【摘要】：鼻咽癌是中國南方和東南亞等地域廣泛流行的高度惡性腫瘤，具有發(fā)病部位隱蔽和易于發(fā)生遠(yuǎn)處轉(zhuǎn)移的特點(diǎn)。遠(yuǎn)處轉(zhuǎn)移是鼻咽癌治療失敗的主要原因之一。目前對(duì)于局部晚期鼻咽癌主要使用以順鉑為基礎(chǔ)的聯(lián)合化療，但是有30%以上的患者由于發(fā)生遠(yuǎn)處轉(zhuǎn)移而導(dǎo)致治療失敗，其5年總生存率僅為25%-30%�？笶GFR單克隆抗體Cetuximab聯(lián)合化療可以一定程度提高復(fù)發(fā)或轉(zhuǎn)移性鼻咽癌患者的總生存率，，但尚存在著人抗鼠抗體（HAMA）反應(yīng)導(dǎo)致的顯著毒副作用問題。與小分子化療藥物相比，基于納米載體的藥物靶向運(yùn)輸具有延長藥物的體內(nèi)循環(huán)周期、提高藥物的利用率、增加藥物的細(xì)胞內(nèi)攝取能力等優(yōu)點(diǎn)，并在一定程度上降低了藥物的毒副作用。因此，研制高效的鼻咽癌靶向納米藥物，對(duì)轉(zhuǎn)移性鼻咽癌的治療具有重要的臨床應(yīng)用價(jià)值。本研究取得如下創(chuàng)新結(jié)果： （1）發(fā)明了一種八價(jià)多肽納米熒光探針，可用于靶向多肽的在體評(píng)價(jià)，由此鑒定了一條鼻咽癌高特異性的靶向多肽（TTP）。具體方法是，將靶向多肽基因序列偶聯(lián)到四聚體遠(yuǎn)紅色熒光蛋白（tfRFP）基因序列的碳、氮兩端，通過tfRFP成熟后的四聚化效應(yīng)自組裝形成一種八價(jià)多肽納米熒光探針Octa-FNP。在體熒光成像證實(shí)，它對(duì)鼻咽癌5-8F皮下腫瘤具有高特異性的靶向能力、增強(qiáng)的富集能力和高對(duì)比度的腫瘤顯像能力，因此利用Octa-FNP可以快速靈敏地評(píng)價(jià)多肽的靶向能力。本文利用此方法鑒定出一條鼻咽癌高特異性的靶向多肽LTVSPWY（TTP），并通過對(duì)125I標(biāo)記的Octa-FNP進(jìn)行活體放射成像，同樣發(fā)現(xiàn)其能夠高效蓄積于腫瘤組織內(nèi)； （2）發(fā)明了一種新型的同時(shí)具有鼻咽癌協(xié)同靶向增強(qiáng)能力和殺傷能力的多肽R4F-TTP。具體方法是將鑒定的TTP多肽與兩親性螺旋多肽R4F親水端進(jìn)行偶聯(lián)。熒光顯微成像和流式細(xì)胞檢測結(jié)果均證實(shí)，F(xiàn)ITC標(biāo)記的R4F-TTP被5-8F細(xì)胞攝取的量是FITC標(biāo)記的TTP的3.1倍，是FITC標(biāo)記的R4F的10.4倍。此結(jié)果表明R4F-TTP多肽對(duì)5-8F細(xì)胞確實(shí)具有協(xié)同增強(qiáng)的靶向能力。碘化丙啶（PI）染色結(jié)果證實(shí)，R4F-TTP對(duì)5-8F細(xì)胞還具有選擇性殺傷能力。 （3）成功研制了一種對(duì)鼻咽癌5-8F腫瘤具有高選擇性靶向能力和良好治療效果的超小粒徑納米顆粒。透射電鏡和納米粒度儀結(jié)果顯示，NP-TTP是一種直徑約為11nm的球形納米顆粒。流式細(xì)胞檢測結(jié)果表明，NP-TTP被5-8F細(xì)胞攝取的量是納米顆粒NP（不含多肽TTP）的20.5倍，是NP-scrTTP（scrTTP為TTP的隨機(jī)序列多肽）的3.0倍。NP-TTP表現(xiàn)出明顯增強(qiáng)的鼻咽癌細(xì)胞特異性攝取能力，提示多肽TTP被呈現(xiàn)在納米顆粒的表面。整體熒光成像結(jié)果證實(shí)，近紅外熒光染料DiR-BOA標(biāo)記的NP-TTP能夠高效蓄積于鼻咽癌5-8F腫瘤內(nèi)，并有效地?cái)U(kuò)散到整個(gè)腫瘤組織中，DiR-BOA的熒光信號(hào)在腫瘤組織與肝、腎等正常臟器之間具有極高的對(duì)比度。通過尾靜脈連續(xù)兩次隔天給藥，NP-TTP對(duì)5-8F腫瘤的生長抑制率為88±7%，對(duì)5-8F-mRFP轉(zhuǎn)移性腫瘤也具有明顯的抑制效果。NP-TTP對(duì)腫瘤生長的這種抑制作用，被證明與其誘導(dǎo)細(xì)胞凋亡和自噬體形成密切相關(guān)。當(dāng)NP-TTP同步裝載姜黃素后，表現(xiàn)出更強(qiáng)的抑制鼻咽癌5-8F-mRFP腫瘤轉(zhuǎn)移的能力，明顯延長了實(shí)驗(yàn)小鼠的生存期。 綜上所述，本文基于四聚體熒光蛋白發(fā)明了一種八價(jià)多肽納米熒光探針，鑒定了一條鼻咽癌高特異性的靶向多肽TTP。將TTP與兩親性螺旋多肽R4F偶聯(lián)所形成的雜交多肽R4F-TTP，不僅具有協(xié)同增強(qiáng)鼻咽癌靶向攝取和選擇性殺傷的能力，還能控制脂質(zhì)納米顆粒形成及其功能。利用R4F-TTP形成的脂質(zhì)納米顆粒，具有鼻咽癌高對(duì)比度靶向成像的能力和極低毒副作用的靶向治療效果。本研究為靶向和治療性多肽的在體評(píng)價(jià)、腫瘤的靶向成像和診斷以及多策略治療提供了新方法。
[Abstract]:Nasopharyngeal carcinoma (NPC) is a highly malignant tumor, such as China's South and Southeast Asia, which is characterized by the hidden and easy occurrence of distant metastasis. The distant metastasis is one of the main causes of the failure of the treatment of nasopharyngeal carcinoma. Currently, for locally advanced nasopharyngeal carcinoma, combined chemotherapy based on cisplatin is mainly used, but more than 30% of patients have failed due to distant metastasis, and the 5-year