【作者】 陈虎；

【导师】 夏虹；

【作者基本信息】 南方医科大学 ， 外科学（骨外）（专业学位）， 2021， 硕士

【摘要】 目的:针对脓毒症治疗中清除致病菌和抑制炎症过度反应的临床需求,构建同时具备良好抗菌及抗炎特性的氧化铜/抗菌多肽/细胞膜(CuO-Tcp25-CM)纳米材料,并分别评价其生物相容性、体外抗菌性能、体外抗炎性能以及对小鼠脓毒症模型的干预效果进行全面评估。方法:1.利用氯化亚铜(Cucl)、聚乙烯吡咯烷酮(PVP)和谷胱甘肽(GSH)合成氧化铜(CuO),然后将抗菌多肽(Tcp-25)通过1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)/N-羟基琥珀酰亚胺(NHS)接枝到材料表面,并包被小鼠骨髓间充质干细胞的细胞膜,最终合成CuO-Tcp25-CM。使用高分辨率的透射电镜(TEM)、傅立叶变换红外吸收光谱仪(FTIR)、X射线衍射仪(XRD)、Zeta电位测量仪检等测试材料的理化特性。2.通过CCK-8法、细胞死活染色法、溶血实验评估材料的生物相容性。3.通过材料分别与金黄色葡萄球菌(S菌)、大肠杆菌(E菌)以及耐甲氧西林金黄色葡萄球菌(M菌)共培养后涂板结果评估CuO-Tcp25-CM材料的抗菌性能。4.通过实时荧光定量PCR(qPCR)和酶联免疫吸附剂测定(ELISA)技术评估材料对炎症因子(TNF-α、IL-6、IL-10和IL-1 β)影响,通过qPCR和蛋白免疫印迹(WB)技术评估材料对巨噬细胞极化的影响,通过ELISA和免疫荧光染色评估材料对E-Selectin表达的影响。5.通过构建脓毒症小鼠模型,评估材料的体内抗对脓毒症的干预效果。结果:1.本实验成功构建CuO-Tcp25-CM纳米颗粒。通过电镜扫描可见CuO呈梭形状态,FTIR显示材料的官能区位于3300-3600cm-1,表明存在羟基(-OH),指纹区位于400-700 cm-1。XRD图谱提示样品的晶体特征峰与CuO的标准卡片特征峰位置基本重合。Zeta电位结果为-12mV,表明CuO-Tcp25-CM纳米材料倾向于凝结,抗菌肽及细胞膜成功包被在CuO表面。2.CuO-Tcp25-CM纳米颗粒,具有良好的生物相容性。细胞毒性与浓度呈现正相关,当浓度为10 μg/mL时,在共培养1天及7天对小鼠成纤维细胞无明显毒性。体外溶血实验发现,浓度在200 μg/mL时,材料无急性溶血反应。3.CuO-Tcp25-CM纳米颗粒体外具有良好的抗菌效能。抗菌效果与浓度呈现正相关。抗菌种类来说,对E菌抑制作用最强,S菌次之,M菌最差。当浓度为 10 μ g/mL 时,抑菌率分别为:S 菌:97.90±1.74%、E 菌:99.55±0.45%、M菌:90.75±2.99%,均具有较强抑菌效果。4.CuO-Tcp25-CM纳米颗粒体外具有良好的抗炎效能。10 μg/mL的材料能有效下调促炎因子(TNF-α、IL-1β、IL-6)的表达,上调抑炎因子(IL-10)的表达。此外,材料还可以有效的抑制E-Selectin的表达,影响下游的炎症反应。可促进巨噬细胞从M1型极化向M2型极化,从而影响炎症反应状态。5.体内实验发现CuO-Tcp25-CM纳米材料能有效的提高脓毒症小鼠的生存率,对炎症反应有一定抑制作用,减少肝肾功能损伤。重要器官切片观察表明该材料无明显生物毒性,具有良好的体内生物安全性。结论:CuO-Tcp25-CM纳米颗粒具有良好的生物相容性,同时具备良好的体外抗菌及抗炎功能,在体内小鼠脓毒症模型中能显著提高小鼠生存率,减轻全身炎症反应。本研究为抗脓毒症生物材料的研究提供了新的思路,为临床脓毒症的治疗提供了新的策略。更多还原

