本文選題：聯(lián)合轉(zhuǎn)運(yùn)　切入點(diǎn)：表阿霉素　出處：《青島大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：在腫瘤治療中,內(nèi)源性或獲得性的多藥耐藥是化療過程中的主要障礙,其降低藥物在細(xì)胞內(nèi)的積累或增強(qiáng)腫瘤細(xì)胞DNA損傷修復(fù)機(jī)制,導(dǎo)致單種藥物治療腫瘤的效率降低�；蛩幬锟梢砸种萍�(xì)胞多藥耐藥的發(fā)生,但是遞送過程面臨較多困難,因此選擇合適的載體,將基因與化療藥物聯(lián)合共遞送至腫瘤組織,以發(fā)揮藥效,是治療腫瘤的一個(gè)新策略。本課題制備了一種聯(lián)合轉(zhuǎn)運(yùn)si RNA與表阿霉素(EPI)的p H敏感長循環(huán)逆轉(zhuǎn)多藥耐藥多功能信封式納米載體(Multifunction Envelop-type Nano Device,MEND)。MEND是以壓縮的DNA、si RNA等為核心,用含有功能性配體的脂質(zhì)體包裹而成的類似信封式外殼的納米微球,兼具了納米粒和脂質(zhì)體的優(yōu)點(diǎn)。選用聚乙二醇(PEG)連接至磷脂分子二硬脂�；字Ｒ掖及�(DSPE)上形成的共聚物DSPE-PEG,實(shí)現(xiàn)長循環(huán)的目的。通過邁克爾加成反應(yīng),合成具有酸敏感性能的含縮酮鍵的聚β氨基酯結(jié)構(gòu)(KPAE),其通過正負(fù)電結(jié)合作用,縮合si RNA;在細(xì)胞酸性環(huán)境中,KPAE的縮酮建斷裂,實(shí)現(xiàn)si RNA的智能釋放。BCL-2-si RNA通過調(diào)低P糖蛋白(P-gp)的表達(dá)和促進(jìn)細(xì)胞凋亡,逆轉(zhuǎn)細(xì)胞多藥耐藥的發(fā)生,協(xié)同表阿霉素起到治療腫瘤的作用。各成分通過核磁共振、凝膠滲透色譜進(jìn)行結(jié)構(gòu)鑒定;本實(shí)驗(yàn)選用薄膜分散法制備復(fù)合物載體,EPI包裹在脂質(zhì)體薄膜的磷脂雙分子層疏水端,KPAE/si RNA縮合物溶液水化脂質(zhì)體薄膜后,得到EPI/si RNA-MEND。利用透射電鏡觀察其形態(tài);馬爾文激光粒度儀測定其粒徑及電位;瓊脂糖凝膠電泳實(shí)驗(yàn)考察復(fù)合物對的si RNA縮合能力;分別采用濾膜法-高效液相色譜法和超濾離心法,測定EPI和si RNA的包封率;實(shí)驗(yàn)細(xì)胞選用人肝癌Hep G_2細(xì)胞,選用BCL-2-si RNA(si BCL-2)制備EPI/si BCL-2-MEND,采用MTT法,考察EPI/si BCL-2-MEND對肝癌Hep G_2細(xì)胞存活率的影響;采用熒光標(biāo)記法,分別用FAM標(biāo)記si RNA,Lyso Tracker-Blue DND-22標(biāo)記溶酶體,Hoechst 33342標(biāo)記細(xì)胞核,考察EPI/FAM-si RNA-MEND在肝癌Hep G_2細(xì)胞的分布以及溶酶體逃逸現(xiàn)象;采用流式細(xì)胞法,考察EPI/FAM-si RNA-MEND對肝癌Hep G_2細(xì)胞的轉(zhuǎn)染效率;用EPI/si BCL-2-MEND,對Hep G_2細(xì)胞轉(zhuǎn)染,考察P-糖蛋白(P-gp)的表達(dá)。采用小動(dòng)物活體成像技術(shù),考察EPI/Cy5-si RNA-MEND的體內(nèi)分布。結(jié)果表明,成功合成了所需的各目標(biāo)產(chǎn)物;MEND呈規(guī)則的球形且具有明顯的脂質(zhì)雙分子層結(jié)構(gòu)和指紋結(jié)構(gòu);EPI/si RNA-MEND平均粒徑為121.6 nm,平均電位為+41.5 m V;載體復(fù)合物對EPI和si RNA的包封率分別為86.13%和97.07%;MEND在一定給藥劑量范圍內(nèi)毒性較小,對細(xì)胞存活率幾乎無影響;EPI/si BCL-2-MEND高于同濃度下單獨(dú)藥物的腫瘤細(xì)胞抑制率,并具有較高的轉(zhuǎn)染效率;相比于游離的EPI組和EPI-MEND組,EPI/si BCL-2-MEND可顯著下調(diào)P-gp的表達(dá);EPI/Cy5-si RNA-MEND相比單獨(dú)藥物更容易富集于肝臟中,腎臟內(nèi)幾乎無分布,證明其具有長循環(huán)性能。所制備的載體復(fù)合物能夠有效地將si RNA與EPI共遞送至腫瘤細(xì)胞中,實(shí)現(xiàn)高效率的轉(zhuǎn)染,并實(shí)現(xiàn)si RNA在細(xì)胞內(nèi)的智能釋放,具有良好的使用前景。
[Abstract]:In the treatment of cancer, or endogenous multidrug resistance is the main obstacle in the process of chemotherapy, reduce the accumulation of intracellular drug or enhance repair mechanisms of tumor cell DNA damage, which leads to the decrease of the efficiency of single drug therapy. Gene therapy can inhibit cell multidrug resistance, but the delivery process is facing more difficult, so choose the appropriate carrier gene