【作者】 张潇；

【导师】 宋淑亮；

【作者基本信息】 山东大学 ， 药学， 2025， 硕士

【摘要】 恶性肿瘤对全球人类的健康构成了严重威胁,是导致人类死亡的主要原因之一。化学药物治疗作为治疗恶性肿瘤的有效方法,在临床中被广泛应用。但化疗药物依然存在着细胞毒性过高、靶向性较差的缺陷,在杀伤患者体内肿瘤细胞的同时容易损害患者的免疫系统,降低免疫细胞存活率,从而减弱机体的抵抗力。脂多糖(lipopolysaccharides,LPS)是革兰氏阴性菌外膜的组成部分,可激活机体免疫细胞对抗外来物质的强烈免疫反应。本课题首次发现在肿瘤微环境中,化疗药物联合LPS可以在不影响化疗药物杀伤肿瘤细胞的同时,减轻化疗药物对巨噬细胞的损伤,提升巨噬细胞的存活率,这对于提高患者的免疫力具有重要意义。本课题延续前期的研究基础,旨在探究LPS对化疗药物损伤巨噬细胞保护作用的构效关系、作用途径和位点。主要实验内容及结果如下:1.脂质A是LPS发挥保护化疗药物损伤巨噬细胞作用的主要结构。通过CCK-8实验探究不同浓度的LPS对化疗药物损伤的不同细胞的作用,以及LPS的构效关系。研究表明,100-1000 ng/mLLPS均可对阿霉素(adriamycin,ADR)/长春新碱(vincristine,VCR)损伤的小鼠单核巨噬细胞RAW264.7有保护作用。LPS对ADR损伤的大鼠肺泡巨噬细胞NR8383、原代小鼠腹腔巨噬细胞等巨噬细胞具有保护作用,且不影响ADR对小鼠乳腺癌细胞4T1、小鼠结肠癌细胞MC38等肿瘤细胞的杀伤作用,肿瘤微环境中也验证了此结果。研究表明,不同来源具有共同脂质A结构的LPS对ADR损伤的RAW264.7巨噬细胞具有保护作用。高压蒸汽灭活失去脂质A活性的LPS对ADR损伤的RAW264.7巨噬细胞保护作用下降。单独添加外源性的脂质A对ADR损伤的RAW264.7巨噬细胞具有LPS样保护作用,且与LPS的保护作用相比无显著性差异。综上,脂质A是LPS发挥保护化疗药物损伤巨噬细胞作用的主要结构。2.LPS通过吞噬和胞饮途径进入胞内发挥对化疗药物损伤巨噬细胞的保护作用。将FITC标记LPS,通过胞吞抑制剂检测FITC荧光,探究LPS进入胞内的途径。研究表明,能量抑制前,激光共聚焦显微镜和流式细胞仪均可检测到RAW264.7巨噬细胞内的FITC荧光,即LPS可以进入到胞内且过程种需要能量。吞噬抑制剂Cytochalasin D能够显著降低RAW264.7巨噬细胞的吞噬作用、LPS对ADR/VCR损伤的RAW264.7巨噬细胞的保护作用和胞内的LPS含量。四种不同作用机制的胞饮抑制剂(CPZ、MβCD、Dynasore和EIPA)也能够不同程度降低LPS对ADR/VCR损伤的RAW264.7巨噬细胞的保护作用和胞内的LPS含量,其中MβCD的抑制作用较强。综上,LPS通过吞噬和胞饮途径进入胞内发挥对化疗药物损伤巨噬细胞的保护作用。3.溶酶体是LPS发挥对化疗药物损伤巨噬细胞的保护作用的位点。微管蛋白抑制剂Colchicine显著减弱LPS的保护作用,即LPS在胞内通过微管蛋白分布和运输。通过转录组测序、Western Blot和荧光共定位探究LPS发挥保护作用的位点。研究表明,LPS使胞吞作用、溶酶体相关基因发生了显著变化,FITC标记的LPS与Lyso Tracker Red标记的溶酶体荧光共定位散点图和皮尔森系数表明二者有较大的相关性。qRT-PCR和Western Blot检测到LPS显著上调溶酶体相关基因和蛋白如NPC1、NPC2、M6PR、VATP(ATP6v1h 和 ATP6v0b)、LAMP2 等。溶酶体抑制剂 Chloroquine显著减弱LPS的保护作用。由此说明,LPS发挥保护化疗药物损伤的RAW264.7巨噬细胞的作用位点为溶酶体。进一步实验证明吞噬、胞饮抑制剂和溶酶体抑制剂能够显著降低LPS与溶酶体的共定位皮尔森系数和溶酶体相关蛋白的表达。综上,LPS在胞内经微管蛋白运输至溶酶体发挥对化疗药物损伤巨噬细胞的保护作用。综上,LPS在肿瘤微环境中对ADR损伤的RAW264.7巨噬细胞具有保护作用,且不影响ADR对肿瘤细胞的杀伤作用。脂质A是LPS发挥保护化疗药物损伤巨噬细胞作用的主要结构。LPS经吞噬和胞饮途径进入RAW264.7巨噬细胞内,经微管蛋白运输至溶酶体,激活溶酶体相关蛋白发挥对化疗药物损伤的RAW264.7巨噬细胞的保护作用。本课题对LPS保护作用的构效关系、途径和位点进行了研究,为深入寻找LPS保护作用的具体靶点提供基础理论支持,表明其在恶性肿瘤化疗中提高患者免疫力的应用前景。更多还原

