摘要：本研究制备了一种氧化石墨烯（Graphene oxide，GO）纳米载药微粒，并将其作用于脂多糖（Lipopolysaccharide，LPS）诱导的奶牛乳腺上皮细胞，探究了该载药微粒对炎症的影响。实验首先在GO表面修饰TLR4抗体蛋白、羟基喜树碱和荧光素标记的胱天蛋白酶（Caspase）-3特异性底物肽（Pep-FAM）等材料合成纳米载药微粒并对其进行紫外光谱表征；之后将其作用于LPS诱导的奶牛乳腺上皮细胞炎症模型进行相关炎症因子的检测，并结合流式细胞和激光共聚焦荧光成像等技术，探究了该纳米载药微粒对炎症细胞的影响。实验结果证实我们成功制备出Caspase-3特异性识别的GO纳米载药微粒，该药物对炎症细胞有显著的促凋亡作用，能显著降低炎症细胞IL-6、TNF-α的分泌以及上清中NAGase、LDH酶的活性，对奶牛乳腺上皮细胞的炎症有一定程度的缓解作用。

关键词：GO纳米载药微粒；奶牛乳腺上皮细胞；LPS；炎症；细胞凋亡

Preparation of graphene oxide drug-loaded nanoparticles and its effect on inflammation of epithelial cells in bovine mammary gland

Abstract：In this study, a kind of graphene oxide drug-loaded nanoparticles was prepared, and its effect on lipopolysaccharide (LPS)-induced bovine mammary gland epithelial cells (MAC-T) was investigated. Firstly, TLR4 antibody protein, hydroxycamptothecin and fluorescein labeled Caspase-3 specific substrate peptide (Pep-FAM) were modified on the surface of graphene oxide and the prepared drug-loaded nanoparticles were characterized by UV spectroscopy. Then, we established an inflammation model of MAC-T based on the stimulation of LPS. After incubation of MAC-T, the effect of the prepared drug-loaded nanoparticles on inflammatory cells and the expression levels of various inflammatory factors in cell supernatants were explored at last by flow cytometry, laser confocal fluorescence imaging and other methods. Experimental results confirmed that graphene oxide drug-loaded nanoparticles specifically recognized Caspase-3 were successfully synthetized. They had a significant apoptosis-promoting effect on inflammatory cells, and can obviously reduce the secretion of IL-6 and TNF-α in inflammatory cells and the activities of NAGase and LDH in cell supernatants. As a result, we consider that graphene oxide drug-loaded nanoparticle prepared in this study have a certain degree of alleviation of inflammation reaction of MAC-T.

Key words: Graphene oxide drug-loaded nanoparticles, Bovine mammary gland epithelial cells, Lipopolysaccharide, Inflammation, Apoptosis

目  录

摘要 2

关键词 2

Abstract 2

Key words 2

1 绪论 3

1.1 奶牛乳腺炎概述 3

1.2 由脂多糖（LPS）引起的奶牛乳腺炎 3

1.2.1 LPS 3

1.2.2 模式识别受体 4

1.3 TLR4/NF-κB炎症信号通路 4

1.3.1 TLR4信号通路 4

1.3.2 NF-κB信号通路 6

1.4 氧化石墨烯（GO）纳米载药微粒 7

1.5 本文研究 8

2 材料与方法 9

2.1 主要材料和仪器 9

2.2 GO纳米载药微粒的制备及修饰 9

2.3 GO纳米载药微粒表征及检测 9

2.4 奶牛乳腺上皮细胞炎症模型的建立 .10

2.5 药物作用后流式细胞术检测细胞凋亡 10

2.6 药物作用后激光共聚焦荧光显微镜观察细胞状态 .....10

2.7 药物作用后细胞上清中炎症指标检测 .10

2.8 数据统计分析 11

3 结果与分析 11

3.1 GO纳米载药微粒表征及检测的结果与分析 11

3.2 奶牛乳腺上皮细胞炎症模型建立的结果与分析 .12

3.3 药物作用后流式细胞术检测细胞凋亡的结果与分析 .13

3.4 药物作用后激光共聚焦荧光显微镜细胞状态的观察结果与分析 ...14

3.5 药物作用后细胞上清中炎症指标检测的结果与分析 15

4 讨论 16

致谢 17

参考文献 17

氧化石墨烯纳米载药微粒的制备及其对奶牛乳腺上皮细胞炎症的影响