鈦表面含雙重炎性因子復(fù)合涂層及其調(diào)控巨噬細(xì)胞極化
發(fā)布時(shí)間：2018-08-20 15:49
【摘要】：炎性反應(yīng)中促炎M1巨噬細(xì)胞-抑炎M2巨噬細(xì)胞序列事件在創(chuàng)傷愈合過(guò)程中起著至關(guān)重要的作用。生物材料在宿主體內(nèi)構(gòu)成的微環(huán)境決定了巨噬細(xì)胞M1型或M2型的轉(zhuǎn)化,干擾素-γ(interfcron-γ,IFN-γ)等促炎因子和白細(xì)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(xì)胞。鈦及其合金由于良好的力學(xué)性質(zhì)和生物相容性已普遍用于骨植入材料,納米管因其特殊結(jié)構(gòu)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(chǔ)器,制備了一種新型免疫調(diào)節(jié)生物材料,通過(guò)體外釋放與降解試驗(yàn)、細(xì)胞相容性評(píng)價(jià)、巨噬細(xì)胞培養(yǎng)試驗(yàn),探究改性后的鈦材料能否誘導(dǎo)M2型巨噬細(xì)胞的激活和炎癥反應(yīng)的消退。本工作制備免疫調(diào)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(nèi)初期炎癥反應(yīng),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(jié)果顯示,京尼平成功地將CMCS交聯(lián),且干燥后獲得結(jié)構(gòu)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(shí)驗(yàn)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(guò)在PBS中的浸泡實(shí)驗(yàn),采用酶聯(lián)免疫吸附測(cè)定,研究了 IL-4和IFN-y的釋放趨勢(shì),結(jié)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(xì)胞毒性試驗(yàn)表明,凝膠層具有良好的細(xì)胞相容性,且納米管和凝膠層均對(duì)巨噬細(xì)胞的有較微弱的激活作用。最后,進(jìn)行了材料與體外巨噬細(xì)胞共培養(yǎng)。細(xì)胞形貌觀察、炎性因子分泌及其基因表達(dá)結(jié)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(xì)胞且大量表達(dá)促炎因子,后期(4-10天)釋放的IL-4調(diào)控巨噬細(xì)胞向M2方向轉(zhuǎn)化且大量表達(dá)抑炎因子促炎癥消退,細(xì)胞尺寸變大且伸出多個(gè)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(xì)胞大量表達(dá)抑炎因子。細(xì)胞培養(yǎng)7天時(shí),兩種材料上都發(fā)生細(xì)胞融合形成異物巨細(xì)胞(Foreign Body Giant Cells,FBGCs),這標(biāo)志著炎癥反應(yīng)被抑制,進(jìn)入愈合期。其中,雙炎性因子涂層材料用于構(gòu)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(xì)胞,后釋放的抑炎因子IL-4使M1型巨噬細(xì)胞轉(zhuǎn)化為M2型,進(jìn)而抑制炎癥、進(jìn)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(nèi)研究和作為植入體臨床應(yīng)用提供了可能性。綜上所述,本研究提供了一種能夠有效調(diào)節(jié)M1/M2型巨噬細(xì)胞平衡的新型免疫調(diào)節(jié)生物材料,本工作可為組織對(duì)生物材料的免疫應(yīng)答及創(chuàng)傷組織修復(fù)的研究提供實(shí)驗(yàn)基礎(chǔ),這對(duì)于骨替換材料的開(kāi)發(fā)具有重要意義,對(duì)于其他藥物控釋體系的研究也有參考價(jià)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(xué)位授予單位】：西南交通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：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(xué)位授予單位】：西南交通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R318.08
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 周善宇;蔡霞;路超;;姜黃素對(duì)巨噬細(xì)胞的極化調(diào)節(jié)作用研究進(jìn)展[J];國(guó)際免疫學(xué)雜志;2016年04期
2 韓建業(yè);羅錦華;袁思波;皇甫強(qiáng);余森;劉春潮;;口腔用鈦及鈦合金材料的研究現(xiàn)狀[J];鈦工業(yè)進(jìn)展;2016年03期
3 張U，
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