【作者】 高丽丽；

【导师】 冯波；

【作者基本信息】 西南交通大学 ， 生物医学工程（专业学位）， 2017， 硕士

【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TiO₂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TiO₂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。更多还原

【Abstract】 Phenotypic and functional polarization of macrophage,characterized as pro inflammatory（M1）or anti-inflammatory（M2）macrophage,play a crucial role in wound healing.Ml macrophages dominate the early inflammatory response,while M2 macrophages initiate the tissue repair and healing process.The microenvironment presented by biomaterials in the host could regulate the polarization of macrophage to Ml or M2.Cytokines,such as proinflammatory factor interferon-y（IFN-y）and anti-inflammatory factor interleukin-4（IL-4）,can promote the M1 phenotype and M2 phenotype.Therefore,in this work,a new type of immunomodulatory biomaterial was prepared to sequentially release IFN-γ and IL-4 to mediate the sequential M1 and M2 polarization of macrophages.Titania（TiO₂）nanotubes with surface coating were applied as the delivery system because of their special structure and excellent biological activity in drug controlled release.The in vitro degradation and release test,and macrophage culture were carried out to explore whether the modified titanium material can induce the activation of M2 macrophages and the regression of inflammatory response.The preparation of immunomodulatory biomaterials:firstly,IL-4,expected to release in the late stage,was loaded into the TiO₂ nanotubes（TNTs）;secondly,carboxymethyl chitosan（CMCS）gel was prepared on the nanotube surface by genipin cross-linking,named as TNTs/IL-4/GP;finally,IFN-γ was loaded into the CMCS gel to achieve an early release to initial inflammatory response.This loading system was named as TNTs/IL-4/GP/IFN-γ.Fourier-infrared spectroscopy and scanning electron microscopy revealed that genipin successfully crosslinked the CMCS,and a uniform gel layer was obtained after drying.Degradation test in phosphate buffer solution（PBS）and lysozyme solution showed that the degradation rate of gel layer was slow in the first 3 days,and then followed by a faster degradation.The gel layer became thinner and many holes and pits appeared.The release of IL-4 and IFN-y in PBS was detected by enzyme-linked immunosorbent assay（ELISA）.In the early stage（0-3 days）IFN-y was mainly released while only a small amount of IL-4 was detected.The release in the late stage（4-10 days）was dominated by IL-4.Finally,TNTs,TNTs with gel layer（TNTs/GP）,TNTs/IL-4/GP and TNTs/IL-4/GP/IFN-γ,were cocultured with macrophages.Macrophage viability,adhesion,cytokine secretion and gene expression were evaluated.Macrophages on all samples presented the M1-like phenotype at 3 days,and then the M2-like phenotype at 7 days.The TNTs/GP showed a little effect of inhibition on the macrophage viability and cytokine expression,compared to the TNTs.TNTs/IL-4/GP promoted the M2 polarization of macrophages,with higher expression of IL-10 and lower expression of IL-6 and tumor necrosis factor-α（TNF-α）than TNTs/GP.TNTs/IL-4/GP/IFN-γupregulated both the expression of proinflammatory and anti-inflammatory factors,compared to TNTs/IL-4/GP.The high expression of IL-10 in the late stage was conducive to the downregulation and solve of the inflammatory response.In conclusion,the novel immunomodulatory biomaterial provided in this study can effectively regulate phenotypic transition of macrophages through the controlled release of IFN-γ and IL-4.It provides an experimental basis for the study of tissue immune response to biomaterials.The development of alternative materials is of great significance and can be used as a reference for other drug controlled release systems.更多还原