發(fā)布時(shí)間：2018-11-17 20:33

【摘要】： 背景與目的:機(jī)體抗腫瘤的免疫機(jī)制十分復(fù)雜,它涉及多種免疫成分,包括體液免疫和細(xì)胞免疫。它們互相協(xié)同殺傷腫瘤細(xì)胞。一般認(rèn)為,以T細(xì)胞、NK細(xì)胞、巨噬細(xì)胞和樹(shù)突狀細(xì)胞為主的細(xì)胞免疫發(fā)揮著重要的作用。如果能研究一種可靶向樹(shù)突狀細(xì)胞或者巨噬細(xì)胞的給藥系統(tǒng),則有望提高抗原靶向樹(shù)突狀細(xì)胞或者巨噬細(xì)胞的能力,增加抗原提呈,促進(jìn)免疫應(yīng)答,增強(qiáng)抗腫瘤作用。本文根據(jù)樹(shù)突狀細(xì)胞和巨噬細(xì)胞表面有甘露糖受體的表達(dá),制備了含有模型蛋白(卵清蛋白,OVA)的甘露糖修飾的殼聚糖包衣的乳酸-羥基乙酸共聚物(PLGA)納米粒,研究其靶向樹(shù)突狀細(xì)胞和巨噬細(xì)胞的能力。 方法:合成了甘露糖修飾的殼聚糖之后,采用復(fù)乳法制備了含有OVA的PLGA納米粒,殼聚糖(CS)以及甘露糖修飾的殼聚糖(MAN-CS)包衣的PLGA納米粒。研究了不同因素,對(duì)納米粒粒徑、載藥量和體外釋放的影響。接著考察了納米粒的濃度和孵育時(shí)間對(duì)于巨噬細(xì)胞的毒性的影響,以及通過(guò)熒光酶標(biāo)儀、熒光顯微鏡、流式細(xì)胞儀和共聚焦顯微鏡考察不同孵育溫度、時(shí)間、濃度,甘露糖對(duì)于巨噬細(xì)胞攝取的影響,和納米粒在細(xì)胞內(nèi)的定位。最后分離小鼠骨髓的單個(gè)核細(xì)胞,體外誘導(dǎo)其分化形成樹(shù)突狀細(xì)胞;通過(guò)流式細(xì)胞儀分析樹(shù)突狀細(xì)胞對(duì)納米粒的攝取,以及激光共聚焦顯微鏡(CLSM)考察納米粒在細(xì)胞內(nèi)的定位。 結(jié)果:通過(guò)紅外光譜、核磁圖以及元素分析確認(rèn)了已合成出甘露糖修飾的殼聚糖。PLGA納米粒制備的最優(yōu)處方為內(nèi)水相pH值4.0,PLGA濃度為30mg·mL-1,外水相PVA濃度為5%,油水相比為1:3,超聲次數(shù)為120次。使用最優(yōu)處方制得的納米粒粒徑在255nm左右,OVA的包封率約為70%,載藥量約為8%。CS包衣納米粒的粒徑和zeta電位,隨著CS濃度的增加而增加。在內(nèi)水相中加入PEG1000、外水相添加蔗糖、CS和MAN-CS包衣均能較少PLGA納米粒的突釋。納米粒對(duì)巨噬細(xì)胞基本不產(chǎn)生毒性。4℃時(shí),巨噬細(xì)胞的吞噬受到抑制;8h時(shí),巨噬細(xì)胞對(duì)于納米粒的攝取已接近飽和;在實(shí)驗(yàn)的濃度范圍內(nèi),巨噬細(xì)胞對(duì)于游離的FITC-OVA和納米粒的攝取是逐漸增加的。甘露糖能抑制了MAN-CS包衣的PLGA納米粒的攝取。熒光酶標(biāo)儀、流式細(xì)胞儀結(jié)果表明,巨噬細(xì)胞對(duì)MAN-CS包衣的納米粒攝取最多;CLSM表明納米粒的主要定位在細(xì)胞質(zhì)內(nèi)。小鼠骨髓的單個(gè)核細(xì)胞在體外用GM-CSF和IL-4誘導(dǎo)可分化為未成熟樹(shù)突狀細(xì)胞,其表面高表達(dá)CD11c;流式細(xì)胞儀的結(jié)果表明,樹(shù)突狀細(xì)胞對(duì)含有FITC-OVA納米粒的攝取比游離的FITC-OVA多,且對(duì)MAN-CS包衣的納米粒攝取最多。 結(jié)論:本文通過(guò)單因素考察確定了PLGA納米粒制備的最優(yōu)處方,使用此處方制備納米粒重現(xiàn)性好;以PLGA納米粒制備的最優(yōu)處方為基礎(chǔ),采用復(fù)乳法成功地制備出含有OVA的CS以及MAN-CS包衣的PLGA納米粒。MTT實(shí)驗(yàn)表明納米粒的毒性較低。體外細(xì)胞實(shí)驗(yàn)表明MAN-CS包衣的PLGA納米粒能有效地被樹(shù)突狀細(xì)胞和巨噬細(xì)胞攝取。
[Abstract]:BACKGROUND & OBJECTIVE: The anti-tumor immune mechanism of the body is very complex, and it involves many kinds of immune components, including humoral immunity and cell immunity. They co-act with each other to kill tumor cells. It is generally believed that cellular immunity, which is dominated by T cells, NK cells, macrophages and dendritic cells, plays an important role. if a drug delivery system is capable of targeting dendritic cells or macrophages, it is expected to improve that ability of the antigen to target the dendritic cell or the macrophage, increase the antigen presentation, promote