本文主要研究内容
作者田泾（2019）在《共载多西紫杉醇和siPLK1的肿瘤靶向外泌体构建及其抗前列腺癌作用研究》一文中研究指出：前列腺癌是大部分西方国家发病率最高的恶性肿瘤。近年来随着国内老龄化和生活方式的改变,我国前列腺癌的发病率逐年上升。对于去势抵抗型的晚期前列腺癌,目前的治疗以化疗为主,治疗手段十分有限。基于纳米技术的化疗药物和基因药物联合使用是当前肿瘤治疗研究的热点,本课题选用巨噬细胞来源的外泌体（Exosome）作为药物载体,选用一线化疗药物多西紫杉醇和基因药物siPLK1,结合基因治疗和纳米药物靶向输送的理念和技术,构建了共载多西紫杉醇和基因药物siPLK1的肿瘤靶向外泌体,并对其抗前列腺癌作用进行了体内外系统性和评价。课题的第一章的研究内容主要包含三个部分:1.利用多肽固相合成法合成了硬脂酰多肽stearyl-H3CR5C（SHR）的合成,并对其进行了制备液相纯化、HPLC纯度检测和和MALDI-TOF质谱法鉴定。将合成的硬脂酰多肽单体通过二硫键连接成聚合物SHRss2,并对其进行了GPC分子量表征和1H-NMR鉴定。采用超声自乳化的方法将化疗药物DTX包裹在多肽聚合物SHRss2自组装形成的胶束中,随后将自组装形成的多肽胶束利用其正电性与基因药物siPLK1共孵育,制备成共载DTX和siPLK1的多肽聚合物胶束（Co-PMs）,并对Co-PMs进行了表征;2.利用多西紫杉醇和siPLK1共载的硬脂酰多肽胶束转染小鼠巨噬细胞系Raw264.7,采用主动载入的方式一步法直接产生共载多西紫杉醇和siPLK1的外泌体,并对其分离纯化;3.利用共孵育的方法,将DSPE-PEG-FA与外泌体整合至外泌体表面,引入叶酸靶头并将外泌体表面PEG功能化,构建了共载多西紫杉醇和siPLK1的肿瘤靶向外泌体（Co-Exo-FA）并对其进行表征,包括粒径电位测定、透射电镜观察、载药量测定、标识性蛋白检测等内容。课题的第二章考察了共载多西紫杉醇和siPLK1的肿瘤靶向外泌体（Co-Exo-FA）在体外细胞层面的抗前列腺癌细胞增殖作用,研究阐明了其对前列腺癌细胞的靶向能力,对细胞内PLK1蛋白表达的抑制作用以及siPLK1与DTX联合使用的协同作用。我们以去势抵抗型前列腺癌的细胞系PC-3和DU145作为研究对象,细胞摄取实验表明了去势抵抗型前列腺癌细胞对共载尼罗红和FAM-siPLK1的肿瘤靶向外泌体有较高的摄取。激光共聚焦显微镜观察的结果表明Co-Exo-FA携带了大量的FAM-siRNA进入了细胞。最后在细胞层面考察了Co-Exo和Co-Exo-FA对PLK1和BCL-2两种蛋白表达的影响。在增殖和凋亡实验中,我们发现与只载DTX的外泌体相比,共载了DTX和siPLK1的外泌体能够更加显著的抑制PC-3和DU-145细胞的增殖,促进PC-3细胞的凋亡,表明siPLK1与DTX确实产生了协同抗前列腺癌的作用。其中Co-Exo-FA组,细胞增殖最缓慢,发生凋亡的比例最高,表明叶酸靶头的引入增强了共载DTX和siPLK1外泌体的药效。在蛋白印迹实验证实Co-Exo-FA中的siPLK1干扰了蛋白的表达过程。BCL-2蛋白低表达也表明Co-Exo-FA促进了前列腺癌细胞的凋亡进程。课题的第三章章在前期对共载多西紫杉醇和siPLK1的肿瘤靶向外泌体的体外抗前列腺癌作用评价的基础上,通过构建人源前列腺癌裸鼠皮下移植瘤模型对其进行了体内评价。在人源前列腺癌裸鼠皮下移植瘤模型上对共载多西紫杉醇和siPLK1的肿瘤靶向外泌体进行了体内评价。在移植瘤裸鼠体内的分布情况考察中,游离DIR在肿瘤部位分布很少,其肿瘤部位荧光与周围组织没有明显差异,Co-Exo/DIR在肿瘤部位有一定的累积,而Co-Exo-FA/DIR在注射后迅速的大量富集在肿瘤部位,显示出很强的荧光信号,并且能够持续超过24小时,并且在裸鼠肝脏脾脏分布少,表明PEG减少了RES对外泌体的清除。肿瘤药效学评价证实了化疗药物DTX和基因药物siPLK1在体内可以发挥协同作用。抗肿瘤增殖作用排序为:Co-Exo-FA>Co-Exo>DTX-Exo>DTX>Blank Exo=>Saline。组织学研究发现Co-Exo-FA组的肿瘤组织细胞破坏最明显化疗药物DTX和基因药物siPLK1在体内有协同抗肿瘤作用。对体重的跟踪测量发现外泌体及载药后的外泌体全身毒性作用都比较低。DTX组裸鼠的肝脏组织出现一定程度的损伤,其余各组裸鼠没有观察到明显的器官组织病理学改变。安全性评价结果表明,DTX装载于外泌体可以降低全身系统性毒性,Co-Exo-FA具有比较好的体内安全性。通过课题研究独创了化疗药物和基因药物共转染细胞制备共载化疗药物和基因药物的外泌体的方法。我们构建的肿瘤靶向外泌体为去势抵抗型前列腺癌和其他恶性肿瘤的治疗研究提供了新思路和有益借鉴。
Abstract
qian lie xian ai shi da bu fen xi fang guo jia fa bing lv zui gao de e xing zhong liu 。jin nian lai sui zhao guo nei lao ling hua he sheng huo fang shi de gai bian ,wo guo qian lie xian ai de fa bing lv zhu nian shang sheng 。dui yu qu shi di kang xing de wan ji qian lie xian ai ,mu qian de zhi liao yi hua liao wei zhu ,zhi liao shou duan shi fen you xian 。ji yu na mi ji shu de hua liao yao wu he ji yin yao wu lian ge shi yong shi dang qian zhong liu zhi liao yan jiu de re dian ,ben ke ti shua yong ju shi xi bao lai yuan de wai bi ti （Exosome）zuo wei yao wu zai ti ,shua yong yi xian hua liao yao wu duo xi zi sha chun he ji yin yao wu siPLK1,jie ge ji yin zhi liao he na mi yao wu ba xiang shu song de li nian he ji shu ,gou jian le gong zai duo xi zi sha chun he ji yin yao wu siPLK1de zhong liu ba xiang wai bi ti ,bing dui ji kang qian lie xian ai zuo yong jin hang le ti nei wai ji tong xing he ping jia 。