钛表面含双重炎性因子复合涂层及其调控巨噬细胞极化
发布时间：2018-08-20 15:49
【摘要】：炎性反应中促炎M1巨噬细胞-抑炎M2巨噬细胞序列事件在创伤愈合过程中起着至关重要的作用。生物材料在宿主体内构成的微环境决定了巨噬细胞M1型或M2型的转化,干扰素-γ(interfcron-γ,IFN-γ)等促炎因子和白细胞介素-4(interleukin-4,IL-4)等抑炎因子分别激活M1型和M2型巨噬细胞。钛及其合金由于良好的力学性质和生物相容性已普遍用于骨植入材料,纳米管因其特殊结构和优异的生物活性被广泛用于药物控释。因此,本研究以钛为基底,以TiO_2纳米管为存储器,制备了一种新型免疫调节生物材料,通过体外释放与降解试验、细胞相容性评价、巨噬细胞培养试验,探究改性后的钛材料能否诱导M2型巨噬细胞的激活和炎症反应的消退。本工作制备免疫调节生物材料:先将要求后释放的抑炎因子IL-4装载到TiO_2纳米管上,利用京尼平交联羧甲基壳聚糖(CarboxymethylChitosan,CMCS)溶胶制备的水凝胶将其包封后,再将促炎因子IFN-γ装载到凝胶层上以先期释放,模拟体内初期炎症反应,命名为TNTs/IL-4/GP/IFN-γ。傅里叶-红外光谱和扫描电子显微镜结果显示,京尼平成功地将CMCS交联,且干燥后获得结构平整的凝胶层。磷酸盐缓冲液溶液(PBS)和溶菌酶溶液中的降解实验证明,凝胶层前3天降解速率缓慢,4-10天降解速率加快;降解后凝胶的凝胶层不仅厚度变薄表面还出现了很多孔洞和凹坑。通过在PBS中的浸泡实验,采用酶联免疫吸附测定,研究了 IL-4和IFN-y的释放趋势,结果表明,前期(0-3天)以IFN-y的大量释放为主,IL-4仅有少量释放,后期(4-10天)以IL-4释放为主。细胞毒性试验表明,凝胶层具有良好的细胞相容性,且纳米管和凝胶层均对巨噬细胞的有较微弱的激活作用。最后,进行了材料与体外巨噬细胞共培养。细胞形貌观察、炎性因子分泌及其基因表达结果显示,含双炎性因子的材料前期(0-3天)突释的IFN-γ激活M1型巨噬细胞且大量表达促炎因子,后期(4-10天)释放的IL-4调控巨噬细胞向M2方向转化且大量表达抑炎因子促炎症消退,细胞尺寸变大且伸出多个伪足;而仅含抑炎因子的样品后期释放的IL-4激活了 M2型巨噬细胞大量表达抑炎因子。细胞培养7天时,两种材料上都发生细胞融合形成异物巨细胞(Foreign Body Giant Cells,FBGCs),这标志着炎症反应被抑制,进入愈合期。其中,双炎性因子涂层材料用于构建体外初期炎性环境,以便先释放促炎因子IFN-γ,激活M1型巨噬细胞,后释放的抑炎因子IL-4使M1型巨噬细胞转化为M2型,进而抑制炎症、进入愈合期;单炎性因子涂层材料后期抑炎作用虽略低于双炎性因子涂层材料,但也为体内研究和作为植入体临床应用提供了可能性。综上所述,本研究提供了一种能够有效调节M1/M2型巨噬细胞平衡的新型免疫调节生物材料,本工作可为组织对生物材料的免疫应答及创伤组织修复的研究提供实验基础,这对于骨替换材料的开发具有重要意义,对于其他药物控释体系的研究也有参考价值。
[Abstract]:The microenvironment of biomaterials in the host determines the transformation of M1 or M2 macrophages, pro-inflammatory factors such as interferon-gamma (IFN-gamma) and interleukin-4 (IL-4). Titanium and its alloys have been widely used in bone implants because of their excellent mechanical properties and biocompatibility. Nanotubes have been widely used in drug controlled release because of their special structure and excellent biological activity. Therefore, in this study, titanium-based nanotubes were prepared with titanium as the substrate and titanium dioxide nanotubes as the memory. A novel immunomodulatory biomaterial was developed to investigate whether the modified titanium could induce the activation of M 2 macrophages and the regression of inflammation by in vitro release and degradation test, cell compatibility evaluation and macrophage culture test. The nanotubes were encapsulated with Carboxymethyl Chitosan (CMCS) sol and loaded with IFN-gamma to simulate the initial inflammatory reaction in vivo. The results of FTIR and SEM showed that TNTs/IL-4/GP/IFN-gamma was the first time to release IFN-gamma. The degradation experiments in PBS and lysozyme solutions showed that the degradation rate of CMCS was slow in the first three days and accelerated in the fourth to tenth days. The release trend of IL-4 and IFN-y was studied by enzyme-linked immunosorbent assay (ELISA) after soaking in PBS. The results showed that the release of IFN-y was predominant at the early stage (0-3 days), only a small amount of IL-4 was released at the later stage (4-10 days). Cytotoxicity test showed that the gel layer had good cell compatibility, and nanotubes and gels were also released. The results of cell morphology, inflammatory factor secretion and gene expression showed that the M1 macrophages were activated by IFN-gamma, which was suddenly released at the early stage (0-3 days) and expressed a large number of pro-inflammatory factors at the later stage (4-10 days). IL-4 stimulated macrophages to transform to M2 and expressed a large number of anti-inflammatory factors to promote inflammation subsidence, cell size became larger and extended many pseudopods; however, IL-4 released at the later stage of the sample containing only anti-inflammatory factors activated M2 macrophages to express a large number of anti-inflammatory factors. Foreign Body Giant Cells (FBGCs), which indicate that inflammation is inhibited into the healing stage, are used to construct an in vitro initial inflammatory environment in order to release pro-inflammatory factor IFN-gamma, activate M1 macrophages, and then release anti-inflammatory factor IL-4 to convert M1 macrophages into M2, thereby inhibiting inflammation. In conclusion, this study provides a novel immunoregulatory biomaterial that can effectively regulate the balance of M1/M2 macrophages. It can provide experimental basis for the study of tissue immune response to biomaterials and wound tissue repair, which is of great significance for the development of bone replacement materials and for the study of other drug controlled release systems.
