基于巨噬细胞免疫应答机制构建新型多糖复合水凝胶治疗糖尿病溃疡的基础研究

发布时间：2018-06-16 20:44

本文选题：糖尿病溃疡 + 水凝胶　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的随着糖尿病及其主要并发症糖尿病足溃疡的发病率日益增高,为改善其所造成的经济损失及截肢等后果,本研究旨在合成一种类似于细胞外基质的新型功能化复合水凝胶材料,通过负载白细胞介素4(IL-4)对巨噬细胞进行免疫调控,诱导其分化为选择性激活的巨噬细胞即M2亚型,从而分泌抗炎性细胞因子发挥抗炎性作用,以促进糖尿病足溃疡伤口的快速愈合。方法以壳聚糖和葡聚糖为主要原料合成巯基化壳聚糖(CS-SH)和马来酸葡聚糖(Dex-Ma),再通过Michael加成反应制备得到新型功能化复合水凝胶即巯基化壳聚糖/马来酸葡聚糖(CS-SH/Dex-Ma,CSDM)复合水凝胶。通过调节巯基含量与马来酸含量构筑一系列不同比例的水凝胶,利用核磁共振谱仪和傅里叶红外光谱仪分析其结构,运用扫描电子显微镜(SEM)观察该凝胶的微观结构,并对其溶胀动力学和流变学等特性进行测定评价,同时运用四甲基偶氮唑盐(MTT)法评估水凝胶的生物相容性。基于上述关于水凝胶的基本性能评价后,进一步对水凝胶进行负载IL-4药物实验,同时用该载药水凝胶对小鼠单核巨噬细胞白血病细胞(RAW264.7)进行干预评价,实验设立五组,分别为正常对照(NC)组、脂多糖(LPS)组、IL-4组、水凝胶(CSDM)组及水凝胶+IL-4(CSDM+IL-4)组,于显微镜下对干预后的各组细胞形态进行观察并拍照记录,利用总一氧化氮检测试剂盒测定干预后各组细胞上清液中的NO含量,酶联免疫吸附试验(ELISA)检测水凝胶及药物干预细胞后各组上清液中肿瘤坏死因子a(TNF-a)和白细胞介素10(IL-10)的分泌情况,以及通过实时定量多聚酶链反应(qPCR)检测干预后各组的RAW264.7巨噬细胞中诱导型一氧化氮合酶(iNOS)和精氨酸酶-1(Arg-1)的相对表达情况。结果通过调节巯基含量与马来酸含量制备出5种比例的水凝胶,即巯基与双键的摩尔比分别为1:4、1:2、1:1、2:1和4:1(CS1DM4、CS1DM2、CS1DM1、CS2DM1、CS4DM1),通过核磁与红外测试均可证明水凝胶成功合成,SEM下观察水凝胶具有三维多孔状结构,孔径为5μm左右,该结构为水凝胶发挥其功能的基础条件;同时这种多孔结构可使水凝胶的溶胀率在60min内基本达到溶胀平衡,溶胀率高达为400%,随着时间的延长,水凝胶的溶胀率持续升高,最高达700%,结果显示该凝胶具有良好的吸水功能;水凝胶的流变学结果显示,随着角频率的逐渐增长(0.1~100 rad/s),所得水凝胶的G'值均大于G"值,即储存模量大于损耗模量,说明水凝胶的结构稳定;MTT结果表明,水凝胶浸出液分别与NIH3T3细胞和RAW264.7巨噬细胞培养24h和48h后,浸出液浓度4mg/mL的水凝胶浸出液所培养的细胞存活率均在80%以上,说明该凝胶具有良好的生物相容性,毒副作用小。对细胞进行干预后,发现NC组的细胞形态为圆形、小而透亮的正常形态,经LPS诱导后表现为非常态的多边形并有伪足,而IL-4诱导后细胞形态无明显变化,并且CSDM组以及CSDM+IL-4组对RAW264.7巨噬细胞的形态也没有明显的影响;NO测试结果表明CSDM+IL-4组的NO释放量明显低于CSDM组;ELISA检测TNF-a的分泌结果显示CSDM+IL-4组较CSDM组显著降低,CSDM+IL-4组细胞分泌IL-10明显高于CSDM组;通过qPCR检测结果显示CSDM+IL-4组的iNOS相对表达量显著低于CSDM组,而Arg-1的表达量CSDM+IL-4组较CSDM组显著升高。结论CSDM复合水凝胶具有作为伤口敷料的基本条件和良好的生物相容性,不仅为伤口组织提供优良的湿润环境,同时还可以吸收多余的炎性渗液,负载IL-4后可以成功使巨噬细胞诱导分化为M2型巨噬细胞,从而使抗炎性细胞因子IL-10的分泌量显著升高,Arg-1的mRNA表达量也明显增高,以发挥抗炎性作用,促进糖尿病足溃疡的快速愈合。
[Abstract]:Objective with diabetes and its major complications, the incidence of diabetic foot ulcers is increasing. In order to improve the consequences of economic loss and amputation, this study aims to synthesize a new type of functional composite hydrogel, which is similar to the extracellular matrix, and to regulate the immunoregulation of macrophages through the load of interleukin 4 (IL-4). It is induced to differentiate into a selectively activated macrophage, M2 subtype, which secretes anti-inflammatory cytokines to promote the rapid healing of diabetic foot ulcers. Methods chitosan and glucan are used as the main ingredients to synthesize mercapted chitosan (CS-SH) and maleic acid glucan (Dex-Ma), and then Michael addition reaction. A new functional composite hydrogel, mercapto chitosan / CS-SH/Dex-Ma (CSDM) hydrogels, was prepared. By adjusting the content of the sulfhydryl group and the content of maleic acid, a series of different hydrogels were constructed. The structure of the hydrogels were analyzed by NMR and FTIR, and the scanning electron microscope (SEM) was used. The microstructure of the gel was observed and the swelling kinetics and rheological properties were measured and evaluated. The biocompatibility of the hydrogel was evaluated by four methyl azazolide (MTT) method. Based on the evaluation of the basic properties of the hydrogels, the hydrogel was further loaded with IL-4 medicine and the hydrogel was used. The mice mononuclear macrophage leukemia cell (RAW264.7) was evaluated. The experiment set up five groups, which were normal control (NC) group, lipopolysaccharide (LPS) group, IL-4 group, hydrogel (CSDM) group and hydrogel +IL-4 (CSDM+IL-4) group. Under the microscope, the cell morphology of each