CN111744050A - 氧化石墨烯-达托霉素∕表皮生长因子复合敷料制备及创面愈合方法 - Google Patents
氧化石墨烯-达托霉素∕表皮生长因子复合敷料制备及创面愈合方法 Download PDFInfo
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- CN111744050A CN111744050A CN202010680675.0A CN202010680675A CN111744050A CN 111744050 A CN111744050 A CN 111744050A CN 202010680675 A CN202010680675 A CN 202010680675A CN 111744050 A CN111744050 A CN 111744050A
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Abstract
本发明公开了氧化石墨烯‑达托霉素/表皮生长因子复合敷料制备及其对创面愈合影响,主要是一种氧化石墨烯‑达托霉素/表皮生长因子复合敷料制备,包括如下步骤:S1准备氧化石墨烯膜:氧化石墨烯膜购买自南京先锋纳米有限公司(货号:100027,CAS号:7440‑44‑0,参数:尺寸:9x9cm厚度:约25微米);S2制备氧化石墨烯‑多巴胺膜:在100mL去离子水中加入131.14g tris盐酸溶解,得到的tris溶液中加入多巴胺粉200mg,得到tris‑多巴胺溶液,浓度为2mg/mL,pH值为8.5,将S1购买的氧化石墨烯膜浸泡在2mg/ml的多巴胺溶液,37℃恒温箱条件下,以100r/min震荡约2h。本发明成功制备氧化石墨烯膜‑达托霉素/表皮生长因子敷料,样品较好地实现了抗菌效果和促生长性能,对创面愈合有很大的益处。
Description
技术领域
本发明涉及氧化石墨烯技术领域,尤其涉及氧化石墨烯-达托霉素/表皮生长因子复合敷料制备及创面愈合方法。
背景技术
近年来,氧化石墨烯在抗菌方面的优异性能引起了越来越多的关注。已有报道基于氧化石墨烯的纳米复合材料在很大程度上有利于伤口愈合,其抗菌性能的根源在于氧化石墨烯边缘所致的细胞膜紊乱及氧化应激诱导之间的协同作用。此外,氧化石墨烯表面含有丰富的羟基、环氧基团、羧基等表面基团,其亲水性和生物相容性明显增强,同时可以被其他化学物质进一步修饰。因此,我们特意对氧化石墨烯实现功能化,以增强其抗菌作用,同时提高其稳定性。目前,为了防止氧化石墨烯的聚集,已经有几例采用化学材料接枝法,其中交联剂的使用包括聚多巴胺、Fe3O4和银纳米颗(AgNPs),但银纳米颗的细胞毒性值得警惕。达托霉素是一种新的环状脂肽类抗生素,在发酵过程中通过癸酸供给玫瑰孢链霉菌生长培养基而产生,于2003年被FDA 批准用于治疗复杂的皮肤和皮肤结构感染、右侧感染性心内膜炎和菌血症。达托霉素在体外对革兰氏阳性病原体(包括菌株)具有杀菌活性,但杀灭作用有限。此外,达托霉素还被用于治疗儿科感染(如心内膜炎、败血症、菌血症及脑膜炎)。然而,在粪肠球菌和金黄色葡萄球菌中,关于达托霉素耐药性的报道也越来越多。这种耐药性主要是细菌感染的长期治疗过程,它甚至可以在事先没有使用过达托霉素的情况下发生。据报道,各种生长因子(如表皮生长因子、血小板源生长因子,和纤维母细胞生长因子2)可以分别在不同动态阶段发挥关键部分,包括迁移,增殖及血管生成。本发明中,使用表皮生长因子作为模型因子对氧化石墨烯进行修饰,以增强伤口愈合效果。表皮生长因子是细胞增殖的主要促进剂,已被广泛应用于创伤愈合和骨折的修复。考虑到表皮生长因子在正常生理条件下不稳定,可能会快速失活,为了更好的利用,应该将其整合到具有水凝胶等缓释特性的给药系统中。因此,对氧化石墨烯膜和达托霉素/表皮生长因子的综合研究成为热点,不仅因为其具有广泛的应用潜力,而且通过达托霉素抗菌和表皮生长因子促生长的联合作用也很有前景。因此,本技术提出一种氧化石墨烯-达托霉素复合敷料制备及创面愈合方法。
