 透明質(zhì)酸 納米材料 CT成像 造影劑 放療增敏

【摘要】：目的:惡性腫瘤是一種難治性疾病,嚴(yán)重威脅到人類的健康最。放射治療是目前臨床上治療惡性腫瘤最常用的手段之一。然而,由于影像學(xué)在腫瘤類疾病的診斷及定位上的局限性,臨床治療時(shí)常出現(xiàn)放射治療的劑量未達(dá)腫瘤組織的致死劑量、定位不夠精確而對(duì)周圍正常組織造成放療損傷,從而引起放療副反應(yīng);同時(shí)由于各類腫瘤組織自身存在著不同程度的放療抗性,降低了腫瘤細(xì)胞對(duì)放射治療的敏感性,從而進(jìn)一步削弱了放療的臨床治療效果。為了克服在腫瘤類疾病的診療過(guò)程中所出現(xiàn)的影像學(xué)診斷誤差及病灶組織的放療抵抗,本課題旨在利用水熱聚醇法構(gòu)建一種多功能的新型納米顆�！该髻|(zhì)酸功能化三氧化二鉍納米顆粒(HA-Bi_2O_3 NPs),用于腫瘤類疾病的靶向性CT造影成像引導(dǎo)下的放療增敏。方法:利用透明質(zhì)酸鈉、六水氯化鉍(Bi Cl3·6H2O)為前驅(qū)物通過(guò)水熱聚醇法制備HA-Bi_2O_3 NPs;通過(guò)透射電子顯微鏡/高分辨透射電子顯微鏡(TEM/HR-TEM)、傅氏轉(zhuǎn)換紅外線光譜分析儀(FT-IR)、Nano DLS高敏度粒徑分析儀、X射線光電子能譜學(xué)分析儀(XPS)和X射線衍射儀(XRD)揭示HA-Bi_2O_3的理化特性;通過(guò)流式細(xì)胞術(shù)、半定量PCR和細(xì)胞成像來(lái)研究HA介導(dǎo)的HA-Bi_2O_3 NPs細(xì)胞吞噬行為;利用細(xì)胞增值率檢測(cè)、溶血實(shí)驗(yàn)、病理切片和電感耦合等離子體質(zhì)譜(ICP-MS)來(lái)分析HA-Bi_2O_3 NPs的生物相容性;利用ICR小鼠皮下腫瘤模型CT平掃檢測(cè)HA-Bi_2O_3 NPs的腫瘤靶向性CT成像;利用細(xì)胞增值率檢測(cè)、克隆形成實(shí)驗(yàn)、活-死細(xì)胞染色、流式細(xì)胞術(shù)(凋亡與周期)、ICR小鼠皮下腫瘤模型體內(nèi)實(shí)驗(yàn)檢測(cè)HA-Bi_2O_3 NPs的放療增敏作用。結(jié)果:1.HA-Bi_2O_3 NPs的制備及表征利用水熱聚醇法成功制備HA-Bi_2O_3 NPs,Nano DLS高敏度粒徑分析儀顯示HA-Bi_2O_3 NPs粒徑分布均勻,大小約為45nm,在PBS緩沖液中保存8天后HA-Bi_2O_3 NPs粒徑大小無(wú)明顯變化;TEM/HR-TEM提示HA-Bi_2O_3 NPs的水溶性、分散性好,粒徑大小均一,晶格條紋明顯,晶面間距為3.3±0.2?;XRD顯示HA-Bi_2O_3 NPs的物相結(jié)構(gòu)符合Bi_2O_3的特征,為多相異質(zhì)結(jié)構(gòu)。FT-IR提示HA-Bi_2O_3 NPs,表面具有羧基、羥基、羰基等親水基團(tuán);XPS提示HA-Bi_2O_3 NPs主要是由鉍、氧、碳等元素組成。2.透明質(zhì)酸(HA)介導(dǎo)的HA-Bi_2O_3 NPs的細(xì)胞吞噬行為通過(guò)定量PCR提示CD44抗原的表達(dá)在人肝癌細(xì)胞株SMMC-7721中比在人乳腺癌細(xì)胞株MCF7細(xì)胞高,與相關(guān)文獻(xiàn)的報(bào)道相一致;利用流式細(xì)胞術(shù)揭示HA-Bi_2O_3 NPs可被SMMC-7721細(xì)胞和MCF7細(xì)胞吞噬,但在CD44抗原高表達(dá)的SMMC-7721細(xì)胞中,HA-Bi_2O_3 NPs的吞噬效率較CD44抗原低表達(dá)的MCF7細(xì)胞高。3.HA-Bi_2O_3 NPs的生物相容性的表征檢測(cè)細(xì)胞的增殖抑制率顯示,不同濃度的HA-Bi_2O_3 NPs(0-400μg/m L)對(duì)SMMC-7721、VSMCs和MCF7的細(xì)胞活性均無(wú)明顯影響(p0.05);溶血實(shí)驗(yàn)提示,不同濃度的HA-Bi_2O_3 NPs(0-400μg/ml)均不引起明顯的溶血反應(yīng);ICP-MS顯示,ICR小鼠經(jīng)尾靜脈注射HA-Bi_2O_3 NPs后,心、肝、脾、肺、腎對(duì)HA-Bi_2O_3 NPs均有攝取,其中以肝臟(攝取量:27.80mg/g)和腎臟(攝取量:7.40mg/g)對(duì)HA-Bi_2O_3 NPs的攝取為主;組織切片提示HA-Bi_2O_3 NPs對(duì)小鼠重要臟器無(wú)明顯毒性,未引起明顯的病理變化。4.HA-Bi_2O_3 NPs在腫瘤CT靶向性成像的研究體外CT成像結(jié)果提示相較于已用于臨床的CT造影劑碘海醇,HA-Bi_2O_3 NPs具有更加優(yōu)秀的CT造影成像效果。皮下荷瘤小鼠經(jīng)尾靜脈注射HA-Bi_2O_3 NPs,10min后小鼠各臟器及皮下腫瘤的CT造影信號(hào)均有所增強(qiáng),其中腎臟、膀胱及皮下腫瘤處的信號(hào)增強(qiáng)尤為明顯;30min后,腎臟的信號(hào)強(qiáng)度開始下降而膀胱及皮下腫瘤的信號(hào)增強(qiáng)仍十分明顯。5.HA-Bi_2O_3 NPs的放療增敏效果的實(shí)驗(yàn)研究單純HA-Bi_2O_3 NPs對(duì)SMMC-7721細(xì)胞的增殖率的無(wú)顯著影響,而當(dāng)HA-Bi_2O_3 NPs聯(lián)合放射治療時(shí),則明顯抑制了SMMC-7721細(xì)胞的增殖。