本文關(guān)鍵詞： 聚合物量子點(diǎn) 四苯基卟啉 光動(dòng)力療法 光敏劑 胃癌　出處：《吉林大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：背景 胃癌是我國(guó)最常見(jiàn)的惡性腫瘤之一，在我國(guó)發(fā)病率很高。中國(guó)2013年腫瘤登記年報(bào)顯示，2010年胃癌死亡率占惡性腫瘤死亡率的第3位。早期胃癌多無(wú)癥狀或僅有輕微癥狀，當(dāng)臨床癥狀明顯時(shí)，絕大多數(shù)病變已屬中晚期。目前，外科手術(shù)切除、內(nèi)鏡下切除、放射治療及藥物化學(xué)治療仍是主要治療手段。但遺憾的是，有些患者雖然經(jīng)過(guò)上述治療，但對(duì)病灶治療仍不理想。光動(dòng)力療法（Photodynamic therapy，PDT）作為一種微創(chuàng)性治療手段，獲得越來(lái)越多的關(guān)注。這種方法利用特定波長(zhǎng)的光激發(fā)被腫瘤細(xì)胞攝取的光敏劑，產(chǎn)生細(xì)胞毒性因子，導(dǎo)致腫瘤細(xì)胞受損甚至死亡，以達(dá)到治療腫瘤的目的。因此，光敏劑是PDT治療的關(guān)鍵。 有機(jī)半導(dǎo)體聚合物（又稱聚合物量子點(diǎn)）是一類因共軛π電子而產(chǎn)生半導(dǎo)體性質(zhì)的高分子材料，具有非常大的光學(xué)吸收截面、很高的熒光量子效率，在相應(yīng)波長(zhǎng)的光激發(fā)下，可以通過(guò)熒光共振轉(zhuǎn)移將能量轉(zhuǎn)移給光敏劑或其他能夠產(chǎn)生單線態(tài)氧的物質(zhì)。聚合物量子點(diǎn)內(nèi)部的柔性疏水特質(zhì)可以解決許多藥物的水溶性問(wèn)題，外部的親水鏈段在水中溶解起穩(wěn)定作用。另外，半導(dǎo)體聚合物量子點(diǎn)的熒光性能，又能幫助追蹤光敏劑的位置，更好的實(shí)現(xiàn)光敏劑的定位和實(shí)時(shí)監(jiān)測(cè)。這些特性使得聚合物量子點(diǎn)作為光敏劑的納米載體比傳統(tǒng)的需要釋放出裝載的光敏劑的納米載體具有明顯的優(yōu)勢(shì)。 目的： 本實(shí)驗(yàn)擬以聚合物量子點(diǎn)為光敏劑的載體，采用納米再沉淀法將難溶于水的光敏劑封裝在聚合物中，制備出摻雜光敏劑的聚合物納米粒子(以下稱為T(mén)PP-doped PFBT納米粒子)；通過(guò)體外細(xì)胞和體內(nèi)動(dòng)物實(shí)驗(yàn)，，研究TPP-dopedPFBT納米粒子在人胃癌細(xì)胞和裸鼠人胃癌移植瘤模型（簡(jiǎn)稱為荷瘤鼠）的分布、及其介導(dǎo)的PDT對(duì)腫瘤細(xì)胞和腫瘤組織的殺傷效果，探討該納米粒子介導(dǎo)的PDT治療胃癌的可行性。 方法： ⑴采用納米再沉淀方法制備TPP-doped PFBT納米粒子；通過(guò)紫外可見(jiàn)分光光度計(jì)測(cè)其吸收值，計(jì)算出納米粒子溶液的濃度；采用動(dòng)態(tài)光散射，測(cè)量納米粒子的尺寸與粒徑分布；采用透射電鏡，測(cè)定納米粒子的形貌；采用紫外可見(jiàn)分光光度計(jì)和熒光光譜儀對(duì)納米粒子進(jìn)行光譜分析；在50mW/cm2藍(lán)色LED燈光照條件下，采用ADMA檢測(cè)納米粒子溶液中單線態(tài)氧的產(chǎn)生。 ⑵在體外細(xì)胞實(shí)驗(yàn)部分，根據(jù)納米粒子本身的熒光特性，通過(guò)熒光顯微鏡和流式細(xì)胞儀，觀察納米粒子在細(xì)胞內(nèi)定位、攝取；用MTT法檢測(cè)TPP-dopedPFBT納米粒子的暗毒性，及其介導(dǎo)的PDT對(duì)人胃腺癌SGC-7901細(xì)胞的殺傷作用，觀察納米粒子與細(xì)胞相互作用時(shí)間、納米粒子濃度及光照劑量對(duì)PDT效果的影響；光學(xué)顯微鏡及熒光顯微鏡下觀察PDT后細(xì)胞的形態(tài)、細(xì)胞內(nèi)活性氧、線粒體膜電位和細(xì)胞核形態(tài)的改變。 ⑶建立裸鼠人胃癌移植瘤模型，采用小動(dòng)物成像系統(tǒng)觀察TPP-doped PFBT納米粒子在荷瘤鼠內(nèi)的分布；不同的給藥方式，TPP-doped PFBT納米粒子介導(dǎo)的PDT對(duì)荷瘤鼠的腫瘤抑制作用及它們之間的差異。PDT治療結(jié)束后，摘除腫瘤、肝臟、脾、腎、肺和心臟，經(jīng)10%中性福爾馬林緩沖液固定，石蠟包埋切片，常規(guī)HE染色后，使用光學(xué)顯微鏡觀察各組織的形態(tài)學(xué)改變。 