发明内容
基于背景技术存在的技术问题,本发明提出了氧化石墨烯-达托霉素复合敷料制备及创面愈合方法。
本发明提出的氧化石墨烯-达托霉素/表皮生长因子复合敷料制备,包括如下步骤:
S1准备A组氧化石墨烯膜:氧化石墨烯膜购买自南京先锋纳米有限公司,货号:100027,CAS号:7440-44-0,参数:尺寸:9x9cm厚度:约25微米;
S2制备氧化石墨烯-多巴胺膜:
S21在100mL去离子水中加入131.14g tris盐酸溶解,得到的tris溶液中加入多巴胺粉200mg,得到tris-多巴胺溶液,浓度为2mg/mL,pH值为8.5;
S22将S1中的氧化石墨烯膜浸泡在上述溶液中12h后,将混合物转移到振动筛中,在37℃下以100r/min的速度振动;
S3制备B组氧化石墨烯-达托霉素样品:
先将达托霉素粉溶解于去离子水中,制备10mg/mL的达托霉素水溶液,再将S1中的氧化石墨烯样品,经去离子水仔细清洗,孵育在达托霉素水溶液中,其中孵育温度37℃;震动速度:100r/min;孵化时间,12h。
S4制备C组氧化石墨烯膜-表皮生长因子:将S2中制备好的氧化石墨烯-多巴胺膜,溶解在10μg/ml的表皮生长因子溶液中,孵育温度37℃;震动速度:100r/min;孵化时间,12h;
S5制备D组氧化石墨烯膜-达托霉素/表皮生长因子:将氧化石墨烯膜-多巴胺加入到S3和S4步骤中相同浓度的达托霉素+表皮生长因子混合物中,孵育温度37℃;震动速度:100r/min;孵化时间,12 h,制备得氧化石墨烯膜-达托霉素/表皮生长因子膜。
氧化石墨烯-达托霉素/表皮生长因子复合敷料的表征及其对创面愈合的影响,包括如下步骤:
S1扫描电镜观察材料表面结构:将A组和D组样品进行喷金,并彻底干燥,然后用扫描电镜仪器在真空中观察得到的薄膜,详细地拍摄孔径结构;
S2傅里叶红外光谱观察材料合成成分:将制备的样品在600cm-1 -4000cm-1波数范围内,通过傅里叶红外光谱测量表征其化学结构;
S3接触角测试仪观察材料亲疏水性:ABCD四组样本被放置水平,每个材料表面均滴1μL去离子水,封闭静置12h,用形成的液滴测量接触角,每个样品测试三次,得到平均角度值;
S4评估材料体外抗菌性:将大肠杆菌(E.coli)和金黄色葡萄球菌(S.aureus)菌体培养、扩增至1×109CFU/mL的密度,再经LB稀释至1×104CFU/mL的密度,提取100mL菌液。采用96孔板,将ABCD 每个样本放置3个孔,每孔滴入200μL菌液,孵化24小时,37℃孵化温度。采用分光光度计评估菌液变化,细菌原液标准OD值设为 0.7,24h后再次行OD值检测,观察不同材料对细菌原液影响。
S5评估材料体外细胞毒性:原代成纤维细胞来源于正常的新生小鼠,并进一步将细胞传代至第二代和第三代。采用96孔板进行细胞计数及培养,2000个细胞/孔,将每组样品与血管内皮细胞共同培养,每组3孔。第1、3、5、7天依次检测,37℃条件下孵化后加上LB培养基溶液中(150μL/孔),上述操作一式三份;
