【作者】 徐向上；

【导师】 龚建平；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【摘要】 肿瘤的治疗一直是临床医生和肿瘤研究专家所面临的难点,虽然现在以手术治疗、化疗以及放疗所结合的治疗手段可以延长肿瘤病人的生存率,但是其长期生存率仍然不理想,生物靶向治疗可能是一种很好的手段,自从RNA干扰技术在蠕虫身上被发现开启了RNAi的研究之后,一系列的研究在哺乳动物身上证明这种RNA干扰是通过siRNA实现的,这推动了基因调控理论的发展,也为基因研究提供了有力工具。逐渐的,RNAi技术开始应用于动物疾病模型的体内实验并且将其用于药物开发的步伐也明显加快。RNAi技术是通过激活选择性的mRNA的剪切以达到有效的基因沉默,虽然现在已经有一些基于RNAi技术的临床实验药物的出现,但不可否认,这几种尚未上市的药物都是局部使用的,而且具有各种各样的缺陷存在。目前存在于RNAi,应用面前主要的难题是采用什么样的载体来靶向的将siRNA运送到细胞里面以及这种载体的安全性问题。面对上面的三个问题,在本研究中我们设计了一种新的siRNA的载体系统,即aptamer-protamine-siRNA纳米复合物,我们首先通过合成带有FAM的无靶向基因的siRNA制成aptamer-protamine-FAMsiRNA来验证了该复合物是可以靶向的进入到细胞中去的；然后合成了针对EGFP基因的siRNA,制成aptamer-protamine-siEGFP复合物,将其加入到转染了pEGFP质粒的细胞中,发现细胞表达绿色荧光明显减少,EGFP蛋白的表达量也明显减少；然后我们合成了aptamer-protamine-sisurvivin复合物来针对凋亡抑制蛋白家族成员survivin进行干扰,发现经该复合物干扰之后的细胞survivin在mRNA和蛋白质水平都有明显的降低,并且survivin降低后细胞呈现明显的生长变慢、凋亡出现等现象；接着,我们合成了针对细胞周期M期重要激酶的CDK1的aptamer-protamine-siCDKl,发现其对于细胞有明显的细胞阻滞效应,细胞主要阻滞在G2M期,生长速度变慢；在上述的体外实验中我们证实了合成的aptamer-protamine-siRNA是一种有效的siRNA载体之后,我们通过裸鼠成瘤模型来进一步验证了该复合物是一种有效的靶向干扰制剂,对于成瘤的裸鼠进行aptamer-protamine-sisurvivin注射,可以减缓肿瘤的生长,抑制肿瘤的增殖,同时我们对于生长激素是否可以对于生物靶向治疗具有影响在肿瘤模型实验中做了部分证实,我们发现同时注射生长激素组,肿瘤的成瘤更小,这可能和生长激素可以促进肿瘤细胞的增殖进而细胞容易被靶向治疗所干预有关。通过我们的研究,我们合成了一种新的siRNA的载体,并且在体内外的实验中验证了其靶向性和有效性,这种载体有可能成为肿瘤靶向治疗的新手段。更多还原

【Abstract】 Cancer therapy is still a difficult subject for doctors and pharmacists, although some radical operations could prolong some cancer patients’5 years or 10 years survival rates, the recurrence of most cancers still need us to deal with, maybe chemotherapy and radiotherapy could make benefits for some patients, but the side effects of them, for example, the normal tissues toxicities of chemotherapy and regional tissues adhesions of radiotherapy still can not be avoided. RNAi might be a good choice for cancer therapy. RNAi therapy is a new and hot way to treat disease by targeting specified genes strongly associated with disease. From the day of discovering RNAi in Caenorhabditis elegans and subsequently the exploration of siRNA in mammalian cells, these years’researches on RNAi have acquired large achievements. It has been not only a tool of gene research in laboratory, but also a way in clinical trials for some diseases therapy. But there are still many problems in RNAi applications, the main of which is the delivery. In recent times, formulations of RNAi in vivo research are mainly as conjugated with cholesterol or aptamer, embedded by liposome or lipoplexes, complexed with peptides or polymers and fused with antibodies. Although these methods of using siRNA could meet the results of silencing specified genes, but there are still some problems to be resolved, such as lack of targeted cell characteristics or high cell membrane toxicities. So, this gives us the enthusiasm to find a safe and effective way to deliver siRNA into targeted cells.Here we designed a way to use siRNA, which is complexed with protamine and aptamer, so we called aptamer-protamine-siRNA nanoparticle. Then we performed four parts of experiments to verify the potency of the nanoparticle:(1) aptamer-parotamine-FAMsiRNA could be uptaken by cells, (2) aptamer-protamin-siEGFP could downrelgulate the expression of EGFP, (3) aptamer-protamine-sisurvivin could inhibit the expression of survivin, and the cell growth was obviously slower than the control group, some cells went to apoptosis. (4) in vivo test of the interfering effect of aptamer-protamine-siRNA in nude mice, we found that the tumors in aptamer-protamine-sisurvivin nanoparticle group were significantly smaller than vehicle group, and the expression of survivin could also be inhibited in vivo, the cell proliferation in nanoparticle group was significantly inhibited by the detection of PCNA and Ki67, also in this research we explored that growth hormone might have the ability to promote cancer cell’s proliferation which could increase the sensitivity of cancer cells to targeted therapy.Our research showed that aptamer-protamine-siRNA nanoparticle was an effective delivery to carry siRNA into cells and the siRNA could take part in the gene-silence activities. It could not only be a good delivery of siRNA in vitro tests but also be proved to be effective in vivo experiments. This nanoparticle could recognize targeted cells which express aptamer conjunctive antigens by aptamer, the siRNA it carried could inhibit the expression of specified gene, so aptamer-protamine-siRNA nanoparticle could act as a targeted RNAi delivery. This nanoparticle could be used not only on breast cancer but also on other cancers and other diseases just by changing the aptamer to another specified antigen conjunctive aptamer and replacing the siRNA to another gene siRNAs, so aptamer-protamine-siRNA nanoparticle might become a good method for gene silencing research and even for clinical diseases therapy.更多还原