【Abstract】 OBJECTIVE:To construct nanomaterials with good antibacterial and anti-inflammatory prope Objective:In response to the clinical needs of removing pathogenic bacteria and inhibiting excessive inflammation in the treatment of sepsis,we aimed to construct copper oxide/antibacterial peptide/cell membrane(CuO-Tcp25-CM)nanomaterials with both good antibacterial and anti-inflammatory properties.Its characterization,biocompatibility,in vitro antibacterial properties,in vitro anti-inflammatory properties preliminary efficacy in mice sepsis model were done respectively.Methods:1.Using cuprous chloride(Cucl),polyvinylpyrrolidone(PVP)and glutathione(GSH)to synthesize copper oxide(CuO),and then complex the antibacterial polypeptide(Tcp-25)through EDC/NHS is grafted onto the surface of the material and coated on the cell membrane of mouse bone marrow mesenchymal stem cells(BMSC).Using TEM,FTIR,XRD and Zeta potential to measure its characterization.2.Evaluate the its biocompatibility by CCK-8 method,cell live/death staining and hemolysis test.3.Evaluate the antibacterial properties by co-cultivating the materials with Staphylococcus aureus(S),Escherichia coli(E)and methicillin-resistant Staphylococcus aureus(M)and coating results.4.Evaluate the influence on inflammatory factors(TNF-α,IL-6,IL-10 and IL-1β)by real-time fluorescent quantitative PCR(qPCR)and enzyme-linked immunosorbent assay(ELISA)techniques.And qPCR and Western Blot(WB)was used to evaluate the influence on the polarization of macrophages.The influence on the expression of E-Selectin was evaluated by ELISA and immunofluorescence staining.5.By constructing a sepsis mouse model,to assess the material’s in vivo anti-intervention effect on sepsis.Results:1.This experiment successfully constructed CuO-Tcp25-CM nanoparticles.HRTEM showed that CuO was in a fusiform state.FTIR showed that the functional area of the material was located at 3300-3600 cm-1,indicating the presence of hydroxyl(-OH),and the fingerprint area was located at 400-700 cm-1.The XRD pattern indicated that the crystal characteristic peak of the sample basically coincides with the characteristic peak position of the CuO standard card.The Zeta potential result was-12mV,indicating that the CuO-Tcp25-CM nanomaterials tend to condense,and the antimicrobial peptides and cell membranes were successfully coated on the CuO surface.2.CuO-Tcp25-CM nanoparticles had good biocompatibility.Cytotoxicity was positively correlated with concentration.When the concentration was 10 μg/mL,there was no obvious toxicity to mouse fibroblasts after 1 day and 7 days of co-cultivation.In vitro hemolysis experiments found that when the concentration was 200μg/mL,the material had no acute hemolysis reaction.3.CuO-Tcp25-CM nanoparticles had good antibacterial effect in vitro.The antibacterial effect was positively correlated with the concentration.In terms of antibacterial species,the inhibitory effect on bacteria E was the strongest,S was the second,and M was the worst.When the concentration was 10μg/mL,the antibacterial rates were respectively:S:97.90±1.74%,E:99.55±0.45%,M:90.75±2.99%,all of which had strong antibacterial effects.4.CuO-Tcp25-CM nanoparticles had good anti-inflammatory effects in vitro.10μg/mL material can effectively down-regulate the expression of pro-inflammatory factors(TNF-α,IL-1β,IL-6)and up-regulate the expression of anti-inflammatory factors(IL-10).In addition,the material can also effectively inhibit the expression of E-Selectin and affect the downstream inflammatory response.It can promote the polarization of macrophages from M1 type to M2 type,thereby affecting the state of inflammatory response.5.In vivo experiments found that CuO-Tcp25-CM nanomaterials can effectively improve the survival rate of septic mice,had a certain inhibitory effect on inflammation,and reduced liver and kidney damage.Observation of important organ slices showed that the material has no obvious biological toxicity and has good biological safety in vivo.Conclusion:CuO-Tcp25-CM nanoparticles had good biocompatibility,and also have good in vitro antibacterial and anti-inflammatory functions.It can significantly improve the survival rate of mice and reduce systemic inflammation in the in vivo murine sepsis model.This study provides new ideas for the study of anti-sepsis biomaterials and new strategies for the treatment of clinical sepsis更多还原