S6评估材料促细胞迁移能力:将血管内皮细胞接种于24孔板(2 ×104/孔)后,使用DMEM培养基培养,使用枪头划出一道划痕,并记录时间为0h,将ABCD四组材料与该细胞进行共培养,用活细胞工作站显微镜进行24小时观察,在单个实验中,每组设置6个重复,使用ImageJ 1.48V软件(美国NIH公司)进行具体测量,上述操作一式三份;
S7小鼠创面模型建立及不同材料对创面愈合影响:小鼠的腹腔内注射戊巴比妥钠(1%,70μL/g)麻醉,然后使用打孔器建立全层皮肤缺损模型,缺损面积直径0.6厘米,每个创面滴加细菌溶液(5μL 108/毫升)培养大肠杆菌和金黄色葡萄球菌,用75%的酒精对材料进行消毒,用磷酸缓冲盐溶液漂洗彻底去除杂质,然后,在伤口上涂上准备好的薄膜,然后用粘胶毛巾固定,伤后1、3、5、7天拍照更换材料,采用山东贝诺医药生物科技有限公司【国家发明专利号 ZL200620082586.1】采购的市售甲壳素敷料(CCD)作为阳性对照;
S8创面愈合计算:比较创面愈合前后创面面积,计算愈合率,采用IPP6.0软件辅助,根据“感兴趣区域”(AOI)功能选择目标创面面积,我们用“大小计数”方法测量像素面积,伤口面积可按伤口愈合率=(创面面积-愈合一定时间后的创面面积)/创面面积×100%的公式计算;
S9通过Origin软件,采用单因素方差分析和双因素方差分析分别分析两组之间和两组以上的显著性差异,实验数据以均数±标准差表示,P<0.05被认为有统计学意义。
本发明中,所述氧化石墨烯-达托霉素复合敷料制备及创面愈合方法,成功制备了氧化石墨烯膜-达托霉素/表皮生长因子敷料,其较好地实现了抗菌效果和促生长性能,对创面愈合有很大的益处。
附图说明
图1a为氧化石墨烯膜实物图,b代表A组氧化石墨烯膜和D组氧化石墨烯膜-达托霉素/表皮生长因子的扫描电镜图像;
图2为接触角(θ)的A组(氧化石墨烯膜)、B(氧化石墨烯-达托霉素),C(氧化石墨烯-表皮生长因子)、D(氧化石墨烯-达托霉素/ 表皮生长因子)图;
图3为抗菌活性测定图;
图4a为在0~24h内观察不同分组对细胞迁移的影响,b为各群体的迁移百分比图,c为细胞增殖统计图;
图5为感染创面愈合的影响a创面愈合图,b为创面愈合3,7天的统计数据图,c为创面完全愈合所需的时间图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。
参照图1-5,氧化石墨烯-达托霉素/表皮生长因子复合敷料制备,包括如下步骤:
S1准备A组氧化石墨烯膜:氧化石墨烯膜购买自南京先锋纳米有限公司(货号:100027,CAS号:7440-44-0,参数:尺寸:9x9cm厚度:约25微米);
S2制备氧化石墨烯-多巴胺膜:
S21在100mL去离子水中加入131.14gtris盐酸溶解,得到的tris溶液中加入多巴胺粉200mg,得到tris-多巴胺溶液,浓度为2mg/mL,pH值为8.5;
S22将S1中的氧化石墨烯膜浸泡在上述溶液中12h后,将混合物转移到振动筛中,在37℃下以100r/min的速度振动;
S3制备B组氧化石墨烯-达托霉素样品:
先将达托霉素粉溶解于去离子水中,制备10mg/mL的达托霉素水溶液,再将S1中的氧化石墨烯样品,经去离子水仔细清洗,孵育在达托霉素水溶液中,其中孵育温度37℃;震动速度:100r/min;孵化时间,12h。
S4制备C组氧化石墨烯膜-表皮生长因子:将S2中制备好的氧化石墨烯-多巴胺膜,溶解在10μg/ml的表皮生长因子溶液中,孵育温度37℃;震动速度:100r/min;孵化时间,12h;
