本文主要研究内容
作者郑先珍(2019)在《一种多功能靶向自组装纳米粒的构建及其抗肿瘤活性研究》一文中研究指出:恶性肿瘤的治疗一直是个难以攻克的世界难题,究其原因主要有两个方面,一方面是因为传统的细胞毒类抗肿瘤药物缺乏选择性,易诱导肿瘤细胞产生耐药性;另一方面是因为肿瘤干细胞(Cancer stem cells,CSCs)的存在增加了肿瘤转移复发的机率。CSCs是一类具有高致瘤性,保留了类干细胞样无限增殖能力和多向分化潜能的细胞,此类细胞虽然在肿瘤组织中数量较少,却在肿瘤的发生发展中起到关键作用。为了杀灭CSCs和克服肿瘤细胞耐药,目前临床多将不同作用机制的抗肿瘤药物共同给药,利用药物之间的协同作用提高抗肿瘤疗效。但由于每种药物理化性质差异巨大,给药途径也不完全相同,单纯的联合给药方案易导致患者耐受性差,依从性不高,经济负担重。因此,寻找一种同时具有多种作用机制的新型多功能抗肿瘤药物成为当下药学领域的研究热点。本研究结合肿瘤细胞的信号传导特点和抗肿瘤药物的理化特征,在课题组前期研究基础上以全反式维甲酸(All-trans retinoic acid,ATRA)、达沙替尼(Dasatinib,DAS)为模型药物,设计并制备出多功能靶向自组装纳米粒(DAS/ATRA-NPs),构建了不仅具有抗普通肿瘤细胞能力和抗CSCs潜力双重作用机制,理论上还具有肿瘤被动靶向作用和肿瘤小分子蛋白主动靶向作用的多功能肿瘤靶向治疗策略。前言部分主要阐述了ATRA和DAS在肿瘤治疗学方面的研究背景及其应用价值,为本课题提供了充分的理论支持。本文第一章初步考察了ATRA对高致瘤性小鼠黑色素瘤B16F10细胞生物行为学的影响。MTT实验发现ATRA能呈剂量依赖性地抑制B16F10细胞的增殖能力;用中浓度(16μmol/L)的ATRA处理B16F10细胞24 h后,细胞划痕实验显示ATRA能有效抑制B16F10细胞的体外迁移能力;镜下可观察到细胞形态明显发生改变,DAPI核染色可见凋亡小体,流式细胞仪测得其细胞凋亡率为(37.27±4.42)%;体内成瘤实验进一步提示ATRA能显著抑制B16F10细胞的致瘤性,这为本课题选择ATRA作为抗CSCs靶向药物提供了实验依据。本文第二章结合DAS、ATRA药物理化性质,通过超声乳化溶剂蒸发法合成制备出了多功能靶向自组装纳米粒(DAS/ATRA-NPs)。实验先通过单因素处方分析考察了DAS、ATRA的投药比、有机相中DCM与EtOH的比例、有机相与水相的比例、超声功率以及超声时间对DAS/ATRA-NPs的Size、PDI、Zeta Potential、药物包封率(Encapsulation efficiency,EE%)的影响。优化了制备工艺后所制得的DAS/ATRA-NPs呈淡黄色,外观均匀透亮,无肉眼可见颗粒物。透射电镜下观察到纳米粒呈大小约100200 nm的均一椭球形颗粒,激光粒度分析仪测得DAS/ATRA-NPs的Size为(172.57±1.64)nm,PDI为(0.26±0.04),Zeta Potential为(32.10±0.65)mV。HPLC测得DAS/ATRA-NPs中DAS、ATRA的包封率分别(83.61±4.45)%、(95.21±1.09)%。将新制DAS/ATRA-NPs置于4℃条件下储存4周,连续监测Size、PDI和Zeta Potential的变化,发现DAS/ATRA-NPs在4℃储存条件下各参数无明显变化,稳定性良好。实验采用透析法考察DAS/ATRA-NPs体外释放速度,用含20%乙醇的PBS(PH=7.4)作释放介质,发现DAS/ATRA-NPs中的DAS与ATRA累积释药量均小于游离DAS和游离ATRA,表明DAS/ATRA-NPs体外具有一定的缓释能力。本文第三章以人肝癌HepG2细胞为研究对象,着重考察了DAS/ATRA-NPs的体外抗肿瘤效果。实验首先用香豆素-6对DAS/ATRA-NPs进行标记,利用香豆素-6的荧光特点在激光共聚焦下观察到DAS/ATRA-NPs主要存在于HepG2细胞的胞质中。在MTT细胞毒性实验中发现DAS/ATRA-NPs能呈浓度依赖性地抑制HepG2体外增殖,24 h的IC50为12.72μmol/L,药物协同指数CI50为0.96,表明DAS和ATRA两者药效具有协同作用。继以含12.5μmol/L药物浓度的培养基处理细胞,发现DAS/ATRA-NPs能显著抑制HepG2细胞的侵袭迁移能力。给药24 h后,各组HepG2细胞开始发生凋亡,流式细胞仪测得游离ATRA、游离DAS、DAS+ATRA物理混合组、DAS/ATRA-NPs组的细胞凋亡率分别为(14.68±3.25)%、(21.55±1.63)%、(26.98±3.42)%、(45.77±3.30)%,相比各对照组,差异具有统计学意义(P<0.05)。随后,线粒体膜电位实验和Caspase凋亡蛋白荧光实验则表明DAS/ATRA-NPs促凋亡的机制可能与线粒体凋亡途径有关。细胞侧群实验发现正常HepG2细胞中侧群细胞比例(SP)为(1.61±0.12)%,DAS和ATRA两药分别给药和混合给药处理HepG2细胞24 h后,SP比例均有减少,分别为(1.11±0.12)%、(0.89±0.03)%、(0.58±0.02)%,而DAS/ATRA-NPs组的SP比例为(0.35±0.00)%,虽稍弱于阳性对照维拉帕米组(0.21±0.05)%,但DAS/ATRA-NPs对SP的抑制作用明显优于DAS、ATRA分别给药和混合给药组,说明DAS/ATRA-NPs中两药应具有协同抗CSCs的作用。本文第四章将DiD标记过的DAS/ATRA-NPs经尾静脉注射入SD大鼠体内,通过检测不同时间点血浆中DiD的荧光强度而得出DAS/ATRA-NPs在SD大鼠体内药动学曲线并计算药动学参数。实验发现游离DiD体内消除半衰期为4.61 h,标记后的DAS/ATRA-NPs消除半衰期为20.86 h,提示DAS/ATRA-NPs在体内同样具有缓释效果。裸鼠体内成瘤实验发现DAS/ATRA-NPs组肿瘤致瘤率比对照组低,且DAS/ATRA-NPs组肿瘤体积显著小于对照组肿瘤体积(P<0.05),说明DAS/ATRA-NPs能显著抑制HepG2细胞体内成瘤能力,在抗CSCs方面具有一定研究价值。综上所述,本文探索了一种新的恶性肿瘤靶向治疗方案,不仅在理论上丰富了恶性肿瘤治疗领域的研究内容,所构建的多功能自组装纳米粒(DAS/ATRA-NPs)也有望为多功能靶向抗肿瘤药物的研发提供新思路。