ke ti de di yi zhang de yan jiu nei rong zhu yao bao han san ge bu fen :1.li yong duo tai gu xiang ge cheng fa ge cheng le ying zhi xian duo tai stearyl-H3CR5C（SHR）de ge cheng ,bing dui ji jin hang le zhi bei ye xiang chun hua 、HPLCchun du jian ce he he MALDI-TOFzhi pu fa jian ding 。jiang ge cheng de ying zhi xian duo tai chan ti tong guo er liu jian lian jie cheng ju ge wu SHRss2,bing dui ji jin hang le GPCfen zi liang biao zheng he 1H-NMRjian ding 。cai yong chao sheng zi ru hua de fang fa jiang hua liao yao wu DTXbao guo zai duo tai ju ge wu SHRss2zi zu zhuang xing cheng de jiao shu zhong ,sui hou jiang zi zu zhuang xing cheng de duo tai jiao shu li yong ji zheng dian xing yu ji yin yao wu siPLK1gong fu yo ,zhi bei cheng gong zai DTXhe siPLK1de duo tai ju ge wu jiao shu （Co-PMs）,bing dui Co-PMsjin hang le biao zheng ;2.li yong duo xi zi sha chun he siPLK1gong zai de ying zhi xian duo tai jiao shu zhuai ran xiao shu ju shi xi bao ji Raw264.7,cai yong zhu dong zai ru de fang shi yi bu fa zhi jie chan sheng gong zai duo xi zi sha chun he siPLK1de wai bi ti ,bing dui ji fen li chun hua ;3.li yong gong fu yo de fang fa ,jiang DSPE-PEG-FAyu wai bi ti zheng ge zhi wai bi ti biao mian ,yin ru xie suan ba tou bing jiang wai bi ti biao mian PEGgong neng hua ,gou jian le gong zai duo xi zi sha chun he siPLK1de zhong liu ba xiang wai bi ti （Co-Exo-FA）bing dui ji jin hang biao zheng ,bao gua li jing dian wei ce ding 、tou she dian jing guan cha 、zai yao liang ce ding 、biao shi xing dan bai jian ce deng nei rong 。ke ti de di er zhang kao cha le gong zai duo xi zi sha chun he siPLK1de zhong liu ba xiang wai bi ti （Co-Exo-FA）zai ti wai xi bao ceng mian de kang qian lie xian ai xi bao zeng shi zuo yong ,yan jiu chan ming le ji dui qian lie xian ai xi bao de ba xiang neng li ,dui xi bao nei PLK1dan bai biao da de yi zhi zuo yong yi ji siPLK1yu DTXlian ge shi yong de xie tong zuo yong 。wo men yi qu shi di kang xing qian lie xian ai de xi bao ji PC-3he DU145zuo wei yan jiu dui xiang ,xi bao she qu shi yan biao ming le qu shi di kang xing qian lie xian ai xi bao dui gong zai ni luo gong he FAM-siPLK1de zhong liu ba xiang wai bi ti you jiao gao de she qu 。ji guang gong ju jiao xian wei jing guan cha de jie guo biao ming Co-Exo-FAxie dai le da liang de FAM-siRNAjin ru le xi bao 。zui hou zai xi bao ceng mian kao cha le Co-Exohe Co-Exo-FAdui PLK1he BCL-2liang chong dan bai biao da de ying xiang 。zai zeng shi he diao wang shi yan zhong ,wo men fa xian yu zhi zai DTXde wai bi ti xiang bi ,gong zai le DTXhe siPLK1de wai bi ti neng gou geng jia xian zhe de yi zhi PC-3he DU-145xi bao de zeng shi ,cu jin PC-3xi bao de diao wang ,biao ming siPLK1yu DTXque shi chan sheng le xie tong kang qian lie xian ai de zuo yong 。