S5制备D组氧化石墨烯膜-达托霉素/表皮生长因子:将氧化石墨烯膜-多巴胺加入到S3和S4步骤中相同浓度的达托霉素+表皮生长因子混合物中,孵育温度37℃;震动速度:100r/min;孵化时间,12 h,制备得氧化石墨烯膜-达托霉素/表皮生长因子膜。
氧化石墨烯-达托霉素/表皮生长因子复合敷料的表征及其对创面愈合的影响,包括如下步骤:
S1扫描电镜观察材料表面结构:将A组和D组样品进行喷金,并彻底干燥,然后用扫描电镜仪器在真空中观察得到的薄膜,详细地拍摄孔径结构;
S2傅里叶红外光谱观察材料合成成分:将制备的样品在600cm-1 -4000cm-1波数范围内,通过傅里叶红外光谱测量表征其化学结构;
S3接触角测试仪观察材料亲疏水性:ABCD四组样本被放置水平,每个材料表面均滴1μL去离子水,封闭静置12h,用形成的液滴测量接触角,每个样品测试三次,得到平均角度值;
S4评估材料体外抗菌性:将大肠杆菌(E.coli)和金黄色葡萄球菌(S.aureus)菌体培养、扩增至1×109CFU/mL的密度,再经LB稀释至1×104CFU/mL的密度,提取100mL菌液。采用96孔板,将ABCD 每个样本放置3个孔,每孔滴入200μL菌液,孵化24小时,37℃孵化温度。采用分光光度计评估菌液变化,细菌原液标准OD值设为 0.7,24h后再次行OD值检测,观察不同材料对细菌原液影响。
S5评估材料体外细胞毒性:原代成纤维细胞来源于正常的新生小鼠,并进一步将细胞传代至第二代和第三代。采用96孔板进行细胞计数及培养,2000个细胞/孔,将每组样品与血管内皮细胞共同培养,每组3孔。第1、3、5、7天依次检测,37℃条件下孵化后加上LB培养基溶液中(150μL/孔),上述操作一式三份;
S6评估材料促细胞迁移能力:将血管内皮细胞接种于24孔板(2 ×104/孔)后,使用DMEM培养基培养,使用枪头划出一道划痕,并记录时间为0h,将ABCD四组材料与该细胞进行共培养,用活细胞工作站显微镜进行24小时观察,在单个实验中,每组设置6个重复,使用ImageJ 1.48V软件(美国NIH公司)进行具体测量,上述操作一式三份;
S7小鼠创面模型建立及不同材料对创面愈合影响:小鼠的腹腔内注射戊巴比妥钠(1%,70μL/g)麻醉,然后使用打孔器建立全层皮肤缺损模型,缺损面积直径0.6厘米,每个创面滴加细菌溶液(5μL 108/毫升)培养大肠杆菌和金黄色葡萄球菌,用75%的酒精对材料进行消毒,用磷酸缓冲盐溶液漂洗彻底去除杂质,然后,在伤口上涂上准备好的薄膜,然后用粘胶毛巾固定,伤后1、3、5、7天拍照更换材料,采用山东贝诺医药生物科技有限公司【国家发明专利号 ZL200620082586.1】采购的市售甲壳素敷料(CCD)作为阳性对照;
S8创面愈合计算:比较创面愈合前后创面面积,计算愈合率,采用IPP6.0软件辅助,根据“感兴趣区域”(AOI)功能选择目标创面面积,我们用“大小计数”方法测量像素面积,伤口面积可按伤口愈合率=(创面面积-愈合一定时间后的创面面积)/创面面积×100%的公式计算;
S9通过Origin软件,采用单因素方差分析和双因素方差分析分别分析两组之间和两组以上的显著性差异,实验数据以均数±标准差表示,P<0.05被认为有统计学意义。
本发明:扫描电镜成像:氧化石墨烯膜的原理图如图1a所示。可以看出,A组氧化石墨烯膜中完整的氧化石墨烯结构保存较好,在高倍镜下可以清晰地观察到结构。D组氧化石墨烯膜-达托霉素/表皮生长因子样品中,D1仍保留片状结构,D2发生纹理变化,D3进一步显示出嵌入的达托霉素/多巴胺。白色和红色箭头分别表示达托霉素球和多巴胺层。