【学位授予单位】：西南交通大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.08
[Abstract]:The microenvironment of biomaterials in the host determines the transformation of M1 or M2 macrophages, pro-inflammatory factors such as interferon-gamma (IFN-gamma) and interleukin-4 (IL-4). Titanium and its alloys have been widely used in bone implants because of their excellent mechanical properties and biocompatibility. Nanotubes have been widely used in drug controlled release because of their special structure and excellent biological activity. Therefore, in this study, titanium-based nanotubes were prepared with titanium as the substrate and titanium dioxide nanotubes as the memory. A novel immunomodulatory biomaterial was developed to investigate whether the modified titanium could induce the activation of M 2 macrophages and the regression of inflammation by in vitro release and degradation test, cell compatibility evaluation and macrophage culture test. The nanotubes were encapsulated with Carboxymethyl Chitosan (CMCS) sol and loaded with IFN-gamma to simulate the initial inflammatory reaction in vivo. The results of FTIR and SEM showed that TNTs/IL-4/GP/IFN-gamma was the first time to release IFN-gamma. The degradation experiments in PBS and lysozyme solutions showed that the degradation rate of CMCS was slow in the first three days and accelerated in the fourth to tenth days. The release trend of IL-4 and IFN-y was studied by enzyme-linked immunosorbent assay (ELISA) after soaking in PBS. The results showed that the release of IFN-y was predominant at the early stage (0-3 days), only a small amount of IL-4 was released at the later stage (4-10 days). Cytotoxicity test showed that the gel layer had good cell compatibility, and nanotubes and gels were also released. The results of cell morphology, inflammatory factor secretion and gene expression showed that the M1 macrophages were activated by IFN-gamma, which was suddenly released at the early stage (0-3 days) and expressed a large number of pro-inflammatory factors at the later stage (4-10 days). IL-4 stimulated macrophages to transform to M2 and expressed a large number of anti-inflammatory factors to promote inflammation subsidence, cell size became larger and extended many pseudopods; however, IL-4 released at the later stage of the sample containing only anti-inflammatory factors activated M2 macrophages to express a large number of anti-inflammatory factors. Foreign Body Giant Cells (FBGCs), which indicate that inflammation is inhibited into the healing stage, are used to construct an in vitro initial inflammatory environment in order to release pro-inflammatory factor IFN-gamma, activate M1 macrophages, and then release anti-inflammatory factor IL-4 to convert M1 macrophages into M2, thereby inhibiting inflammation. In conclusion, this study provides a novel immunoregulatory biomaterial that can effectively regulate the balance of M1/M2 macrophages. It can provide experimental basis for the study of tissue immune response to biomaterials and wound tissue repair, which is of great significance for the development of bone replacement materials and for the study of other drug controlled release systems.
【学位授予单位】：西南交通大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.08
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