隨著提高HA-Bi_2O_3 NPs的濃度或加大放射治療的強(qiáng)度,SMMC-7721細(xì)胞增殖率所受到的抑制越發(fā)明顯,與劑量呈正相關(guān)性,尤其是在400μg/m L濃度的HA-Bi_2O_3 NPs聯(lián)合9Gy的放療劑量時(shí),SMMC-7721細(xì)胞的增值率顯著降低至23.5%(p0.05);克隆形成實(shí)驗(yàn)提示,單純HA-Bi_2O_3 NPs組與對(duì)照組的細(xì)胞增值率無(wú)明顯差異,單純放療組在6Gy的放療劑量下,細(xì)胞增值率為46.7%,而實(shí)驗(yàn)組在6Gy的放療劑量及200μg/m L濃度的HA-Bi_2O_3 NPs下,細(xì)胞增值率顯著降低至29.8%;活-死細(xì)胞染色實(shí)驗(yàn)中,相較于對(duì)照組、單純HA-Bi_2O_3 NPs組和單純放療組,HA-Bi_2O_3 NPs聯(lián)合放療組中可被FDA染色(綠色)的存活細(xì)胞明顯減少,并與HA-Bi_2O_3 NPs濃度成負(fù)相關(guān),可被PI染色(紅色)的凋亡細(xì)胞明顯增多,并與HA-Bi_2O_3 NPs濃度成正相關(guān);如流式細(xì)胞術(shù)(凋亡)所示,對(duì)照度及單純HA-Bi_2O_3 NPs組中的早期凋亡率及晚期凋亡率無(wú)明顯差異,單純放療組中,早期凋亡率升高至30.4%,晚期凋亡率升至5.89%,而實(shí)驗(yàn)組在6Gy的放療劑量聯(lián)合200μg/m L濃度的HA-Bi_2O_3 NPs下,早期和晚期凋亡率均明顯升高,分別達(dá)到41.2%和14.1%;流式細(xì)胞術(shù)(周期)提示,相比于對(duì)照組與單純HA-Bi_2O_3 NPs組,單純放療組(6Gy)引起G2/M期阻滯(19.87%),Sub-G1期峰值升高至2.34%,實(shí)驗(yàn)組在6Gy的放療劑量下,隨著HA-Bi_2O_3 NPs濃度的升高,G2/M期阻滯越發(fā)明顯、Sub-G1期峰值逐漸升高,特別是HA-Bi_2O_3 NPs的濃度為200μg/m L時(shí),G2/M期升高為33.00%,Sub-G1期峰值為21.59%;ICR小鼠體內(nèi)實(shí)驗(yàn)提示,對(duì)照組和單純HA-Bi_2O_3 NPs組荷瘤小鼠的皮下腫瘤體積在10d時(shí)增長(zhǎng)至270%,生存曲線提示對(duì)照組和單純HA-Bi_2O_3 NPs組小鼠的存活時(shí)間中位數(shù)分別為為17d和20d;單純放療組的腫瘤體積增長(zhǎng)受到抑制,在10d時(shí)縮小了約14.6%,存活時(shí)間中位數(shù)為24d;HA-Bi_2O_3 NPs聯(lián)合放射治療則明顯抑制了腫瘤生長(zhǎng),第10d時(shí)腫瘤體積縮小了33.9%,存活時(shí)間中位數(shù)為35d。結(jié)論:1.本研究以六水氯化鉍作為鉍源材料,利用透明質(zhì)酸進(jìn)行修飾,通過(guò)水熱聚醇法成功構(gòu)建了HA-Bi_2O_3 NPs。2.HA-Bi_2O_3 NPs的水溶性、分散性和穩(wěn)定性良好,粒徑大小均一;物相結(jié)構(gòu)為多層異質(zhì)性,晶格條紋明顯,晶格間距為3.3±0.2?,符合Bi_2O_3的特征;HA-Bi_2O_3 NPs主要是由鉍、氧、碳等元素組成,顆粒表面官能團(tuán)主要為羧基、甲基、羰基等;生物相容性良好。3.HA-Bi_2O_3 NPs相較于已用于臨床的CT造影劑碘海醇,具有更加優(yōu)秀的CT造影成像效果,同時(shí),納米材料具有EPR效應(yīng)和體內(nèi)滯留時(shí)間長(zhǎng)等特性,使得HA-Bi_2O_3 NPs可以用于腫瘤靶向性影像診斷。4.HA-Bi_2O_3 NPs可以增強(qiáng)放射射線對(duì)腫瘤的抑制和殺傷作用,具有成為臨床放療增敏劑的潛力。
[Abstract]:Objective: malignant tumor is a refractory disease, which is a serious threat to human health. Radiation therapy is one of the most commonly used methods to treat malignant tumors at present. However, due to limitations in the diagnosis and localization of tumor diseases on imaging, clinical treatment often dose not tumor lethal dose, positioning is not accurate and the surrounding normal tissue caused by radiation damage, causing the side effect of radiotherapy; at the same time, because of many kinds of tumors has different resistance to radiotherapy the degree of reduced tumor cell sensitivity to radiation therapy, so as to further weaken the curative effect of radiotherapy. In order to diagnose errors and radiotherapy lesions appeared overcome in the process of diagnosis and treatment of diseases such as tumor imaging resistance, the purpose of this study is to construct a new nanoparticles by hydrothermal multifunctional polyol method of hyaluronic acid functionalized three oxidation two bismuth nanoparticles (HA-Bi_2O_3 NPs), radiotherapy for cancer diseases the targeted CT imaging guided sensitization. Methods: using chloride sodium hyaluronate, six water (Bi