CN114948861A - 一种促进放射性皮肤损伤愈合的多功能水凝胶及其制备方法和应用 - Google Patents
一种促进放射性皮肤损伤愈合的多功能水凝胶及其制备方法和应用 Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
本发明公开了一种促进放射性皮肤损伤愈合的多功能水凝胶及其制备方法和应用。将氧化石墨烯加入到Tris缓冲液中，水浴超声处理后，再加入多巴胺，室温下搅拌反应，反应结束后离心收集固体颗粒，洗涤、干燥后，得到PDA@GO；将海藻酸钠溶解，加入PDA@GO和IFI6蛋白粉，室温下搅拌至凝胶状，即得到IFI6‑PDA@GO/SA水凝胶。本发明将IFI6与GO、SA、PDA进行复合制备得到一种水凝胶创面修复材料具有抗ROS和皮肤创面愈合的双重功能。同时还具有良好的粘附性和生物相容性，以及抗菌、抗活性氧和抗缺氧等性能。
Description
技术领域
本发明涉及水凝胶技术领域，具体涉及一种促进放射性皮肤损伤愈合的多功能涂水凝胶及其制备方法和应用。
背景技术
许多癌症患者和恶性肿瘤患者都接受放射治疗，放射治疗在癌症治疗中具有关键作用。然而，放疗反应伴随着许多并发症，限制了放疗剂量和治疗效果。在接受放射治疗的癌症患者中，约95％的患者出现皮肤反应，近10％的患者出现严重的皮肤损伤。放射性皮肤损伤(RISI)的确切机制尚不清楚，目前尚无规范、统一的RISI防治方法，已报道的水凝胶大多缺乏抗菌和抗ROS性能，导致创面感染。常规水凝胶虽然可以保护创面，但需要对创面进行缝线固定，由于缺乏粘连能力，不可避免地对创面造成二次损伤。因此，这些水凝胶存在的缺陷促使我们探索更好的损伤缺陷修复策略。
氧化石墨烯(GO)是材料科学领域的重要新材料。近年来，石墨烯作为一种创伤材料受到了极大的关注。这些都有助于通过氢键和π键的相互作用在聚合物的制备中成键。GO材料广泛应用于伤口材料中，利用无毒无害的生物聚合物对其表面进行改性，以提高其吸附性能。聚多巴胺(PDA)表面含有丰富的活性基团邻苯二酚和胺，对重金属的去除非常有效。在弱碱性环境中，多巴胺通过自聚得到，这使得聚多巴胺能够吸附在几乎所有固体物质的表面，形成PDA薄膜。此外，吸附在材料表面的PDA还可以通过迈克尔加成或席夫碱反应作为反应“桥”与含有亲核基团的试剂进一步反应，从而在材料表面引入其他官能团。结果表明，经PDA改性后的聚合物具有良好的吸附性能、抗菌、抗活性氧等积极作用。
α-干扰素诱导蛋白6(IFI6)是一种Ⅰ型干扰素刺激基因，调节细胞凋亡和免疫应答。IFI6 可诱导登革热和乙型肝炎病毒感染，减少血管内皮细胞和乙型肝炎病毒特异性CD8+T细胞凋亡，发挥其抗病毒作用。激活转录因子3(ATF3)下调其新靶点IFI6，抑制舌鳞癌细胞的生长和迁移。最新的有意义的发现表明，mRNA表达谱显示50个上调基因和13个下调基因，其中(IFI6)上调。IFI6的过度表达促进细胞增殖，减少细胞凋亡和活性氧(ROS)的产生；相反，这种过度表达增加了HaCaT和人真皮成纤维细胞(WS1)的放射敏感性。另外，海藻酸钠(SA)也是一种天然多糖，由于其具有良好的粘附性和生物相容性，在生物医学组织工程中得到了广泛的应用。有研究表明SA类水凝胶可诱导炎症期巨噬细胞M2极化，减少14～15期皮肤再生过程中的纤维化或瘢痕形成。但如何将IFI6与GO、SA、PDA结合起来应用于创面修复材料，是需要解决的问题。因此针对放射性皮肤损伤的愈合，需要一种含有IFI6的水凝胶创面修复材料，具有促进Hacat细胞增殖和迁移的能力，使其具有体内外协同抗辐射能力；同时具有抗ROS和皮肤创面愈合的双重功能，为RISI患者的载体治疗提供了方便。
发明内容