結(jié)果： ⑴成功制備了TPP-doped PFBT納米粒子，球形，直徑約24nm，吸收波長(zhǎng)460nm，發(fā)射波長(zhǎng)625-750nm。460nm藍(lán)光激發(fā)時(shí)，TPP-doped PFBT納米粒子中聚合物PFBT通過(guò)熒光共振轉(zhuǎn)移將能量傳遞給光敏劑TPP，產(chǎn)生紅色熒光。分析ADMA檢測(cè)納米粒子溶液中的單線態(tài)氧發(fā)現(xiàn)，在光照條件下，該納米粒子產(chǎn)生的單線態(tài)氧含量與光照時(shí)間呈正相關(guān)，約在13min到達(dá)平臺(tái)期。 ⑵體外細(xì)胞實(shí)驗(yàn)部分，通過(guò)對(duì)熒光顯微鏡和流式細(xì)胞儀的結(jié)果分析發(fā)現(xiàn)，TPP-doped PFBT納米粒子可以通過(guò)SGC-7901細(xì)胞的細(xì)胞膜，定位于細(xì)胞質(zhì)；隨著納米粒子的濃度增加，SGC-7901細(xì)胞攝取的納米粒子增加，攝取納米粒子的細(xì)胞數(shù)目也在增加。分析MTT檢測(cè)結(jié)果發(fā)現(xiàn)，無(wú)光照條件下，TPP-doped PFBT納米粒子對(duì)腫瘤細(xì)胞和正常細(xì)胞均無(wú)暗毒性；TPP-doped PFBT納米粒子介導(dǎo)的PDT對(duì)人胃腺癌SGC-7901細(xì)胞的殺傷作用，與細(xì)胞相互作用時(shí)間、納米粒子濃度及光照劑量呈正相關(guān)；光學(xué)顯微鏡及熒光顯微鏡下觀察發(fā)現(xiàn)，PDT后細(xì)胞呈凋亡和壞死狀態(tài)，如細(xì)胞皺縮變圓、體積變小、出現(xiàn)胞質(zhì)內(nèi)空泡、細(xì)胞間隙增大、折光性減弱、貼壁能力下降；細(xì)胞核內(nèi)染色質(zhì)固縮，有很多顆粒樣物質(zhì)和碎片；PDT后細(xì)胞內(nèi)活性氧的增加，線粒體膜電位遭到破壞。 ⑶體內(nèi)實(shí)驗(yàn)結(jié)果顯示，靜脈注射TPP-doped PFBT納米粒子主要分布于荷瘤鼠的肝臟和脾，腫瘤組織內(nèi)較少分布，而在腫瘤組織局部注射的納米粒子，則主要蓄積于腫瘤組織。TPP-doped PFBT納米粒子介導(dǎo)的光動(dòng)力療法對(duì)荷瘤鼠的腫瘤有明顯的抑制和殺傷作用，包括腫瘤內(nèi)部大面積的凝固性壞死、腫瘤細(xì)胞呈團(tuán)塊狀分布、炎癥細(xì)胞浸潤(rùn)、微血管減少，尤其是局部注射納米粒子。靜脈注射納米粒子對(duì)荷瘤鼠的肝臟有損傷作用，其他臟器未見(jiàn)損傷。結(jié)論： ⑴TPP-doped PFBT納米粒子尺寸均一、粒徑小、無(wú)細(xì)胞暗毒性，其中聚合物可為內(nèi)部光敏劑提供能量，產(chǎn)生紅色熒光和單線態(tài)氧，具有良好的開(kāi)發(fā)應(yīng)用前景。 ⑵TPP-doped PFBT納米粒子介導(dǎo)的PDT對(duì)人胃癌細(xì)胞和荷瘤鼠的腫瘤具有明顯的治療作用，可引起腫瘤細(xì)胞的凋亡和壞死。 ⑶TPP-doped PFBT納米粒子具有被動(dòng)靶向性，但靜脈給藥時(shí)對(duì)荷瘤鼠的肝臟有損傷作用，應(yīng)該在后續(xù)的試驗(yàn)中進(jìn)一步改善納米粒子在體內(nèi)的代謝動(dòng)力學(xué)特性，增加主動(dòng)靶向性，提高PDT療效，降低毒副作用。
[Abstract]:background
Gastric cancer is one of the most common malignant tumors in China. The morbidity is very high in our country. China 2013 cancer registry annual report shows that in 2010, the mortality of gastric cancer accounted for third. The mortality of malignant tumor in early gastric cancer without symptoms or only mild symptoms, when clinical symptoms, most lesions had been advanced. At present. Surgical resection, endoscopic resection, radiotherapy and chemotherapy drugs is the main treatment. Unfortunately, some patients even after the treatment, but the treatment of the