接触角数据:图2表明,接触角(θ)A组平均(101.18±11.42)°、 B组(86.53±9.17)°,C组(76.13±8.43)°,D组(67.99±7.60)°, 减少值表示材料疏水性逐渐增加。
抗菌活性:结果图3给出了D组≈B组>C组≈A组>与对照组的抗菌活性排序(P>0.05)。
材料的细胞毒性:从细胞增殖抑制试验收集的数据来看,C组和D组在第1-7天内均能有效促进细胞增殖(图4,P<0.05)。体外细胞迁移的比活力顺序与对照组D组≈C组>B组≈A>组一致(P<0.05)。
对感染性伤口愈合的影响:损伤后创面愈合率见图5,对照组、 A组、B组、C组、D组创面愈合率分别为40.3%、53.0%、67.8%、62.7%、 75.4%,创面愈合后7D组创面愈合率呈上升趋势(P<0.05)。同时,这五组患者的平均完全恢复时间分别为11.3、10.1、9.1、9.6和8.5 天。
当严重伤害发生在皮肤上时,一个有效的伤口闭合是至关重要的,因为它有利于防止微生物入侵和能量、电解质或体液的损失。伤口敷料具有广泛的功能,如加速伤口愈合,重建皮肤屏障,保护或准备后续手术的[33]。优良材料应具有良好的生物相容性,对水蒸气具有足够的渗透性,并具有较强的机械性能。此外,这些材料应该为伤口愈合过程创造无菌、适宜的微环境,以避免炎症或感染[34]带来的不良影响。最近有报道说氧化石墨烯纳米薄片具有抗菌活性。尽管如此,由于水溶液中分子层间的相互作用,从而阻止了氧化石墨烯纳米片更广泛的应用。此外,也有研究报道氧化石墨烯对抗菌性能有微弱甚至不利的影响[17-19]。为此,本研究通过多巴胺凝胶反应制备氧化石墨烯-达托霉素/表皮生长因子纳米复合敷料,证实氧化石墨烯 -达托霉素/表皮生长因子纳米复合敷料的抗菌活性。
图1a是氧化石墨烯膜的示意图,可以看出,原始的氧化石墨烯结构在A组氧化石墨烯膜中得到了完整的保存,在高倍镜下观察到清晰的纹理,D组氧化石墨烯膜-达托霉素/表皮生长因子样品中,D1仍可见片状结构,D2中规则和纹理发生变化,D3中嵌入的达托霉素/多巴胺层进一步析出,此外,白色和红色箭头分别表示达托霉素和多巴胺层。这是氧化石墨烯膜结构随反应前的变化而变化的结果,使达托霉素层有效地附着在膜上,平均接触角(θ)A组(101.18± 11.42)°、B组(86.53±9.17)°,C组(76.13±8.43)°,D组(67.99 ±7.60)°.显然,亲水性的样品逐渐增加,受益很多达托霉素去膜的抗菌改进,根据早期的研究,氧化石墨烯通过破坏细胞膜或诱导氧化应激而获得较高的抗菌活性,而达托霉素可以通过扰乱细胞膜达到同样的效果。上述实验结果显示,用纳米复合材料处理的细菌细胞膜受损。然后,我们想知道氧化石墨烯-达托霉素样品是否能达到同等甚至促进的抗菌效果。由于革兰氏阴性菌对氧化石墨烯-达托霉素的耐药性通常高于革兰氏阳性菌,因此选择大肠杆菌和耐甲氧西林金黄色葡萄球菌进行后续抗菌机制的研究。结果表明,氧化石墨烯纳米片与达托霉素配合使用,可获得较好的抗菌性能。具体来说,一方面,氧化石墨烯薄片可以与目标细菌相互作用,包裹住它们的细胞膜;另一方面,氧化石墨烯使接枝的达托霉素充分接触到菌膜,使更多的达托霉素能够集中在靶菌周围,24小时培养金黄色葡萄球菌和大肠杆菌导致测定样品的抗菌活性是按照以下顺序:D组≈B组>C组≈A组(P> 0.05,图3)。这项研究证明氧化石墨烯-达托霉素有一个强大的抗菌活性;因此,抗药菌株几乎不可能存在。