本文選題：SELEX　切入點(diǎn)：適配體-藥物偶聯(lián)物　出處：《廣西醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：癌癥是人類健康的最大威脅之一,伴隨醫(yī)學(xué)的發(fā)展,腫瘤治療策略已經(jīng)趨于多樣化,有聯(lián)合治療、免疫療法,甚至精準(zhǔn)醫(yī)療,但在部分腫瘤患者中化療依舊作為一線治療方案被使用。如何盡量克服、避免化療藥物因非靶向性帶來(lái)的毒副作用,一直是臨床關(guān)注的熱點(diǎn)之一。構(gòu)建靶向遞送系統(tǒng),可提高靶組織局部藥物濃度,減少全身毒副作用,實(shí)現(xiàn)藥物的高效、安全遞送,是一種很有前景的給藥方法。目前已有兩種抗體藥物偶聯(lián)物(Antibody-drug conjugates,ADC)經(jīng)FDA批準(zhǔn)上市且獲得良好藥效。但在抗體-藥物制備過程中的DAR(Drug-anbibody ratio)均一性、裸抗體的免疫原性等問題還未得到良好控制。適配體(Aptamer,Apt)是一種獨(dú)特的單鏈寡核苷酸,其功能類似抗體,有著靶分子范圍廣泛、高特異性及親和性以及免疫原性低等優(yōu)勢(shì)。除可作為藥物(例如FDA批準(zhǔn)上市的Macugen)單獨(dú)應(yīng)用外,更多可作為藥物遞送系統(tǒng)中的靶向載體。本論文關(guān)注化療藥物的靶向遞送系統(tǒng)的概念性驗(yàn)證研究,構(gòu)建適配體-藥物偶聯(lián)物遞送系統(tǒng)(Aptamer-drug conjugate,Ap DC),證明其顯著提高藥物治療指數(shù),降低藥物毒副作用的應(yīng)用前景。本文首先應(yīng)用指數(shù)富集的配體系統(tǒng)進(jìn)化技術(shù)(Systematic evolution of ligands by exponential enrichment,SELEX),以人的表皮生長(zhǎng)因子受體3(human epidermal growth factor receptor 3,HER3)的胞外結(jié)構(gòu)域(extracellular domain,ECD)為靶目標(biāo),篩選獲得具有高親和力和高特異性的候選Apt。選擇阿霉素(Doxorubicin,DOX)為遞送對(duì)象,薄膜分散法制備脂質(zhì)體阿霉素(liposome-doxorubicin,Lip-DOX)。通過“后插入法”連接Apt,構(gòu)建穩(wěn)定的適配體-脂質(zhì)體-阿霉素(Aptamer-liposome-doxorubicin,Apt-Lip-DOX)。通過包封率、粒徑大小、zeta電勢(shì)、釋放度等理化性質(zhì)指標(biāo),考察并優(yōu)化Apt-Lip-DOX制備工藝。體外實(shí)驗(yàn)中,在MCF-7 HER3+、BT474 HER3+細(xì)胞及陰性對(duì)照293THER3-細(xì)胞中考察該Ap DC系統(tǒng)的入胞情況及半數(shù)抑制濃度(half maximal inhibitory concentration,IC50)并對(duì)內(nèi)吞途徑進(jìn)行初步探索。體內(nèi)實(shí)驗(yàn)中,選擇MCF-7HER3+荷瘤小鼠模型,考察Ap DC針對(duì)其他對(duì)照組在靶向性、有效性及安全性方面的優(yōu)勢(shì)。第一部分HER3ECD Apt的篩選和鑒定。選擇HER3ECD質(zhì)粒瞬時(shí)轉(zhuǎn)染293E細(xì)胞,表達(dá)回收HER3ECD蛋白,作為篩選的靶蛋白。采用經(jīng)優(yōu)化的“QPCR-磁珠分選”SELEX方法,經(jīng)四輪十次篩選,獲得靶向HER3ECD的候選Apt。通過酶聯(lián)免疫吸附測(cè)定(enzyme linked immunosorbent assay,ELISA)并擬合親和力曲線,得出Apt#7及#13親和力常數(shù)較佳,Kd值分別為116±23 n M、98±9.7 n M。通過細(xì)胞流式實(shí)驗(yàn)及激光共聚焦結(jié)果顯示,該適配體針對(duì)高表達(dá)HER3的乳腺癌細(xì)胞系具有較高的親和力及特異性。第二部分Ap DC遞送系統(tǒng)的構(gòu)建及優(yōu)化。優(yōu)化脂質(zhì)體阿霉素的制備工藝,通過DSPE-PEG2000連接Apt,采用“后插入法”制備穩(wěn)定的Ap DC,即Apt-Lip-DOX�？疾霢pt-Lip-DOX的理化性質(zhì),粒徑為170±25 nm,zeta電勢(shì)為-45.9±6.31 m V,結(jié)構(gòu)穩(wěn)定。超濾法結(jié)合高效液相色譜(High Performance Liquid Chromatography,HPLC)測(cè)定DOX藥物包封率為55±5%。模擬體內(nèi)環(huán)境藥物釋放度結(jié)果顯示:同等條件下,游離的DOX在1h釋放度接近100%,而Apt-Lip-DOX 24h釋放度僅為20%,充分證明了該遞送系統(tǒng)緩釋遞送的優(yōu)勢(shì)。第三部分Ap DC系統(tǒng)體外胞吞機(jī)制和藥效實(shí)驗(yàn)。細(xì)胞水平上,選擇MCF-7 HER3+細(xì)胞及陰性對(duì)照293THER3-,比較Apt的介導(dǎo)是否增加遞送系統(tǒng)的入胞量。并通過不同內(nèi)吞途徑抑制劑結(jié)合活細(xì)胞觀測(cè)站,初步探討該Ap DC入胞途徑機(jī)制。結(jié)果表明Ap DC的入胞依賴于HER3的表達(dá)及Apt的存在,突出HER3 Apt的優(yōu)勢(shì),同時(shí)該Ap DC主要通過網(wǎng)格蛋白介導(dǎo)的內(nèi)吞作用完成內(nèi)吞。進(jìn)一步選擇MCF-7 HER3+、BT474HER3+兩株細(xì)胞系經(jīng)實(shí)時(shí)細(xì)胞檢測(cè)(real time cell assay,RTCA)考察DOX不同遞送形式的IC50值,同時(shí)加入293THER3-細(xì)胞作為對(duì)照。結(jié)果顯示,在三株細(xì)胞系中,Lip-DOX針對(duì)游離DOX的IC50值降低,且均具有統(tǒng)計(jì)學(xué)差異(P0.05),體現(xiàn)脂質(zhì)體遞送系統(tǒng)的優(yōu)勢(shì)。Apt-Lip-DOX在HER3高表達(dá)的MCF-7、BT474中較游離DOX組出現(xiàn)顯著性統(tǒng)計(jì)學(xué)差異(P0.01),在293T細(xì)胞中未表現(xiàn)出該優(yōu)勢(shì)。證明所篩選適配體在遞送系統(tǒng)中發(fā)揮一定的靶向優(yōu)勢(shì),增加體外靶向腫瘤細(xì)胞的殺傷力,為后續(xù)體內(nèi)實(shí)驗(yàn)奠定基礎(chǔ)。第四部分Ap DC系統(tǒng)荷瘤小鼠體內(nèi)腫瘤靶向性和藥效、毒性實(shí)驗(yàn)。