针对上述现有技术，本发明的目的是提供一种促进放射性皮肤损伤愈合的多功能涂水凝胶及其制备方法和应用。本发明将IFI6与GO、SA、PDA进行复合制备得到一种水凝胶创面修复材料具有抗ROS和皮肤创面愈合的双重功能。同时还具有良好的粘附性和生物相容性，以及抗菌、抗活性氧和抗缺氧等性能。
为实现上述目的，本发明采用如下技术方案：
本发明的第一方面，提供一种促进放射性皮肤损伤愈合的多功能涂水凝胶的制备方法，包括以下步骤：
(1)将氧化石墨烯(GO)加入到Tris缓冲液中，水浴超声处理后，再加入多巴胺(DA)，室温下搅拌反应，反应结束后离心收集固体颗粒，洗涤、干燥后，得到PDA@GO；
(2)将海藻酸钠(SA)溶解，加入步骤(1)制备的PDA@GO和IFI6蛋白粉，室温下搅拌至凝胶状，即得到IFI6-PDA@GO/SA水凝胶。
优选的，步骤(1)中，所述Tris缓冲液的pH值为8.5，所述Tris缓冲液的浓度为10mM。
优选的，所述氧化石墨烯与Tris缓冲液的加入量之比为30mg:10mL；所述氧化石墨烯与多巴胺的质量比为1：1。
优选的，步骤(1)中，所述水浴超声的温度为60℃、时间为2小时；所述搅拌的时间为48h。
优选的，步骤(1)中，所述离心的速度为8000rpm/min、时间为10min；所述洗涤为用水乙醇洗涤3次；所述干燥为40℃干燥12h。
优选的，步骤(2)中，所述海藻酸钠溶解后的浓度为10mg/mL。
优选的，溶解后的海藻酸钠、PDA@GO和IFI6蛋白粉的质量比为10：10：1。
搅拌时间为5～15min。
本发明的第二方面，提供上述制备方法制备得到的多功能IFI6-PDA@GO/SA水凝胶。
优选的，所述水凝胶具有抗ROS和皮肤创面愈合的双重功能，以及具有促进Hacat细胞增殖和迁移的能力。
本发明的第三方面，提供多功能IFI6-PDA@GO/SA水凝胶在如下1)～2)中的用途：
1)促进放射性皮肤损伤的愈合；
2)制备治疗放射性皮肤损伤的喷涂药剂。
本发明的有益效果：
(1)本发明成功地制备了可喷涂的IFI6-PDA@GO/SA复合水凝胶，喷涂的 IFI6-PDA@GO/SA水凝胶具有操作简单、立即可用的特点，适用于RISI伤口等紧急情况下的伤口处理。为RISI患者的载体治疗提供了方便，PDA@GO/SA水凝胶有利于RISI患者的治疗。
(2)本发明中IFI6与PDA@GO/SA之间具有相互促进关系，制备的PDA@GO/SA水凝胶具有促进Hacat细胞增殖和迁移的能力，使其具有体内外协同抗辐射能力。还具有抗ROS 和皮肤创面愈合的双重功能。同时还具有良好的粘附性和生物相容性，以及抗菌、抗活性氧和抗缺氧能力；从而进一步抑制癌细胞增殖、转移。
附图说明
图1：实施例1制备IFI6-PDA@GO/SA的过程及其在RISI愈合中的应用示意图；
图2：IFI6-PDA@GO/SA的特征。(A)IFI6-PDA@GO/SA的SEM(左)和EDS(右) 图像。(B)IFI6-PDA@GO/SA的XPS。(C)IFI6-PDA@GO/SA凝胶电泳结果。 (D)IFI6-PDA@GO/SA的FTIR。(E)IFI6-PDA@GO/SA的紫外光谱。(F)IFI6-PDA@GO/SA 的EDS，测试元素包括O，N，Na，CL，Ca，C(。G)粒度和电位分析。D＝162nm,PDI＝0.19,zeta 电位＝-14.64mV。
图3：IFI6-PDA@GO/SA的生物相容性和抗菌活性。(A)HaCaT细胞在 IFI6-PDA@GO/SA上培养7天后的FITC/DAPI染色图像。(B)IFI6-PDA@GO/SA对 G+(MRSA)和G-(大肠杆菌)细菌的抗菌活性。(C)在IFI6-PDA@GO/SA上培养的HaCaT细胞第7天进行CCK-8测定。(D)细菌计数*P<0.05，横坐标由左到右依次为control、IFI6、 PDA@GO/SA、IFI6-PDA@GO/SA；