Abstract
e xing zhong liu de zhi liao yi zhi shi ge nan yi gong ke de shi jie nan ti ,jiu ji yuan yin zhu yao you liang ge fang mian ,yi fang mian shi yin wei chuan tong de xi bao du lei kang zhong liu yao wu que fa shua ze xing ,yi you dao zhong liu xi bao chan sheng nai yao xing ;ling yi fang mian shi yin wei zhong liu gan xi bao (Cancer stem cells,CSCs)de cun zai zeng jia le zhong liu zhuai yi fu fa de ji lv 。CSCsshi yi lei ju you gao zhi liu xing ,bao liu le lei gan xi bao yang mo xian zeng shi neng li he duo xiang fen hua qian neng de xi bao ,ci lei xi bao sui ran zai zhong liu zu zhi zhong shu liang jiao shao ,que zai zhong liu de fa sheng fa zhan zhong qi dao guan jian zuo yong 。wei le sha mie CSCshe ke fu zhong liu xi bao nai yao ,mu qian lin chuang duo jiang bu tong zuo yong ji zhi de kang zhong liu yao wu gong tong gei yao ,li yong yao wu zhi jian de xie tong zuo yong di gao kang zhong liu liao xiao 。dan you yu mei chong yao wu li hua xing zhi cha yi ju da ,gei yao tu jing ye bu wan quan xiang tong ,chan chun de lian ge gei yao fang an yi dao zhi huan zhe nai shou xing cha ,yi cong xing bu gao ,jing ji fu dan chong 。yin ci ,xun zhao yi chong tong shi ju you duo chong zuo yong ji zhi de xin xing duo gong neng kang zhong liu yao wu cheng wei dang xia yao xue ling yu de yan jiu re dian 。ben yan jiu jie ge zhong liu xi bao de xin hao chuan dao te dian he kang zhong liu yao wu de li hua te zheng ,zai ke ti zu qian ji yan jiu ji chu shang yi quan fan shi wei jia suan (All-trans retinoic acid,ATRA)、da sha ti ni (Dasatinib,DAS)wei mo xing yao wu ,she ji bing zhi bei chu duo gong neng ba xiang zi zu zhuang na mi li (DAS/ATRA-NPs),gou jian le bu jin ju you kang pu tong zhong liu xi bao neng li he kang CSCsqian li shuang chong zuo yong ji zhi ,li lun shang hai ju you zhong liu bei dong ba xiang zuo yong he zhong liu xiao fen zi dan bai zhu dong ba xiang zuo yong de duo gong neng zhong liu ba xiang zhi liao ce lve 。qian yan bu fen zhu yao chan shu le ATRAhe DASzai zhong liu zhi liao xue fang mian de yan jiu bei jing ji ji ying yong jia zhi ,wei ben ke ti di gong le chong fen de li lun zhi chi 。ben wen di yi zhang chu bu kao cha le ATRAdui gao zhi liu xing xiao shu hei se su liu B16F10xi bao sheng wu hang wei xue de ying xiang 。