動(dòng)物水平上,選擇MCF-7荷瘤小鼠,考察體內(nèi)Ap DC的腫瘤靶向性、有效性及安全性。1)腫瘤靶向性:按照Ap DC制備工藝,選擇包裹量子點(diǎn)(QD),制備Apt-Lip-QD,同時(shí)Lip-QD為對(duì)照組,尾靜脈注射MCF-7荷瘤小鼠1h后進(jìn)行活體成像,觀察熒光分布判斷藥物的體內(nèi)分布。結(jié)果顯示Ap DC表現(xiàn)出良好的靶向性,解剖瘤體與Lip-QD相比,具有明顯的統(tǒng)計(jì)學(xué)差異(P0.01)。2)體內(nèi)抑制腫瘤藥效實(shí)驗(yàn):設(shè)置空白對(duì)照組(Blank,等體積生理鹽水)以及實(shí)驗(yàn)組DOX、Lip-DOX、Apt-Lip-DOX共四個(gè)組別,實(shí)驗(yàn)組的DOX給藥量為5mg/kg,每三天尾靜脈注射給藥。結(jié)果顯示:給藥18天后,Apt-Lip-DOX抑瘤效果針對(duì)Blank組具有非常顯著的統(tǒng)計(jì)學(xué)差異(P0.01,P0.001),同時(shí)較DOX組也表現(xiàn)出較大的優(yōu)勢(shì)(P0.05)。3)體內(nèi)毒性考察:分別考察小鼠給藥期間體重狀態(tài),60天存活率,組織病理切片。結(jié)果顯示:給藥期間Apt-Lip-DOX組小鼠狀態(tài)、體重維持在正常水平,DOX組則在20天出現(xiàn)體重急劇下降的現(xiàn)象。不同組別(DOX、Lip-DOX、Apt-Lip-DOX)小鼠60天以內(nèi)的存活率分別為0、40%、100%。小鼠死亡后對(duì)各臟器做HE病理切片觀察病變損傷情況,在DOX組中,心臟切片顯示出現(xiàn)脂肪變性,散在出血點(diǎn),血管擴(kuò)張出血等病變;肝臟切片出現(xiàn)局灶肝細(xì)胞壞死。在Lip-DOX組中也出現(xiàn)輕微病變,Apt-Lip-DOX組所有組織均正常。綜上體內(nèi)實(shí)驗(yàn)結(jié)果,充分證明了該Ap DC的靶向性、高效性及安全性。綜上,本研究建立并完善SELEX技術(shù)路線,為其他靶點(diǎn)篩選提供參考;靶向HER3的治療策略將會(huì)為腫瘤靶向治療或逆轉(zhuǎn)EGFR抑制劑耐藥帶來(lái)新的思路;以化療藥(DOX)為例,通過構(gòu)建Ap DC遞送系統(tǒng),對(duì)其靶向性、抑瘤作用等進(jìn)行研究,為實(shí)現(xiàn)化療藥的高效低毒遞送提供參考。
[Abstract]:Cancer is one of the biggest threat to human health, along with the development of medicine, therapeutic strategies have tended to be diverse, with combined therapy, immunotherapy, and accurate medical treatment, but still chemotherapy as first-line therapy is used in patients with tumor. How to overcome and avoid chemotherapy for non targeting of the side role is one of clinical focus. Construction of targeted delivery system, can improve the local drug concentration of target tissue, reduce systemic toxicity, drug efficiency, safe delivery, is a promising drug delivery method. There are two kinds of antibody drug conjugates (Antibody-drug, conjugates, ADC) by approved by the FDA and get a good effect. But the antibody drug in the preparation process of DAR (Drug-anbibody ratio) uniformity, immunogenicity and other issues have not been good naked antibody aptamer (Aptam control. Er, Apt) is a unique single stranded oligonucleotide, its function is similar with the target molecule antibody, wide range, high specificity and affinity and lower immunogenicity. In addition can be used as a drug (such as the FDA approved Macugen) alone, more can be used as a drug delivery system targeting carrier this paper focuses on. Chemotherapy drugs targeting research proof of concept delivery system, construct the aptamer conjugates drug delivery system (Aptamer-drug conjugate, Ap DC), that significantly improve the therapeutic efficacy, reduce the side effect of the drug application. This paper firstly applied evolution of ligands by exponential enrichment (Systematic evolution system of ligands by exponential enrichment, SELEX), human epidermal growth factor receptor 3 (human epidermal growth factor receptor 3, HER3) of the extracellular domain (extracellular, domain, ECD) as the target Target candidate Apt. with high affinity and high specificity for selection of adriamycin (Doxorubicin, DOX) for screening the delivery object by film dispersion liposomal