图4：IFI6-PDA@GO/SA的体外细胞学研究。(A)流式细胞术；(B)总凋亡率结果。
图5：IFI6-PDA@GO/SA的体外细胞学研究。(A)用IFI6-PDA@GO/SA对HaCaT细胞划伤迁移的影响。(B)IFI6的蛋白印迹图。(C)细胞克隆形成实验。(D)图A细胞迁移率的统计数据，*表示P<0.05。(E)图B蛋白印迹图的统计数据，横坐标由左到右依次为A 组、B组、C组、D组、E组，*表示P<0.05。
图6：IFI6-PDA@GO/SA的体内小鼠研究。(A)医用电子直线加速器及其在小鼠辐射致皮肤损伤模型中的操作过程。(B)第1、14天的伤口照片和第14天的HE染色。(C)第 14天各组创面面积。(D)完全愈合时间。(E)第14天辐射致皮肤损伤等级。(F-G)第14天肉芽组织厚度及创面微血管密度，*P<0.05。其中，D-E图中横坐标由左到右依次为B组、 C组、D组、E组；F图中横坐标由左到右依次为A组、B组、C组、D组、E组。
图7：抗ROS表达，横坐标由左到右依次为A组、B组、C组、D组、E组。
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本申请提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
正如背景技术部分介绍的，IFI6在照射后的皮肤细胞内高表达，并且显著增加皮肤细胞的辐射抗性，具体表现为促进照后皮肤细胞的增殖能力，降低辐射诱导的细胞凋亡水平。与此同时，IFI6在照后的皮肤细胞内向细胞核聚集，与另一入核蛋白SSBP1发生相互作用，显著影响内质网应激通路中热休克调节因子1(HSF1)的转录活性。所以，IFI6可以通过调控HSF1介导的内质网应激过程从而抑制辐射诱导的皮肤细胞损伤，降低皮肤细胞的辐射敏感性。如果将IFI6制备成便于携带的喷涂药剂用于RISI伤口等紧急情况下的伤口处理，将为RISI患者的载体治疗提供了方便。但还需要克服药剂的生物相容性、粘附性、抗菌、抗氧化性等多个问题。
基于此，本发明的目的是提供一种促进放射性皮肤损伤愈合的多功能涂水凝胶及其制备方法和应用。本发明以IFI6与GO、SA、PDA复合制得创面修复材料，IFI6-PDA@GO/SA的制备过程如图1所示。IFI6与PDA@GO/SA之间具有相互促进关系，使得制备得到的水凝胶具有抗ROS和皮肤创面愈合的双重功能。较单独使用IFI6对创面进行修复相比，本发明制备的水凝胶还具有抗菌、抗活性氧和抗缺氧能力，皮肤修复后可促进创面愈合。本发明制备的IFI6-PDA@GO/SA中PDA作为生物胶具有良好的抗活性氧作用和良好的生物相容性，GO具有比表面积大、官能团丰富、易修饰、抗菌等优点。因此，PDA@GO 的包封促进了IFI6向角质层的渗透和向皮肤的输送。IFI6促进RISI创面细胞增殖和迁移； IFI6具有良好的抗凋亡作用。SA具有保湿性，潮湿的环境有利于RISI的创面修复，所以本发明制备的IFI6-PDA@GO/SA水凝胶有利于RISI患者的治疗。
IFI6是被包覆在PDA@GO/SA形成的水凝胶网络，实际应用中IFI6也是从复合海绵中释放出来。IFI6-PDA@GO/SA除了IFI6的防细胞辐射功能，还有PDA的抗氧化应激作用，GO的抗菌作用，SA的水凝胶保湿作用，各种组分互相辅助，协同促进创面愈合。且将IFI6结合到PDA@GO/SA上有利于IFI6的释放。
为了使得本领域技术人员能够更加清楚地了解本申请的技术方案，以下将结合具体的实施例详细说明本申请的技术方案。
本发明实施例中所用的试验材料均为本领域常规的试验材料，均可通过商业渠道购买得到。