ji zhong Co-Exo-FAzu ,xi bao zeng shi zui huan man ,fa sheng diao wang de bi li zui gao ,biao ming xie suan ba tou de yin ru zeng jiang le gong zai DTXhe siPLK1wai bi ti de yao xiao 。zai dan bai yin ji shi yan zheng shi Co-Exo-FAzhong de siPLK1gan rao le dan bai de biao da guo cheng 。BCL-2dan bai di biao da ye biao ming Co-Exo-FAcu jin le qian lie xian ai xi bao de diao wang jin cheng 。ke ti de di san zhang zhang zai qian ji dui gong zai duo xi zi sha chun he siPLK1de zhong liu ba xiang wai bi ti de ti wai kang qian lie xian ai zuo yong ping jia de ji chu shang ,tong guo gou jian ren yuan qian lie xian ai luo shu pi xia yi zhi liu mo xing dui ji jin hang le ti nei ping jia 。zai ren yuan qian lie xian ai luo shu pi xia yi zhi liu mo xing shang dui gong zai duo xi zi sha chun he siPLK1de zhong liu ba xiang wai bi ti jin hang le ti nei ping jia 。zai yi zhi liu luo shu ti nei de fen bu qing kuang kao cha zhong ,you li DIRzai zhong liu bu wei fen bu hen shao ,ji zhong liu bu wei ying guang yu zhou wei zu zhi mei you ming xian cha yi ,Co-Exo/DIRzai zhong liu bu wei you yi ding de lei ji ,er Co-Exo-FA/DIRzai zhu she hou xun su de da liang fu ji zai zhong liu bu wei ,xian shi chu hen jiang de ying guang xin hao ,bing ju neng gou chi xu chao guo 24xiao shi ,bing ju zai luo shu gan zang pi zang fen bu shao ,biao ming PEGjian shao le RESdui wai bi ti de qing chu 。zhong liu yao xiao xue ping jia zheng shi le hua liao yao wu DTXhe ji yin yao wu siPLK1zai ti nei ke yi fa hui xie tong zuo yong 。kang zhong liu zeng shi zuo yong pai xu wei :Co-Exo-FA>Co-Exo>DTX-Exo>DTX>Blank Exo=>Saline。zu zhi xue yan jiu fa xian Co-Exo-FAzu de zhong liu zu zhi xi bao po huai zui ming xian hua liao yao wu DTXhe ji yin yao wu siPLK1zai ti nei you xie tong kang zhong liu zuo yong 。dui ti chong de gen zong ce liang fa xian wai bi ti ji zai yao hou de wai bi ti quan shen du xing zuo yong dou bi jiao di 。DTXzu luo shu de gan zang zu zhi chu xian yi ding cheng du de sun shang ,ji yu ge zu luo shu mei you guan cha dao ming xian de qi guan zu zhi bing li xue gai bian 。an quan xing ping jia jie guo biao ming ,DTXzhuang zai yu wai bi ti ke yi jiang di quan shen ji tong xing du xing ,Co-Exo-FAju you bi jiao hao de ti nei an quan xing 。tong guo ke ti yan jiu du chuang le hua liao yao wu he ji yin yao wu gong zhuai ran xi bao zhi bei gong zai hua liao yao wu he ji yin yao wu de wai bi ti de fang fa 。wo men gou jian de zhong liu ba xiang wai bi ti wei qu shi di kang xing qian lie xian ai he ji ta e xing zhong liu de zhi liao yan jiu di gong le xin sai lu he you yi jie jian 。
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论文作者分别是来自中国人民解放军海军军医大学的田泾，发表于刊物中国人民解放军海军军医大学2019-07-01论文，是一篇关于多西紫杉醇论文,肿瘤靶向论文,外泌体论文,前列腺癌论文,中国人民解放军海军军医大学2019-07-01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，资料来自中国人民解放军海军军医大学2019-07-01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：多西紫杉醇论文; 肿瘤靶向论文; 外泌体论文; 前列腺癌论文; 中国人民解放军海军军医大学2019-07-01论文;