doxorubicin (liposome-doxorubicin, Lip-DOX) method. Apt connection through the post insertion method, construct the aptamer liposome doxorubicin (Aptamer-liposome-doxorubicin, Apt-Lip-DOX) stable through. Encapsulation efficiency, particle size, zeta potential, release of physical and chemical properties, investigate and optimize the preparation process of Apt-Lip-DOX. In vitro experiments, in MCF-7 HER3+, BT474 HER3+ cell and negative control cell of the Ap into the DC system in 293THER3- cells and the half inhibitory concentration (half maximal inhibitory concentration, IC50) and the endocytic pathways were investigated. In vivo, MCF-7HER3+ tumor bearing mice model of Ap DC for the other control group to the target, effectiveness and safety The advantage of the first part. Screening and identification of HER3ECD Apt. HER3ECD plasmid was transfected to 293E cells, the expression of HER3ECD protein recovery, as the target protein screening. Using the optimized "QPCR- multisort" SELEX method, after four rounds of the ten screening, get targeted candidate Apt. HER3ECD by ELISA (enzyme linked immunosorbent assay, ELISA) and the Apt#7 curve fitting affinity, and the affinity constant #13 is better, Kd = 116 + 23 n M + 9.7 n, 98 M. by flow cytometry and confocal laser experiments showed that the affinity and specificity of the adapter body of high expression of breast cancer cell lines HER3 has a higher Ap DC. The second part delivery of construction and optimization of the system. Optimization of the preparation of liposomal doxorubicin, Apt connection through the DSPE-PEG2000, the "post insert method" the preparation of stable Ap DC, namely Apt-Lip-DO X. Apt-Lip-DOX on the physical and chemical properties, particle size of 170 + 25 nm, zeta potential is -45.9 + 6.31 m V, stable structure. Ultrafiltration combined with high performance liquid chromatography (HPLC High Performance Liquid Chromatography, DOX) determination of drug entrapment efficiency shows 55 + 5%. for simulating the in vivo environment drug release results: the same conditions next, the free DOX in the 1H release rate close to 100%, while the Apt-Lip-DOX 24h release rate is only 20%, it is proved that this delivery system sustained release delivery advantage. The third part of the Ap DC system in vitro endocytosis mechanism and experiment. The level of cell, MCF-7 HER3+ cell and negative control 293THER3-, Apt mediated whether increase the delivery system into the cell. And through the different endocytic pathway inhibitors combined with living cell observation station, preliminary study of the Ap DC into the cell mechanism. The results showed that the expression of Apt and Ap in DC cell dependent on the presence of HER3 HER3 A, prominent The advantages of the Pt and the Ap DC endocytosis through clathrin mediated endocytosis. Further complete the selection of MCF-7 HER3+ and BT474HER3+ two cell lines by real-time cell detection (real time cell assay, RTCA DOX) on different delivery form of the IC50 value, while adding 293THER3- cells as control. The results showed that in three cell lines, Lip-DOX for free DOX