其中，动物实验经陆军医科大学动物伦理委员会批准进行。
昼夜节律(12h)，相对湿度(50％)，环境温度(25℃)。
HaCat和VEGF细胞由陆军医科大学西南医院肿瘤科提供，
雄性BALB/c小鼠(25g)由陆军医科大学实验动物系提供，
IFI6蛋白粉购自SAB公司(美国马里兰州)，
DA购自Sigma公司(美国)；
GO片购自西安瑞禧生物科技有限公司。
实施例
合成PDA@GO:30mg GO片，加入pH值8.5的10mm Tris缓冲液，水浴超声2小时，再加入30mg DA，室温搅拌48小时，8000rpm离心10分钟，用水乙醇洗涤3次，40℃干燥12小时。
IFI6-PDA@GO/SA的合成：SA在100ml烧杯中溶解，然后PDA@GO加入烧杯混合物，加入50μL H202溶液(浓度为20mM)和50μL辣根过氧化物酶，然后将3mg IFI6蛋白粉加入烧杯中，在室温(25摄氏度)下搅拌1小时至混合物为水凝胶。
用动态光散射法测定了纳米粒子的粒径和zeta电位。用扫描电镜观察了 IFI6-PDA@GO/SA的形貌。用Nicolet 6700FTIR光谱仪(4000-600cm-1)记录傅里叶变换红外(FTIR)光谱。紫外可见近红外光谱测量材料的紫外光谱仪/近红外光吸收效应。能量色散谱(EDS)/X射线光电子能谱(XPS)分析了材料的元素组成。用Western印迹法测定材料的蛋白质组成。
IFI6-PDA@GO/SA材料为半固态，扫描电镜显示海藻酸钠衬底为片状。SEM图像显示SA水凝胶在微米尺度上具有孔结构，IFI6-PDA@GO颗粒主要分布在孔壁内侧(图2A)。
EDS和XPS测量的元素图谱表明，O、N、Na、Cl、Ca、C元素在IFI6-PDA@GO/SA 中分布均匀(图2B，2F)。IFI6中含有C、N、O，SA中含有C、O，而PDA@GO中含有 Na、Cl和Ca，说明IFI6、SA和PDA@GO均存在于IFI6-PDA@GO/SA水凝胶中。
C峰(284.08eV)、C-C峰(284.08eV)和O1S峰(531.95eV)占主导地位，表明该复合体系由GO片组成。此外，在286.56和285.79eV处观察到C-O和C-N结合能，表明PDA中含有C-N键。
GO片特征吸收带在1054cm-1(烷氧基)、1224cm-1(环氧基)、1401cm-1(羧基 C-O)和1724cm-1(羧基C＝O)。IFI6-PDA@GO/SA在1579cm-1处的新吸附峰可能是由-N-H 键合的变形振动和-C-N键合的拉伸振动引起的(图2D)，进一步证实了在PDA@GO复合材料的制备过程中，GO的环氧基团与PDA的胺基团之间可能发生了反应。目前还没有关于IFI6特征吸收峰的报道，但我们认为3400cm-1位置可能与IFI6的组成密切相关。
为了进一步测试IFI6的负载，进行了十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析(图2C)。我们发现IFI6-PDA@GO/SA材料表达了IFI6，与Interferon- αinduClble protein 6(IFI6)confers protection against ionizing radiation inskin cells(Jia,H 等J.Dermatol.SCl.2020,100,139-147.)中的IFI6蛋白条带基本一致，表明IFI6测试成功。本实施例制备的IFI6-PDA@GO/SA具有典型的纳米结构，直径约162nm，PDI＝0.19，zeta 电位＝-14.64mV(图2G)，紫外光谱在400-800nm范围内无明显变化(图2E)。