overall survival rate is only 25-30%. The combined chemotherapy of anti-EGFR monoclonal antibody, Cetuximab, can improve the overall survival rate of patients with recurrent or metastatic nasopharyngeal carcinoma to a certain extent, but there are significant toxic and side effects caused by the reaction of human anti-mouse antibody (HAMA). Compared with the small-molecule chemotherapy medicine, the drug-targeted transportation based on the nano-carrier has the advantages of prolonging the in-vivo circulation period of the medicine, improving the utilization rate of the medicine, increasing the intracellular uptake capacity of the medicine, and the like, and reducing the toxic and side effect of the medicine to a certain extent. Therefore, it is of great clinical value to develop high-efficiency target nano-drugs for nasopharyngeal carcinoma and to treat metastatic nasopharyngeal carcinoma. The results of this study are as follows: (1) The invention provides an eight-valent polypeptide nano-fluorescent probe, which can be used for the body evaluation of a target polypeptide, thereby identifying a target polypeptide (TT) with high specificity for nasopharyngeal carcinoma, P). The specific method is that the target polypeptide gene sequence is coupled to the carbon and nitrogen ends of the tetramer far red fluorescent protein (tfRFP) gene sequence, and an eight-valence polypeptide nano-fluorescent probe Octa-F is formed by self-assembly by the tetramerization effect after the tfRFP is mature. NP. It is confirmed by the body fluorescence imaging that it has high specific targeting ability, enhanced enrichment ability and high contrast tumor imaging ability for the 5-8F subcutaneous tumor of the nasopharyngeal carcinoma, so the targeting of the polypeptide can be quickly and sensitively evaluated by using the Octa-FNP. Ability. This method is used to identify a highly specific target polypeptide LtWY (TTP) for nasopharyngeal carcinoma, and it is also found that it can accumulate in the tumor group with high efficiency by carrying out in-vivo radiation imaging of the Octa-FNP labeled with 125I. The invention relates to a novel polypeptide R4 with a synergistic targeting and enhancing capability and an anti-killing capability of nasopharyngeal carcinoma, F-TTP. The specific method is to hydrophilic the identified TTP polypeptide and the amphipathic helical polypeptide R4F. The results of the fluorescence microscopy and flow cytometry confirmed that the FITC-labeled R4F-TTP was taken by the 5-8F cell as a 3. 