IC50 value decreased, and there was significant difference (P0.05), liposome delivery system embodies the advantages of the high expression of.Apt-Lip-DOX in HER3 MCF-7, significant difference between DOX group compared with the free BT474 (P0.01), in 293T cells did not show the advantage. To prove that the screening aptamers in a delivery system play a targeted advantage, increase the in vitro targeting tumor cell destruction, which lays the foundation for further study in vivo. The fourth part of the Ap DC system in vivo tumor targeting and tumor bearing mice The efficacy, toxicity. Animal level, select the MCF-7 tumor bearing mice were investigated in vivo Ap DC tumor targeting, effectiveness and safety of.1) tumor targeting Ap DC: according to the preparation process, select the package of quantum dots (QD), the preparation of Apt-Lip-QD Lip-QD, at the same time as the control group, tail vein injection of MCF-7 mice after 1h in vivo imaging, observation of fluorescence distribution determine the distribution of drugs in the body. The results showed that Ap DC exhibited better targeting tumor anatomy, compared with Lip-QD, the difference is statistically significant (P0.01).2) in inhibiting tumor experiment: blank control group (Blank, volume etc. saline) and experimental group DOX, Lip-DOX, Apt-Lip-DOX a total of four groups, the experimental group of DOX dosage was 5mg/kg, every three days intravenous injection. Results: after 18 days of treatment, Apt-Lip-DOX inhibitory effect in Blank group was statistically significantly different (P0.0 1, P0.001), and compared with the DOX group also showed a greater advantage (P0.05).3) were investigated in vivo toxicity study: mice were administered during weight status, the 60 day survival rate, pathological sections. The results showed that during the administration of Apt-Lip-DOX mice, body weight was maintained at a normal level, DOX group showed weight a sharp decline in the phenomenon in 20 days. Different groups (DOX, Lip-DOX, Apt-Lip-DOX) the survival rate of mice within 60 days were 0,40%, 100%. after the death of mice HE pathological lesions of various organs in the DOX group, the heart slices showed steatosis, scattered hemorrhage, vascular dilatation and hemorrhage other diseases; liver biopsy and focal necrosis of liver cells. Slight lesions in the Lip-DOX group, Apt-Lip-DOX group all were normal. The results of in vivo experiments proved that targeting the Ap of DC, high efficiency and safety. In summary, this research The establishment and improvement of SELEX technology, provide a reference for other target screening; targeted treatment strategies will HER3 for tumor targeted therapy or reversal of EGFR inhibitor resistance brings new ideas; to chemotherapeutic drugs (DOX) as an example, by constructing Ap DC delivery system, the targeting and anti-tumor effect etc. study, provide a reference for the realization of high efficiency and low toxicity of chemotherapeutic drug delivery.

【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R943;R96

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本文編號(hào)：1573606