Cl3 6H2O) bismuth precursor by hydrothermal synthesis of HA-Bi_2O_3 NPs poly alcohols; by transmission electron microscopy and high-resolution transmission electron microscopy (TEM/HR-TEM), Fourier transform infrared spectroscopy (FT-IR), Nano DLS high sensitivity particle size analyzer, X ray photoelectron spectroscopy analyzer (XPS) and X ray diffraction (XRD) reveals the physicochemical characteristics of HA-Bi_2O_3; to study the HA mediated HA-Bi_2O_3 NPs cells by semi quantitative PCR and cell imaging and flow cytometry, cell proliferation rate by phagocytic behavior; test, hemolysis test, pathological sections and inductively coupled plasma mass spectrometry (ICP-MS) to analyze the biological compatibility of HA-Bi_2O_3 NPs; tumor using ICR mice subcutaneous tumor model CT scan detection of HA-Bi_2O_3 NPs to CT imaging; rate of detection, clone formation assay, using live cell proliferation Dead cell staining, flow cytometry (apoptosis and cycle), and ICR mice model of subcutaneous tumor were tested in vivo to detect the sensitization effect of HA-Bi_2O_3 NPs. Results: the prepared HA-Bi_2O_3 NPs 1.HA-Bi_2O_3 preparation and characterization of NPs by hydrothermal polyol method, Nano DLS high sensitivity HA-Bi_2O_3 NPs particle size analyzer showed uniform particle size distribution, the size is about 45nm, save 8 days HA-Bi_2O_3 the particle size of NPs had no obvious change in PBS buffer; TEM/HR-TEM HA-Bi_2O_3 NPs water solubility, good dispersibility, uniform particle size, lattice fringe is obvious, the interplanar spacing is 3.3 + 0.2?; XRD display HA-Bi_2O_3 NPs phase structure with the characteristics of Bi_2O_3 multiphase heterogeneous structure. FT-IR indicates HA-Bi_2O_3 NPs, which has hydrophilic groups such as carboxyl, hydroxyl and carbonyl groups; XPS indicates that HA-Bi_2O_3 NPs is mainly composed of bismuth, oxygen, carbon and other elements. 2. hyaluronic acid (HA) expression of HA-Bi_2O_3 mediated by NPs cell phagocytosis behavior showed CD44 antigen by quantitative PCR in human hepatocellular carcinoma cell line SMMC-7721 than in human breast cancer cell line MCF7 cells, consistent with relevant reports; HA-Bi_2O_3 revealed by NPs flow cytometry by SMMC-7721 cells and MCF7 phagocytosis, but high expression of CD44 antigen in SMMC-7721 cells, HA-Bi_2O_3 NPs phagocytosis low expression efficiency compared with CD44 antigen of MCF7 cells. Characterization of 3.HA-Bi_2O_3 cell compatibility of NPs biological inhibition rate showed that different concentrations of HA-Bi_2O_3 NPs (0-400 g/m L) had no significant effect on cell activity of SMMC-7721, VSMCs and MCF7 (P0.05); hemolysis test indicated that different concentrations of