1-fold of the FITC-labeled TTP, which was the FITC-labeled R4F. The results show that the R4F-TTP polypeptide has a synergistic effect on the 5-8F cells. The results showed that R4F-TTP had the choice of 5-8F cells. (3) successfully developed a highly selective targeting ability and good therapeutic effect on the 5-8F tumor of nasopharyngeal carcinoma. The results of transmission electron microscopy and nano-particle size show that the NP-TTP is a kind of nano-particle with a diameter of about 11n. The results of flow cytometry show that the amount of NP-TTP in the 5-8F cells is 20. 5 times that of the NP (not including the polypeptide TTP), which is the random sequence of the NP-scrTTP (sctp). The 3. 0-fold. NP-TTP of the polypeptide showed enhanced specific uptake of the nasopharyngeal carcinoma cells, suggesting that the polypeptide TTP was presented. The results of the overall fluorescence imaging show that the near-infrared fluorescent dye DiR-BOA-labeled NP-TTP can be efficiently accumulated in the 5-8F tumor of the nasopharyngeal carcinoma and effectively diffuse into the whole tumor tissue, and the fluorescence signal of the DiR-BOA is between the tumor tissue and the normal organs such as the liver and the kidney. With very high contrast, the growth inhibition rate of NP-TTP on 5-8F tumor was 88-7%, and the 5-8F-mRFP metastatic tumor was also used for 5-8F-mRFP. The inhibitory effect of NP-TTP on the growth of the tumor is proved to be related to the induction of apoptosis and self-adaptation. It is closely related to the formation of the autophagy. When the NP-TTP is loaded with the curcumin, the ability to inhibit the metastasis of the 5-8F-mRFP tumor of the nasopharyngeal carcinoma is shown to be significantly prolonged. In conclusion, based on the tetramer fluorescent protein, an eight-valent polypeptide nano-fluorescent probe was developed to identify a nasopharyngeal carcinoma (NPC). the hybrid polypeptide R4F-TTP formed by coupling the TTP with the amphipathic helical polypeptide R4F not only has the capability of cooperatively enhancing the target uptake and the selective killing of the nasopharyngeal carcinoma, but also can control the lipid The formation and the function of the quality nano-particles. The lipid nanoparticles formed by the R4F-TTP have the capability of high-contrast target imaging of the nasopharyngeal carcinoma and the extremely low targeted therapeutic effects of toxic and side effects. The present study provides for targeted and therapeutic polypeptides in vivo evaluation, targeted imaging and diagnosis of tumors, and
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R739.63

【參考文獻(xiàn)】

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1 Jill Wykosky;Tim Fenton;Frank Furnari;Webster K. Cavenee;;Therapeutic targeting of epidermal growth factor receptor in human cancer: successes and limitations[J];癌癥;2011年01期

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