纳米海藻酸钠固体含量从0.04％到0.08％不等，较高浓度的海藻酸钠会产生较大的团聚状溶液体系。通过zeta电位的测量认为SA海绵带负电荷，复合海绵的zeta电位受SA比例的影响，因此小颗粒的主动靶向比大纳米球的被动靶向具有更大的优势。
试验例
1.试验和材料
1.1生物相容性评价及细胞形态学观察
细胞学实验分为5组，A组为正常细胞组，B组为正常细胞+3Gy辐射组，C组为正常细胞+3Gy辐射+PDA@Go/SA水凝胶组，D组为正常细胞+3Gy辐射+IFI6组，E组为正常细胞+3Gy辐射+IFI6-PDA@Go/SA水凝胶组(以下试验中细胞学实验分组均相同，动物学分组均相同)。
HaCat细胞在Dulbecco改良Eagle培养基(DMEM)中以2×103细胞/孔的密度接种到A-E组。将HaCat接种于A-E组培养基上24h，观察细胞骨架形态对细胞活力的影响。当它准备就绪时，细胞然后与DAPI(2-(4-脒苯基)-6-吲哚氨基甲酸二盐酸盐)孵育。用共聚焦激光扫描显微镜(780，Zeiss，德国)获得染色细胞的荧光图像。
1.2细菌共培养
MRSA(耐甲氧西林金黄色葡萄球菌)和大肠杆菌(Escherichia coli)来自陆军医科大学。抽出等量的菌液涂在盘子上。37℃培养24h后，测定细菌数量的变化。
1.3划伤迁移
将HaCat培养到24孔板(2×104/孔)中。用吸液管在单层上划出伤口(0h)，并用蔡司视频显微镜进行24小时检查。具体测量采用ImageJ1.48V软件(美国NIH)。每组试验重复三次。
1.4流式细胞术
根据制造商的说明，使用流式细胞术分析和Annexin V-FITC/PI凋亡检测试剂盒(Dojindo Molecular Technologies，Japan)进行细胞凋亡分析。简单地说，收集细胞并将其悬浮在含有5μL Annexin V-FITC和5μL PI的200μL结合缓冲液中。然后，将细胞在室温黑暗中孵育15分钟。应用流式细胞仪检测RISI创面免疫微环境中的CD4+、CD8+、 NK细胞和M1细胞，并用美国Beckman Coulter公司的荧光激活细胞分选仪(FACS)软件对细胞进行分析。
1.5细胞克隆形成试验
将A-E组的细胞悬液加入到上述低浓度琼脂糖溶液中，以获得1000/ml的最终细胞浓度。每孔照射剂量为3Gy。每孔用0.005％结晶紫(1ml)染色1h，显像后用Image ProPlus6.0 计算群落数。
1.6RISI小鼠模型
每只小鼠腹腔注射戊巴比妥约0.2ml麻醉。该直线加速器发射6Mev电子射线(一次照射30Gy，照射场1cm×1cm，剂量300cGy/min，10min)。源皮肤距离为1米，剩余皮肤用铅板封堵。每组照射后，用材料覆盖照射部位，每隔一天更换一次，共7～14d，分为5组，每组5只。细胞学实验分为5组，A组为正常小鼠组，B组为正常小鼠+30Gy辐射组，C组为为正常小鼠+30Gy辐射+PDA@Go/SA水凝胶组，D组正常小鼠+30Gy辐射+ IFI6组，E组为正常小鼠+30Gy辐射+IFI6-PDA@Go/SA水凝胶组。
采用IPP6.0软件，比较创面愈合前后创面面积，计算创面愈合率。创面-愈合率＝(初始创面面积-愈合特定时间后的创面面积)/初始创面面积×100％。RISI评分遵循道格拉斯和福勒评分法。
1.7苏木精-伊红(HE)染色及组织学分析
每只小鼠伤后14d取创面标本制作石蜡切片进行HE染色。选择高质量图像进行HE染色。几位病理学专家用盲法测量了新生上皮的长度。
1.8蛋白印迹和免疫组化染色检测SBPP1/HSF1的表达
伤后14天，从全层缺损处取10mm×10mm的小方块，包括新表皮和肉芽组织，立即液氮冷冻。严格参照制造商的说明(varioskan Flash；thermo SClentifiClent，美国)。抗IFI6抗体(biuss，China)、抗SBPP1抗体(finetest，China)和抗HSF1抗体(biuss，China) 稀释1:100。用山羊辣根过氧化物酶标记的山羊抗兔二次抗体(中国中山生物)稀释至1:2000，收获PVDF(聚偏氟乙烯)膜送化学发光观察(美国热科学)，免疫组化步骤大致相同。