the immune response, and enhance the anti-tumor effect. according to the expression of the mannose receptor on the surface of the dendritic cells and the macrophages, the lactic acid-glycolic acid copolymer (PLGA) nanoparticles containing the mannose-modified chitosan-coated chitosan coated with the model protein (ovalbumin, OVA) are prepared, The ability to target dendritic cells and macrophages was investigated. Methods: After the chitosan-modified chitosan was synthesized, PLGA nanoparticles, chitosan (CS) and mannoose-modified chitosan (MAN-CS)-coated PLG were prepared by complex emulsion method. A nanoparticles were studied. The particle size, drug loading and in vitro release of the nanoparticles were studied. The effects of the concentration of the nanoparticles and the incubation time on the toxicity of the macrophages were then examined, and the different incubation temperatures, time, concentration, mannose for macrophages were examined by means of a fluorescence enzyme marker, a fluorescence microscope, a flow cytometer, and a confocal microscope. the effect of taking, and the nano-particles in the cell, In vitro, the individual nuclear cells of the bone marrow of the mouse were isolated and their differentiation was induced to form a dendritic cell. The uptake of the nanoparticles by the dendritic cells was analyzed by flow cytometry, and the nano-particles were examined by a laser confocal microscope (CLSM). Internal positioning. Result: The synthesized output is confirmed by the infrared spectrum, the nuclear magnetic diagram, and the element analysis the optimal formulation prepared by the mannoose modified chitosan and the PLGA nano-particle is the internal water phase pH value of 4.0, the PLGA concentration is 30mg/ mL-1, the concentration of the external water phase PVA is 5%, the oil-water ratio is 1: 3, The ultrasonic frequency was 120 times. The particle size of the nanoparticles was about 255nm, the encapsulation efficiency of OVA was about 70%, and the drug loading was about 8%. The particle size and zeta potential of the CS-coated nanoparticles were as follows: The increase of S concentration was increased. The addition of PEG1000 and the addition of sucrose, CS and MAN-CS in the internal water phase could be less P The release of the LGA nanoparticles. The nanoparticles were substantially free of toxicity to the macrophages. At 4 鈩，

本文編號(hào)：2338966