本文選題：主動靶向　切入點：小核酸　出處：《吉林大學》2017年碩士論文　論文類型：學位論文

【摘要】：在過去的數(shù)十年間,隨著對癌癥發(fā)展分子機制的不斷探索,靶向型基因遞送體系應(yīng)運而生,其中一些納米藥物已經(jīng)投入到臨床使用中,其他很多納米基因藥物也已進入臨床試驗階段。為了提高藥物的安全性、可靠性,解決藥物的溶解度低、對身體正常部位產(chǎn)生毒副作用、生物學不穩(wěn)定、無法跨越生理屏障等問題,實現(xiàn)納米體系的主動靶向性,減少網(wǎng)狀內(nèi)皮吞噬系統(tǒng)(RES)對納米載體的捕獲,使納米體系可以通過腫瘤細胞表面的受體進行受體-配體相互作用而進入細胞,實現(xiàn)主動靶向治療成為基因藥物研究的重中之重。基于上述原因,本論文通過EDC/NHS法,將能夠特異性識別癌胚抗原6的單域抗體修飾到高分子納米材料PEN表面,構(gòu)建一種具有腫瘤特異靶向性的高分子納米載體PEN-Nb。該載體對miR-34a有良好的結(jié)合能力,同時可以通過腫瘤細胞表面的癌胚抗原6受體進行受體-配體相互作用而進入細胞,在提高生物學穩(wěn)定性的同時提高轉(zhuǎn)染效率。miR-34a在胞內(nèi)充分發(fā)揮生物學效應(yīng),抑制了腫瘤細胞MCF-7的增殖,促進其產(chǎn)生凋亡,使細胞周期阻滯在G1~S期,同時也抑制了腫瘤細胞的遷移浸潤。為了進一步研究主動靶向性載體在活體水平上的治療效率,我們以BALB/c-nu裸鼠構(gòu)建異種移植瘤肺癌模型,以天然多糖硫酸軟骨素對PAMAM進行修飾合成CD44靶向性納米載體CS-PAMAM,以此遞送小核酸miR-34a,觀察該納米遞送體系在動物水平上的靶向性及對腫瘤的治療效果。CS-PAMAM在細胞水平上有良好的生物相容性及CD44靶向性,在動物實驗結(jié)果中,以CS-PAMAM遞送miR-34a能夠有效抑制腫瘤體積的增長,通過組織切片觀察可以發(fā)現(xiàn)CS-PAMAM/miR-34a組有明顯的原位凋亡的產(chǎn)生,并且該納米體系并沒有對小鼠的其他臟器產(chǎn)生任何的毒副作用。藥物體內(nèi)分布實驗證明CS-PAMAM能夠使miR-34a最大程度的在腫瘤部位蓄積,使其充分發(fā)揮生物學功能。綜上,這兩種腫瘤靶向型納米基因遞送體系均能通與過腫瘤細胞表面的受體進行受體-配體相互作用實現(xiàn)主動靶向,從而實現(xiàn)小核酸miR-34a的高效遞送,充分發(fā)揮小核酸miR-34a的生物學功能,在細胞水平和動物水平上抑制腫瘤的增殖與遷移。以上研究,為構(gòu)建主動靶向型納米基因遞送體系提供了新的策略,證明了靶向型納米基因遞送體系在腫瘤治療領(lǐng)域的巨大潛力,為未來的臨床應(yīng)用奠定了良好的基礎(chǔ)。
[Abstract]:Over the past few decades, as the molecular mechanisms of cancer development have been explored, targeted gene delivery systems have emerged, and some of these nanopharmaceuticals have been put into clinical use. Many other nanocrystalline drugs have also entered the clinical trial stage. In order to improve the safety and reliability of drugs, solve the low solubility of drugs, have toxic side effects on normal parts of the body, biological instability, It is impossible to cross the physiological barrier to achieve the active targeting of nano-system and reduce the capture of nano-carrier by reticular endothelial phagocytosis system (RES). So that the nanoscale system can enter the cell through receptor ligand interaction on the surface of tumor cells and realize active targeting therapy has become the most important part of gene drug research. Based on the above reasons, the EDC/NHS method is adopted in this paper. A novel polymer nano-carrier PEN-Nb, which can specifically recognize carcinoembryonic antigen 6, was constructed by modifying it onto the surface of the polymer nano-material PEN. The carrier has good binding ability to miR-34a. At the same time, the receptor-ligand interaction can be carried out by carcinoembryonic antigen 6 receptor on the surface of tumor cells, which not only improves the biological stability but also increases the transfection efficiency. MiR-34a exerts the full biological effect in the cell. It inhibited the proliferation of MCF-7, promoted its apoptosis, blocked the cell cycle in G1S phase, and also inhibited the migration and infiltration of tumor cells. In order to further study the therapeutic efficiency of active targeting vector at the in vivo level, We used BALB/c-nu nude mice to construct xenotransplantation tumor lung cancer model. PAMAM was modified with natural polysaccharide chondroitin sulfate to synthesize CD44 targeting nano-carrier CS-PAMAM, which was used to deliver small nucleic acid miR-34a. The targeting of the nano-delivery system at the animal level and the therapeutic effect of CS-PAMAM on tumor were observed at the cell level. Good biocompatibility and CD44 targeting. In animal experiments, CS-PAMAM delivery of miR-34a could effectively inhibit the growth of tumor volume, and in situ apoptosis could be found in CS-PAMAM/miR-34a group by histological observation. And the nanoparticles did not produce any toxic side effects on other organs of mice. The drug distribution in vivo showed that CS-PAMAM can make miR-34a accumulate in tumor sites to the greatest extent, so that it can give full play to its biological function. Both of these two tumor targeting nano-gene delivery systems can actively target the receptor ligand interaction with receptors passing through the surface of tumor cells, thus realizing the efficient delivery of small nucleic acid miR-34a and giving full play to the biological functions of small nucleic acid miR-34a. Inhibition of tumor proliferation and migration at the cellular and animal levels provides a new strategy for the construction of active targeting nanogene delivery systems. It is proved that the targeting nano gene delivery system has great potential in the field of tumor therapy, which will lay a good foundation for clinical application in the future.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R730.5

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本文編號：1600971