group was observed and photographed, and the total nitric oxide was detected by the total nitric oxide. The content of NO in the cell supernatant after intervention was measured by the kit, and the enzyme linked immunosorbent assay (ELISA) was used to detect the secretion of tumor necrosis factor A (TNF-a) and interleukin 10 (IL-10) in the supernatant of each group after the hydrogel and drug intervention cells, and the RAW264.7 of each group after intervention by real-time fixed polymerase chain reaction (qPCR). The relative expression of inducible nitric oxide synthase (iNOS) and arginase -1 (Arg-1) in macrophages. Results by adjusting the sulfhydryl content and maleic acid content, 5 kinds of hydrogels were prepared, that is, the mole ratio of the sulfhydryl and double bonds is 1:4,1:2,1:1,2:1 and 4:1 (CS1DM4, CS1DM2, CS1DM1, CS2DM1, CS4DM1), by NMR and IR test. The hydrogels can be successfully synthesized. The hydrogels have a three-dimensional multi pore structure under SEM. The pore size is about 5 mu m. This structure is the basic condition for the hydrogel to play its function. At the same time, this porous structure can make the swelling rate of hydrogel basically reach the swelling balance in 60min, the swelling rate is up to 400%, with the time prolonging, hydrogel. The swelling rate continued to rise, up to 700%. The results showed that the gel had a good water absorption function. The rheological results of the hydrogel showed that the G'value of the hydrogel was greater than the value of G with the gradual increase of the angular frequency (0.1~100 rad/s), that is, the storage modulus was greater than the loss modulus, indicating that the structure of the hydrogel was stable; the MTT results showed that the hydrogel was leached. After 24h and 48h were cultured with NIH3T3 cells and RAW264.7 macrophages respectively, the survival rate of the cells cultured in the leaching solution of 4mg/mL was above 80%, indicating that the gel had good biocompatibility and small side effects. The cell morphology of the NC group was round, small and bright. State, after LPS induction, the expression of the polygon and the pseudo foot, and the cell morphology without obvious change after IL-4 induction, and the CSDM group and CSDM+IL-4 group have no obvious influence on the morphology of RAW264.7 macrophage. The NO test results showed that the NO release of CSDM+IL-4 group was obviously lower than that of the CSDM group; ELISA test TNF-a secretion showed CS. Group DM+IL-4 was significantly lower than group CSDM, and the secretion of IL-10 in group CSDM+IL-4 was significantly higher than that in group CSDM. The relative expression of iNOS in group CSDM+IL-4 was significantly lower than that in group CSDM, and the expression of Arg-1 in CSDM+IL-4 group was significantly higher than that in CSDM group. Conclusion the composite hydrogel has a basic condition and good survival as a wound dressing. The compatibility, not only provides excellent moist environment for wound tissue, but also absorbs superfluous inflammatory infiltration fluid. After loading IL-4, macrophages can be induced to differentiate into M2 type macrophages, thus the secretion of anti-inflammatory cytokine IL-10 is increased significantly, and the expression of mRNA in Arg-1 is also significantly increased in order to play an anti inflammatory effect. It promotes the rapid healing of diabetic foot ulcers.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2

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