MTTshi yan fa xian ATRAneng cheng ji liang yi lai xing de yi zhi B16F10xi bao de zeng shi neng li ;yong zhong nong du (16μmol/L)de ATRAchu li B16F10xi bao 24 hhou ,xi bao hua hen shi yan xian shi ATRAneng you xiao yi zhi B16F10xi bao de ti wai qian yi neng li ;jing xia ke guan cha dao xi bao xing tai ming xian fa sheng gai bian ,DAPIhe ran se ke jian diao wang xiao ti ,liu shi xi bao yi ce de ji xi bao diao wang lv wei (37.27±4.42)%;ti nei cheng liu shi yan jin yi bu di shi ATRAneng xian zhe yi zhi B16F10xi bao de zhi liu xing ,zhe wei ben ke ti shua ze ATRAzuo wei kang CSCsba xiang yao wu di gong le shi yan yi ju 。ben wen di er zhang jie ge DAS、ATRAyao wu li hua xing zhi ,tong guo chao sheng ru hua rong ji zheng fa fa ge cheng zhi bei chu le duo gong neng ba xiang zi zu zhuang na mi li (DAS/ATRA-NPs)。shi yan xian tong guo chan yin su chu fang fen xi kao cha le DAS、ATRAde tou yao bi 、you ji xiang zhong DCMyu EtOHde bi li 、you ji xiang yu shui xiang de bi li 、chao sheng gong lv yi ji chao sheng shi jian dui DAS/ATRA-NPsde Size、PDI、Zeta Potential、yao wu bao feng lv (Encapsulation efficiency,EE%)de ying xiang 。you hua le zhi bei gong yi hou suo zhi de de DAS/ATRA-NPscheng dan huang se ,wai guan jun yun tou liang ,mo rou yan ke jian ke li wu 。tou she dian jing xia guan cha dao na mi li cheng da xiao yao 100200 nmde jun yi tuo qiu xing ke li ,ji guang li du fen xi yi ce de DAS/ATRA-NPsde Sizewei (172.57±1.64)nm,PDIwei (0.26±0.04),Zeta Potentialwei (32.10±0.65)mV。HPLCce de DAS/ATRA-NPszhong DAS、ATRAde bao feng lv fen bie (83.61±4.45)%、(95.21±1.09)%。jiang xin zhi DAS/ATRA-NPszhi yu 4℃tiao jian xia chu cun 4zhou ,lian xu jian ce Size、PDIhe Zeta Potentialde bian hua ,fa xian DAS/ATRA-NPszai 4℃chu cun tiao jian xia ge can shu mo ming xian bian hua ,wen ding xing liang hao 。shi yan cai yong tou xi fa kao cha DAS/ATRA-NPsti wai shi fang su du ,yong han 20%yi chun de PBS(PH=7.4)zuo shi fang jie zhi ,fa xian DAS/ATRA-NPszhong de DASyu ATRAlei ji shi yao liang jun xiao yu you li DAShe you li ATRA,biao ming DAS/ATRA-NPsti wai ju you yi ding de huan shi neng li 。ben wen di san zhang yi ren gan ai HepG2xi bao wei yan jiu dui xiang ,zhao chong kao cha le DAS/ATRA-NPsde ti wai kang zhong liu xiao guo 。shi yan shou xian yong xiang dou su -6dui DAS/ATRA-NPsjin hang biao ji ,li yong xiang dou su -6de ying guang te dian zai ji guang gong ju jiao xia guan cha dao DAS/ATRA-NPszhu yao cun zai yu HepG2xi bao de bao zhi zhong 。zai MTTxi bao du xing shi yan zhong fa xian DAS/ATRA-NPsneng cheng nong du yi lai xing de yi zhi HepG2ti wai zeng shi ,24 hde IC50wei 12.72μmol/L,yao wu xie tong zhi shu CI50wei 0.96,biao ming DAShe ATRAliang zhe yao xiao ju you xie tong zuo yong 。