【Abstract】 Malignant tumors pose a serious threat to the health of the global population and are one of the major causes of death in humans around the world.Chemical therapy is widely used in clinical practice as an effective method to treat malignant tumors.However,chemotherapeutic drugs still have the defects of excessive cytotoxicity and poor targeting.While killing tumor cells in patients,they will also damage the patient’s immune system and reduce the survival rate of immune cells,thereby weakening the body’s resistance.Lipopolysaccharides(LPS)are part of the outer membrane of Gram-negative bacteria and activate the body’s immune cells to resist strong immune responses against foreign substances.This study found for the first time that in the tumor microenvironment,chemotherapy drugs combined with LPS can reduce the damage caused by chemotherapy drugs to macrophages and improve the survival rate of macrophages without affecting the killing of tumor cells by chemotherapeutic drugs.This topic continues the previous research basis and aims to explore the structure-activity relationship,pathway and site of LPS on the protective effect of chemotherapy drugs on macrophages.The main experimental contents and results are as follows:1.Lipid A is the main structure in which LPS plays a role in protecting macrophages damaged by chemotherapy drugs.CCK-8 experiments were used to explore the effects of different concentrations of LPS on different cells injured by chemotherapeutic drugs,and the structure-activity relationship.Studies have shown that 100-1000ng/mL LPS can protect mouse monocyte macrophage RAW264.7 injured by adriamycin(ADR)/vincristine(VCR).LPS has a protective effect on macrophages such as rat alveolar macrophages NR8383 and primary mouse peritoneal macrophages injured by ADR,and does not affect the killing effect of ADR on mouse breast cancer cells 4T1,mouse colon cancer cells MC38,and other tumor cells.This result was also verified in the tumor microenvironment.Studies have shown that LPS with a common lipid A structure from different sources has protective effects on ADR-damaged RAW264.7 macrophages.High-pressure steam inactivates LPS that loses lipid A activity,and reduces the protective effect of RAW264.7 macrophages caused by ADR damage.The addition of exogenous lipid A alone had an LPS-like protective effect on ADR-damaged RAW264.7 macrophages,and there was no significant difference compared with the protective effect of LPS.In summary,lipid A is the main structure that LPS plays a role in protecting macrophages from damage caused by chemotherapeutic drugs.2.LPS enters the cell through phagocytosis and pinocytosis pathways to exert a protective effect on macrophages damaged by chemotherapy drugs.FITC was labeled with LPS,and FITC fluorescence was detected by endocytosis inhibitors to explore the pathways of LPS entering the cell.Studies have shown that before energy inhibition,laser confocal microscopy and flow cytometry can detect FITC fluorescence in RAW264.7 macrophages,that is,LPS can enter the cell and the process species requires energy.The phagocytosis inhibitor CytochalasinD can significantly reduce the phagocytosis of RAW264.7 macrophages,the protective effect of LPS on ADR/VCR-damaged RAW264.7 macrophages,and the intracellular LPS content.Four different mechanisms of action pill cytoplasm inhibitors(CPZ,MβCD,Dynasore and EIPA)can also reduce the protective effect and intracellular LPS content of RAW264.7 macrophages damaged by ADR/VCR to varying degrees,among which MβCD has a strong inhibitory effect.In summary,LPS enters the cell through phagocytosis and pinocytosis to exert a protective effect on macrophages damaged by chemotherapeutic drugs.3.Lysosome is the site where LPS exerts a protective effect on macrophages damaged by chemotherapeutic drugs.The tubulin inhibitor Colchicine significantly attenuates the protective effect of LPS,i.e.LPS is distributed and transported through tubulin intracellularly.The sites where LPS play a protective role were explored by transcriptome sequencing,WesternBlot and fluorescence colocalization.Studies have shown that LPS has significantly changed endocytosis and lysosome-related genes.The FITC-labeled LPS and LysoTrackerRed-labeled lysosome fluorescence colocalization scatter plot and Pearson coefficient show that the two have a large correlation.qRT-PCR and WesternBlot detected that LPS significantly upregulates lysosomerelated genes and proteins such as NPC1,NPC2,M6PR,VATP(ATP6v1h and ATP6v0b),LAMP2,etc.The lysosomal inhibitor Chloroquine significantly attenuates the protective effect of LPS.This shows that the site of action of LPS to protect chemotherapy drugs is lysosomes.Further experiments showed that phagocytosis,pinocytosis inhibitors and lysosomal inhibitor Chloroquine could significantly reduce the Pearson coefficient of colocalization of LPS and lysosomes and the expression of lysosome-related proteins.In summary,LPS transported to lysosomes through tubulin intracellularly plays a protective role in chemotherapy drugs to damage macrophages.In summary,LPS can protect RAW264.7 macrophages damaged by ADR in the tumor microenvironment,and does not affect the killing effect of ADR on tumor cells.Lipid A is the main structure of LPS to protect macrophages from damage caused by chemotherapeutic drugs.LPS can enter the cell through the phagocytosis and pinocytosis of RAW264.7 macrophages,and transport to lysosomes through tubulin,activating lysosomal-related proteins to exert a protective effect on RAW264.7 macrophages damaged by chemotherapeutic drugs.This topic studies the structure-activity relationship,pathway and site of LPS protection,providing basic theoretical support for in-depth search for specific targets of LPS protection,and shows its application prospects for improving patients’ immunity in malignant tumor chemotherapy.更多还原