HA-Bi_2O_3 (NPs 0-400 g/ml) were not obvious hemolytic reaction; ICP-MS display, ICR mice by tail vein injection of HA-Bi_2O_3 after NPs, heart, liver and spleen, lung and kidney of HA-Bi_2O_3 NPs were among the liver uptake (intake: 27.80mg/g) and kidney (intake: 7.40mg/g) of HA-Bi_2O_3 NPs uptake; tissue sections showed that HA-Bi_2O_3 NPs had no obvious toxicity to the important in mice, did not cause obvious pathological changes. The study of 4.HA-Bi_2O_3 NPs in targeted imaging of tumor CT showed that compared with the CT contrast agent used in clinic, CT imaging showed better CT imaging effect than HA-Bi_2O_3 NPs. Subcutaneous tumor bearing mice by intravenous injection of HA-Bi_2O_3 NPs, CT contrast signals are organs and subcutaneous tumors in mice increased after 10min, including kidney, bladder and subcutaneous tumor signal enhancement at 30min after the start, very obvious; signal intensity decreased and the signal of the bladder and kidney skin tumor enhancement is still very obvious. The effect of 5.HA-Bi_2O_3 NPs on the radiosensitizing effect was studied. HA-Bi_2O_3 NPs alone had no significant effect on the proliferation rate of SMMC-7721 cells, but when HA-Bi_2O_3 NPs combined with radiotherapy, it significantly inhibited the proliferation of SMMC-7721 cells. With the increase of the concentration of HA-Bi_2O_3 NPs or increasing the radiation intensity of treatment, the inhibition rate of SMMC-7721 cell proliferation is more and more obvious, and the dose was positively correlated, especially in the radiation dose 400 g/m L concentration of HA-Bi_2O_3 NPs combined with 9Gy, the added value of SMMC-7721 cells significantly reduced to 23.5% (P0.05); cloning experiments suggest simple, no difference in the rate of HA-Bi_2O_3 in NPs group and control group cell proliferation, radiotherapy group in the radiation dose of 6Gy, cell proliferation rate was 46.7%, while the experimental group in the radiotherapy dose of 6Gy and 200 g/m L concentration of HA-Bi_2O_3 NPs, cell proliferation was significantly reduced to 29.8%; the live - dead cell staining in the experiment, compared with control group, HA-Bi_2O_3 NPs group and simple radiotherapy group, radiotherapy group HA-Bi_2O_3 NPs by FDA staining (green) cell survival was significantly reduced, and the concentration of NPs and HA-Bi_2O_3 negative Related by PI staining (red) the number of apoptotic cells was significantly increased, and positively correlated with HA-Bi_2O_3 concentration of NPs; flow cytometry (apoptosis) in control and HA-Bi_2O_3 alone in group NPs
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R730

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本文編號(hào)：1347778