ji yi han 12.5μmol/Lyao wu nong du de pei yang ji chu li xi bao ,fa xian DAS/ATRA-NPsneng xian zhe yi zhi HepG2xi bao de qin xi qian yi neng li 。gei yao 24 hhou ,ge zu HepG2xi bao kai shi fa sheng diao wang ,liu shi xi bao yi ce de you li ATRA、you li DAS、DAS+ATRAwu li hun ge zu 、DAS/ATRA-NPszu de xi bao diao wang lv fen bie wei (14.68±3.25)%、(21.55±1.63)%、(26.98±3.42)%、(45.77±3.30)%,xiang bi ge dui zhao zu ,cha yi ju you tong ji xue yi yi (P<0.05)。sui hou ,xian li ti mo dian wei shi yan he Caspasediao wang dan bai ying guang shi yan ze biao ming DAS/ATRA-NPscu diao wang de ji zhi ke neng yu xian li ti diao wang tu jing you guan 。xi bao ce qun shi yan fa xian zheng chang HepG2xi bao zhong ce qun xi bao bi li (SP)wei (1.61±0.12)%,DAShe ATRAliang yao fen bie gei yao he hun ge gei yao chu li HepG2xi bao 24 hhou ,SPbi li jun you jian shao ,fen bie wei (1.11±0.12)%、(0.89±0.03)%、(0.58±0.02)%,er DAS/ATRA-NPszu de SPbi li wei (0.35±0.00)%,sui shao ruo yu yang xing dui zhao wei la pa mi zu (0.21±0.05)%,dan DAS/ATRA-NPsdui SPde yi zhi zuo yong ming xian you yu DAS、ATRAfen bie gei yao he hun ge gei yao zu ,shui ming DAS/ATRA-NPszhong liang yao ying ju you xie tong kang CSCsde zuo yong 。ben wen di si zhang jiang DiDbiao ji guo de DAS/ATRA-NPsjing wei jing mai zhu she ru SDda shu ti nei ,tong guo jian ce bu tong shi jian dian xie jiang zhong DiDde ying guang jiang du er de chu DAS/ATRA-NPszai SDda shu ti nei yao dong xue qu xian bing ji suan yao dong xue can shu 。shi yan fa xian you li DiDti nei xiao chu ban cui ji wei 4.61 h,biao ji hou de DAS/ATRA-NPsxiao chu ban cui ji wei 20.86 h,di shi DAS/ATRA-NPszai ti nei tong yang ju you huan shi xiao guo 。luo shu ti nei cheng liu shi yan fa xian DAS/ATRA-NPszu zhong liu zhi liu lv bi dui zhao zu di ,ju DAS/ATRA-NPszu zhong liu ti ji xian zhe xiao yu dui zhao zu zhong liu ti ji (P<0.05),shui ming DAS/ATRA-NPsneng xian zhe yi zhi HepG2xi bao ti nei cheng liu neng li ,zai kang CSCsfang mian ju you yi ding yan jiu jia zhi 。zeng shang suo shu ,ben wen tan suo le yi chong xin de e xing zhong liu ba xiang zhi liao fang an ,bu jin zai li lun shang feng fu le e xing zhong liu zhi liao ling yu de yan jiu nei rong ,suo gou jian de duo gong neng zi zu zhuang na mi li (DAS/ATRA-NPs)ye you wang wei duo gong neng ba xiang kang zhong liu yao wu de yan fa di gong xin sai lu 。
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论文详细介绍
论文作者分别是来自重庆医科大学的郑先珍,发表于刊物重庆医科大学2019-09-24论文,是一篇关于多功能靶向自组装纳米粒论文,全反式维甲酸论文,达沙替尼论文,肿瘤干细胞论文,重庆医科大学2019-09-24论文的文章。本文可供学术参考使用,各位学者可以免费参考阅读下载,文章观点不代表本站观点,资料来自重庆医科大学2019-09-24论文网站,若本站收录的文献无意侵犯了您的著作版权,请联系我们删除。
标签:多功能靶向自组装纳米粒论文; 全反式维甲酸论文; 达沙替尼论文; 肿瘤干细胞论文; 重庆医科大学2019-09-24论文;