combined with chemotherapy drugs were delivered to the tumor tissue, to take effect, is a new strategy for the treatment of cancer. The preparation of a joint Si RNA and transport of adriamycin (EPI) multidrug resistance P H sensitive long cycle reversal function envelope type nano carrier (Multifunction Envelop-type Nano Device, MEND.MEND) which is compressed with DNA, Si RNA as the core, nanoparticles similar envelope type shell with liposome containing functional ligands and the both The nanoparticles and liposomes. The advantages of polyethylene glycol (PEG) connected to the phospholipid molecules two stearoyl phosphatidyl ethanolamine (DSPE) copolymer DSPE-PEG on the formation of long circulation purposes. Through Michael addition reaction of poly beta amino ester bond structure containing ketal synthesis with acid sensitive properties (KPAE) through the combination of positive and negative electrical, Si, RNA in cell condensation; in acidic environment, KPAE ketal built fracture, realize the release of.BCL-2-si RNA by reducing the P glycoprotein (P-gp) Si intelligent RNA expression and promote apoptosis of MDR cells, play a synergistic anti-tumor effect of epirubicin. The components of the structures were identified by NMR, gel permeation chromatography; this experiment selected films by complex carrier dispersion method, EPI wrapped in liposome membrane phospholipid bilayer hydrophobic KPAE/si RNA condensate water solution Liposome film, EPI/si RNA-MEND. were observed by transmission electron microscope; Malvin laser particle size analyzer to determine the particle size and zeta potential; agarose gel electrophoresis of complexes of Si RNA condensation ability; method and centrifugal ultrafiltration HPLC respectively by membrane filtration method, determination of EPI and Si RNA encapsulation the experimental cell rate; human hepatocellular carcinoma Hep G_2 cells, using BCL-2-si RNA (Si BCL-2) EPI/si was prepared by BCL-2-MEND, using the MTT method, the effect of EPI/si BCL-2-MEND survival rate was investigated on hepatocellular carcinoma Hep G_2 cells; using fluorescence labeling, respectively labeled with FAM Si RNA, Lyso Tracker-Blue DND-22 Hoechst 33342 labeled lysosomes, marked nucleus. The effects of EPI/FAM-si RNA-MEND in the distribution of escape phenomenon hepatocellular carcinoma Hep G_2 cells and lysosomes; using flow cytometry, investigate the transfection efficiency of EPI/FAM-si RNA-MEND on hepatocellular carcinoma Hep G_2 cells; EPI/ si BCL-2-MEND,瀵笻ep G_2緇嗚優(yōu)杞煋,鑰冨療P-緋栬泲鐧，

本文編號(hào)：1611879