lesions is still not ideal. Photodynamic therapy (Photodynamic therapy, PDT) as a minimally invasive treatment, is gaining more and more attention. By this method. A specific wavelength of light excitation by the tumor cell uptake of photosensitizer, produce cytotoxic factor, leading to tumor cell damage and even death, in order to achieve the purpose of treatment of cancer. Therefore, the photosensitizer is PDT The key to the treatment.
Organic polymer (also called polymer semiconductor quantum dots) is a kind of polymer material for conjugated electron and semiconductor properties, has a very large optical absorption cross-section, fluorescence quantum efficiency is very high, the corresponding excitation wavelength of light, by fluorescence resonance energy transfer will be transferred to the photosensitizer or other substances produced can singlet oxygen. Flexible hydrophobic characteristics of internal polymer quantum dots can solve many problems of water soluble drugs, hydrophilic external stabilization dissolved in water. In addition, the fluorescence properties of polymer semiconductor quantum dots, and can help track the photosensitizer position, achieve better positioning and real-time monitoring of photosensitizer. The characteristics of the polymer quantum dots as nano carrier photosensitizer than traditional need to release nanoparticles loaded photosensitizer has obvious advantages.
Objective:
This experiment intends to polymer quantum dots as photosensitizer carrier, using nano will be insoluble in water and precipitation of photosensitizer encapsulated in polymers, preparation of polymer nanoparticles doped photosensitizer (hereinafter referred to as TPP-doped PFBT nanoparticles); in vitro and in vivo animal experiment study of TPP-dopedPFBT nanoparticles in human gastric cancer cells and nude mice transplantation human gastric tumor model (referred to as mice) distribution, and its killing effect mediated by PDT on tumor cells and tumor tissues, to explore the feasibility of the nanoparticle mediated PDT therapy for gastric cancer.
Method錛

本文編號(hào)：1547053