1.9实时定量PCR和活性氧测定
使用7500实时PCR系统(应用生物系统仪器)，使用SYBR Green Master Mix(TOYOBO，QPK-201)，对NLRP3和ROS进行实时PCR。用酶联免疫吸附试剂盒测定各组ROS变化。
2.结果和讨论
2.1IFI6-PDA@GO/SA的生物相容性和抗菌活性
如图3A所示，HaCaT细胞与IFI6-PDA@GO/SA共培养7天，FITC/DAPI染色显示细胞形态无明显差异；细胞核和细胞质完整。CCK-8法显示，4-Gy照射后第3d，B组和E 组HaCaT细胞的OD值降低，提示照射明显抑制细胞生长(图3B)。第6天E组OD值明显恢复，且显著高于B组(P<0.05)，提示IFI6-PDA@GO/SA在短期(1-3d)内可能不影响照射细胞的生长，而在长期(>6d)照射细胞的生长曲线恢复正常。
培养5天后，3个样品细胞活力均提高。因此，尽管有研究报道氧化石墨烯具有一定的毒性，但复合水凝胶显示出良好的生物相容性，没有氧化石墨烯的任何毒性作用。因此，本发明制备的PDA@GO/SA复合水凝胶对GO的毒性有较好的屏蔽作用。此外，PDA和 SA都是可生物降解的。因此，这种复合水凝胶可能更适合于治疗RISI。
皮肤创面的细菌感染是众所周知的延迟创面愈合，甚至引起创面恶化。对照组MRSA(G+)和大肠杆菌(G-)生长良好(如图3B+3D所示)，PDA@GO/SA(C组)抑制细菌生长较好，而IFI6-PDA@GO/SA(E组)则表现出更好的抑菌效果。而IFI6没有抑菌作用。说明采用本发明制备的IFI6-PDA@GO/SA可以进一步提高生物相容性和抗菌活性。
2.2IFI6-PDA@GO/SA的体外细胞学研究
用流式细胞术检测IFI6对细胞凋亡的潜在影响。据报道，IFI6目前仅在高等真核生物 11中表达。放疗显著提高细胞凋亡率(图4)。与B组相比，E组的凋亡率较低(P<0.05),而E组的作用明显高于D组。说明IFI6-PDA@GO/SA能显著降低细胞凋亡率，E组在调节细胞凋亡率方面起重要作用。
如图5A+5D所示，将HaCaT细胞与各组中的材料共培养约24小时，然后进行细胞划痕试验。B组24h迁移率明显降低(P<0.05)，可能与辐射对细胞迁移的影响有关，E组的迁移抑制作用明显改善，提示该材料能提高照射后细胞的迁移率(P<0.05)。值得注意的是，E组细胞迁移率高于D组(P<0.05)，提示IFI6蛋白表达增加促进了Hacat细胞的迁移。 IFI6具有重要的抗病毒和抗凋亡功能。但其在电离辐射诱导皮肤细胞应激中的作用尚未见报道。Western blotting显示，IFI6的成功过表达显著促进了5-Gy X射线照射后的增殖。结果表明，IFI6过表达B16 F10细胞的凋亡率和活性氧生成量明显降低。这些结果提示IFI6 在癌细胞中具有放射耐受性，为放射治疗提供了一个新的靶点。免疫印迹分析中IFI6的表达如图5B+5E所示。E组IFI6的表达明显高于B组(P<0.05)和D组(P<0.05)，提示该物质可能通过内吞作用进入HaCaT细胞胞浆，从而发挥相关作用。
为了验证IFI6蛋白对HaCaT细胞的辐射保护作用，我们采用了“金标准”范式。细胞克隆形成实验表明，4-Gy照射后，单纯照射组(B组)的相对克隆数比照射前减少了 45％(P<0.05)。相比之下，E组的相对克隆数仅下降约20％(P<0.05)(图5C)。说明本发明制备的IFI6-PDA@GO/SA增强了细胞的抗辐射能力，有利于细胞克隆的形成。
