鈦表面含雙重炎性因子復合涂層及其調控巨噬細胞極化
發(fā)布時間：2018-08-20 15:49
【摘要】：炎性反應中促炎M1巨噬細胞-抑炎M2巨噬細胞序列事件在創(chuàng)傷愈合過程中起著至關重要的作用。生物材料在宿主體內構成的微環(huán)境決定了巨噬細胞M1型或M2型的轉化,干擾素-γ(interfcron-γ,IFN-γ)等促炎因子和白細胞介素-4(interleukin-4,IL-4)等抑炎因子分別激活M1型和M2型巨噬細胞。鈦及其合金由于良好的力學性質和生物相容性已普遍用于骨植入材料,納米管因其特殊結構和優(yōu)異的生物活性被廣泛用于藥物控釋。因此,本研究以鈦為基底,以TiO_2納米管為存儲器,制備了一種新型免疫調節(jié)生物材料,通過體外釋放與降解試驗、細胞相容性評價、巨噬細胞培養(yǎng)試驗,探究改性后的鈦材料能否誘導M2型巨噬細胞的激活和炎癥反應的消退。本工作制備免疫調節(jié)生物材料:先將要求后釋放的抑炎因子IL-4裝載到TiO_2納米管上,利用京尼平交聯(lián)羧甲基殼聚糖(CarboxymethylChitosan,CMCS)溶膠制備的水凝膠將其包封后,再將促炎因子IFN-γ裝載到凝膠層上以先期釋放,模擬體內初期炎癥反應,命名為TNTs/IL-4/GP/IFN-γ。傅里葉-紅外光譜和掃描電子顯微鏡結果顯示,京尼平成功地將CMCS交聯(lián),且干燥后獲得結構平整的凝膠層。磷酸鹽緩沖液溶液(PBS)和溶菌酶溶液中的降解實驗證明,凝膠層前3天降解速率緩慢,4-10天降解速率加快;降解后凝膠的凝膠層不僅厚度變薄表面還出現(xiàn)了很多孔洞和凹坑。通過在PBS中的浸泡實驗,采用酶聯(lián)免疫吸附測定,研究了 IL-4和IFN-y的釋放趨勢,結果表明,前期(0-3天)以IFN-y的大量釋放為主,IL-4僅有少量釋放,后期(4-10天)以IL-4釋放為主。細胞毒性試驗表明,凝膠層具有良好的細胞相容性,且納米管和凝膠層均對巨噬細胞的有較微弱的激活作用。最后,進行了材料與體外巨噬細胞共培養(yǎng)。細胞形貌觀察、炎性因子分泌及其基因表達結果顯示,含雙炎性因子的材料前期(0-3天)突釋的IFN-γ激活M1型巨噬細胞且大量表達促炎因子,后期(4-10天)釋放的IL-4調控巨噬細胞向M2方向轉化且大量表達抑炎因子促炎癥消退,細胞尺寸變大且伸出多個偽足;而僅含抑炎因子的樣品后期釋放的IL-4激活了 M2型巨噬細胞大量表達抑炎因子。細胞培養(yǎng)7天時,兩種材料上都發(fā)生細胞融合形成異物巨細胞(Foreign Body Giant Cells,FBGCs),這標志著炎癥反應被抑制,進入愈合期。其中,雙炎性因子涂層材料用于構建體外初期炎性環(huán)境,以便先釋放促炎因子IFN-γ,激活M1型巨噬細胞,后釋放的抑炎因子IL-4使M1型巨噬細胞轉化為M2型,進而抑制炎癥、進入愈合期;單炎性因子涂層材料后期抑炎作用雖略低于雙炎性因子涂層材料,但也為體內研究和作為植入體臨床應用提供了可能性。綜上所述,本研究提供了一種能夠有效調節(jié)M1/M2型巨噬細胞平衡的新型免疫調節(jié)生物材料,本工作可為組織對生物材料的免疫應答及創(chuàng)傷組織修復的研究提供實驗基礎,這對于骨替換材料的開發(fā)具有重要意義,對于其他藥物控釋體系的研究也有參考價值。
[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.
【學位授予單位】：西南交通大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：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.
【學位授予單位】：西南交通大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R318.08
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