达托霉素在体外对革兰氏阳性微生物的灭菌活性广泛(例如耐甲氧西林金黄色葡萄球菌及抗链球菌),并被批准治疗链球菌、大肠粪球菌及耐药的金黄色葡萄球菌等。然而,达托霉素对相应感染可能已逐步出现耐药,目前最好的达托霉素量-效关系尚未建立,一些体外研究显示使用剂量10-12mg/Kg时即应避免耐药性发展。细胞增殖抑制实验(图4)表明,C和D组可以有效地促进细胞增殖(P<0.05),体外细胞迁移实验表明,促进迁移效果为D组≈C组>B组≈A组>对照组(P<0.05)。结合表皮生长因子在乳化微球中的作用,可以稳定地将细胞因子传递到受损的表皮中,刺激细胞进行表皮重构。以前的研究已经表明,表皮生长因子通过海藻酸盐或脂质微球和纳米球在糖尿病溃疡的慢性伤口模型中的作用,但表皮生长因子通过载体在急性伤口中的作用还有待阐明。急性放射性皮炎等总是伴随着体表的损伤,即我们的防御屏障被破坏,随之而来的是我们的免疫能力下降。广泛的组织坏死和细菌侵袭会引起创面感染,一直是放疗常见并发症。图5中分别为对照组、a组、B组、C 组、D组的7天创面愈合率分别为40.3%、53.0%、67.8%、62.7%、75.4%;D 组远远好于对照组,差异有统计学意义(P<0.05)。因此,D组、C 组、B组、A组和对照组完全恢复所需的平均霜量分别从8.5天增加到9.1天、9.6天、10.1天和10.3天。在伤口愈合的过程中,伤口会与上皮细胞的形成一起收缩。由于人类是皮肤致密的物种,重新上皮化作用是伤口愈合的主要驱动力。为了解决上述缺点,研究人员将重点放在结合生物材料(如纤维蛋白、胶原蛋白、葡聚糖和壳聚糖) 的胶囊化生长因子开发。该系统无需多个管理过程,即可针对生长因子进行持续释放。在这些,多巴胺是一个多功能载体,因为它可生物可降解,并可与表皮细胞的已知成分结合,在表皮再生过程中经历重塑。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (2)
1.氧化石墨烯-达托霉素/表皮生长因子复合敷料制备,其特征在于,包括如下步骤:
S1准备A组氧化石墨烯膜:氧化石墨烯膜购买自南京先锋纳米有限公司,货号:100027,CAS号:7440-44-0,参数:尺寸:9x9cm厚度:约25微米;
S2制备氧化石墨烯-多巴胺膜:
S21在100mL去离子水中加入131.14g tris盐酸溶解,得到的tris溶液中加入多巴胺粉200mg,得到tris-多巴胺溶液,浓度为2mg/mL,pH值为8.5;
S22将S1中的氧化石墨烯膜浸泡在上述溶液中12h后,将混合物转移到振动筛中,在37℃下以100r/min的速度振动;
S3制备B组氧化石墨烯-达托霉素样品:
先将达托霉素粉溶解于去离子水中,制备10mg/mL的达托霉素水溶液,再将S1中的氧化石墨烯样品,经去离子水仔细清洗,孵育在达托霉素水溶液中,其中孵育温度37℃;震动速度:100r/min;孵化时间,12h;
S4制备C组氧化石墨烯膜-表皮生长因子:将S2中制备好的氧化石墨烯-多巴胺膜,溶解在10μg/ml的表皮生长因子溶液中,孵育温度37℃;震动速度:100r/min;孵化时间,12h;
S5制备D组氧化石墨烯膜-达托霉素/表皮生长因子:将氧化石墨烯膜-多巴胺加入到S3和S4步骤中相同浓度的达托霉素+表皮生长因子混合物中,孵育温度37℃;震动速度:100r/min;孵化时间,12h,制备得氧化石墨烯膜-达托霉素/表皮生长因子膜。
2.氧化石墨烯-达托霉素/表皮生长因子复合敷料的表征及其对创面愈合的影响,包括如下步骤:
S1扫描电镜观察材料表面结构:将A组和D组样品进行喷金,并彻底干燥,然后用扫描电镜仪器在真空中观察得到的薄膜,详细地拍摄孔径结构;