2.3IFI6-PDA@GO/SA对RISI小鼠模型的影响
图6A中，左图为电子束放疗设备，右图为小鼠动物模型定位建模过程。如图6B所示，将所有小鼠剃毛并成像，以建立第1天治疗的模型。第14天，再次对小鼠进行拍照。处死这些小鼠，收集局部皮肤组织进行HE染色(图6C)。与B组相比，D、E组促进创面愈合(p<0.05)，E组作用显著高于D组(p<0.05)，提示IFI6促进表皮细胞迁移增殖，从而促进创面愈合。如图6A所示，各组在第14天均显著观察到炎症。E组表皮逐渐形成，说明本发明制备的IFI6-PDA@GO/SA水凝胶对于RISI具有的治疗作用。在本发明中，通过将 IFI6与PDA@GO/SA复合增强了IFI6的持续效力，形成了IFI6-PDA@GO/SA纳米复合物，并在X射线照射24小时前皮下给药进入皮肤。本发明制备的IFI6与PDA@GO/SA显著提高了IFI6的稳定性，并能从IFI6-PDA@GO/SA中稳定、激活和释放IFI6。
2.4抗ROS研究
ROS的表达与急性放射性皮肤损伤的发生发展密切相关。如图7所示，30Gy辐射后(B组)ROS相对于未照射组(A组)表达显着增加；但是，IFI6(D组)可降低ROS 表达，且IFI6-PDA@GO/SA(E组)能更一步降低ROS表达。
上述试验可以看出，本发明将IFI6与PDA@GO/SA复合，PDA@GO/SA增强IFI6的作用，同时IFI6可以促进PDA@GO/SA的生物相容性和抗菌等性能，两者协同作用，进一步促进抗ROS和皮肤创面的愈合。
以上所述仅为本申请的优选实施例而已，并不用于限制本申请，对于本领域的技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本申请的保护范围之内。
Claims (10)
1.一种促进放射性皮肤损伤愈合的多功能水凝胶的制备方法，其特征在于，包括以下步骤：
(1)将氧化石墨烯加入到Tris缓冲液中，水浴超声处理后，再加入多巴胺，室温下搅拌反应，反应结束后离心收集固体颗粒，洗涤、干燥后，得到PDA@GO；
(2)将海藻酸钠溶解，加入步骤(1)制备的PDA@GO和IFI6蛋白粉，室温下搅拌至凝胶状，即得到IFI6-PDA@GO/SA水凝胶。
2.根据权利要求1所述的制备方法，其特征在于，步骤(1)中，所述Tris缓冲液的pH值为8.5，所述Tris缓冲液的浓度为10mM。
3.根据权利要求2所述的制备方法，其特征在于，所述氧化石墨烯与Tris缓冲液的加入量之比为30mg:10mL；所述氧化石墨烯与多巴胺的质量比为1：1。
4.根据权利要求1所述的制备方法，其特征在于，步骤(1)中，所述水浴超声的温度为60℃、时间为2小时；所述搅拌的时间为48h。
5.根据权利要求1所述的制备方法，其特征在于，步骤(1)中，所述离心的速度为8000rpm/min、时间为10min；所述洗涤为用水乙醇洗涤3次；所述干燥为40℃干燥12h。
6.根据权利要求1所述的制备方法，其特征在于，步骤(2)中，所述海藻酸钠溶解后的浓度为10mg/mL。
7.根据权利要求6所述的制备方法，其特征在于，溶解后的海藻酸钠、PDA@GO和IFI6蛋白粉的质量比为10：10：1。
8.权利要求1～7任一项所述的制备方法制备得到的多功能IFI6-PDA@GO/SA水凝胶。
9.根据权利要求8所述的水凝胶，其特征在于，所述水凝胶具有抗ROS和皮肤创面愈合的双重功能。
10.权利要求8或9所述的多功能IFI6-PDA@GO/SA水凝胶在如下1)～2)中的用途：
1)促进放射性皮肤损伤的愈合；
2)制备治疗放射性皮肤损伤的喷涂药剂。
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