S2傅里叶红外光谱观察材料合成成分:将制备的样品在600cm-1-4000cm-1波数范围内,通过傅里叶红外光谱测量表征其化学结构;
S3接触角测试仪观察材料亲疏水性:ABCD四组样本被放置水平,每个材料表面均滴1μL去离子水,封闭静置12h,用形成的液滴测量接触角,每个样品测试三次,得到平均角度值;
S4评估材料体外抗菌性:将大肠杆菌(E.coli)和金黄色葡萄球菌(S.aureus)菌体培养、扩增至1×109CFU/mL的密度,再经LB稀释至1×104CFU/mL的密度,提取100mL菌液。采用96孔板,将ABCD每个样本放置3个孔,每孔滴入200μL菌液,孵化24小时,37℃孵化温度。采用分光光度计评估菌液变化,细菌原液标准OD值设为0.7,24h后再次行OD值检测,观察不同材料对细菌原液影响;
S5评估材料体外细胞毒性:原代成纤维细胞来源于正常的新生小鼠,并进一步将细胞传代至第二代和第三代。采用96孔板进行细胞计数及培养,2000个细胞/孔,将每组样品与血管内皮细胞共同培养,每组3孔,第1、3、5、7天依次检测,37℃条件下孵化后加上LB培养基溶液中(150μL/孔),上述操作一式三份;
S6评估材料促细胞迁移能力:将血管内皮细胞接种于24孔板(2×104/孔)后,使用DMEM培养基培养,使用枪头划出一道划痕,并记录时间为0h,将ABCD四组材料与该细胞进行共培养,用活细胞工作站显微镜进行24小时观察,在单个实验中,每组设置6个重复,使用ImageJ1.48V软件(美国NIH公司)进行具体测量,上述操作一式三份;
S7小鼠创面模型建立及不同材料对创面愈合影响:小鼠的腹腔内注射戊巴比妥钠(1%,70μL/g)麻醉,然后使用打孔器建立全层皮肤缺损模型,缺损面积直径0.6厘米,每个创面滴加细菌溶液(5μL108/毫升)培养大肠杆菌和金黄色葡萄球菌,用75%的酒精对材料进行消毒,用磷酸缓冲盐溶液漂洗彻底去除杂质,然后,在伤口上涂上准备好的薄膜,然后用粘胶毛巾固定,伤后1、3、5、7天拍照更换材料,采用山东贝诺医药生物科技有限公司【国家发明专利号ZL200620082586.1】采购的市售甲壳素敷料(CCD)作为阳性对照;
S8创面愈合计算:比较创面愈合前后创面面积,计算愈合率,采用IPP6.0软件辅助,根据“感兴趣区域”(AOI)功能选择目标创面面积,我们用“大小计数”方法测量像素面积,伤口面积可按伤口愈合率=(创面面积-愈合一定时间后的创面面积)/创面面积×100%的公式计算;
S9通过Origin软件,采用单因素方差分析和双因素方差分析分别分析两组之间和两组以上的显著性差异,实验数据以均数±标准差表示,P<0.05被认为有统计学意义。
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CN114948861A (zh) * | 2022-05-26 | 2022-08-30 | 中国人民解放军西部战区总医院 | 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 |
CN114948861B (zh) * | 2022-05-26 | 2023-08-25 | 中国人民解放军西部战区总医院 | 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 |
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