CN107802883B - 一种伤口清洗液 - Google Patents
一种伤口清洗液 Download PDFInfo
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Abstract
本发明提供一种伤口清洗液，为包含有羧甲基壳聚糖和多肽的溶液，所述的多肽的氨基酸序列为SEQ ID NO:1。本发明提供的伤口清洗液对伤口无刺激，即可以清洗伤口，又具有杀菌、的作用。而且其中使用的多肽与血液相接触的时候会形成凝胶态，具有止血功能，从而有效的防止处理后的伤口再次出血。
Description
技术领域
本发明属于医学材料技术领域，具体涉及一种伤口清洗液。
背景技术
在医学领域中，伤口分为如下几种类型：
1、擦伤：擦伤是皮肤表层擦破伤中最常见的一种类型，由于钝器(略有粗糙)机械力摩擦的作用，造成表皮剥脱、翻卷为主要表现的损伤。可表现为抓痕、擦痕、撞痕、压痕、压擦痕等，损伤轻微。
2、刺伤：刺伤是由锐器戳刺所致人体的损伤，常见的锐器有针、剪、木刺等，损伤特点为皮肤伤口小，但往往造成深部组织损伤，因引流不畅，易继发化脓性感染或破伤风等。
3、割伤：割伤是锐器作用于人体所致的软组织损伤。常见的锐器有刀刃、玻璃片和竹片等，边缘整齐，周围组织破坏相对轻。切割伤口达到皮肤下的组织。因为损伤比擦伤深，所以更容易发生出血等问题，也有损伤神经和肌腱的可能性。
4、咬蜇伤：通常以猫狗抓咬伤、蜂蜇伤为主。猫狗抓咬伤对组织有切割、撕扯作用，常伴不同程度的组织挫裂伤，由于动物口腔中有大量细菌，可进入伤口，因此切不可忽视。蜂蜇伤是蜂类的尾针刺伤皮肤时将毒液注入皮内所致。常见有蜜蜂蜇伤和黄蜂蜇伤。
5、烧伤与烫伤：多由火焰、热液(水、汤、油等)、蒸汽所引起的组织损害。
在现有的医疗实际操作中，用于清洗伤口的药品主要是双氧水、碘液、酒精、生理盐水等。氧水、碘液、酒精虽然具有强的杀菌消毒的作用，但在清洗伤口的时候会感到非常的疼痛，且没有止血的效果。
发明内容
本发明提供一种伤口清洗液，所述的清洗液包含有设计筛选的自组装多肽和水溶性的羧甲基壳聚糖，该清洗液能够有效的清洗出伤口，避免细菌感染，并且在伤口处形成凝胶状的膜，产生防护效果；从而弥补现有技术的不足。
本发明所提供的清洗液，为包含有羧甲基壳聚糖和多肽的溶液，所述的多肽的氨基酸序列为Asp-Glu-leu-Lys-val-Cys-Cys-Cys-Ile-Ile-Cys-Cys-Cys-Glu-leu-Lys-val-Arg(DELKVCCCIICCCELKVR，SEQ ID NO:1)。
作为优选，所述的清洗液中羧甲基壳聚糖的浓度为2-4g/ml；多肽的浓度为10-50mg/ml。
所述的羧甲基壳聚糖，其分子量为2000-10000，取代度60％或以上。
本发明提供的伤口清洗液对伤口无刺激，即可以清洗伤口，又具有杀菌、的作用。而且其中使用的多肽与血液相接触的时候会形成凝胶态，具有止血功能，从而有效的防止处理后的伤口再次出血。
附图说明
图1：氨基酸序列为SEQ ID NO:1的多肽的原子力显微镜(AFM)形貌图；
图2：氨基酸序列为SEQ ID NO:1的多肽的原子力显微镜(AFM)形貌图；
图3：实施例1制备的清洗液对金黄色葡萄球菌和大肠杆菌的抑制图，其中
A和b平板分别为金黄色葡萄球菌的对照组和实验组；C和D分别为大肠杆菌的对照组和实验组；
图4：清洗液的原子力显微镜(AFM)形貌图。
具体实施方式
下面结合实施例对本发明进行详细的描述。
实施例1：多肽的筛选
1、多肽的设计
本发明设计的短肽通过非共价键相互作用，使该短肽能在缓冲液中自组装形成纤维结构。其氨基酸序列为Asp-Glu-leu-Lys-val-Cys-Cys-Cys-Ile-Ile-Cys-Cys-Cys-Glu-leu-Lys-val-Arg(DELKVCCCIICCCELKVR，SEQ ID NO:1)。
合成时多肽的N端进行乙酰化修饰，C端进行氨基化修饰；合成的纯度为95.5％。合成的初始样品为白色冻干粉，使用前无须经过特殊处理。使用时将精密称量后的短肽冻干粉末加入到超纯水中配制成浓度为100mg/ml的母液，密封并避光储存在4℃冰箱中备用，后续使用时将其直接稀释成相应浓度短肽溶液。
2、凝血活性的测定
首先从志愿者采集血液，血液装入含有0.9mL枸橼酸钠的离心管中，混合均匀后血液样本以3000r/min离心10min得到待测血液。
在96孔微孔板中，采用倍比稀释方法进行。首先在每个孔中加入20μl的10mmol的HEPES缓冲液(pH7.4)，取合成的多肽溶液(初始浓度为10mg/ml)20μl加入到第一实验孔中；混均后，取20μl加入到第二孔中；混合均匀后，再20μl加入到第三孔中，以此类推作倍比稀释；
然后再每孔中加入40μl的待检测血液，添加结束后再混合仪上震动混均5min，室温下放置1小时后，肉眼检测凝集结果，凝血活性单位定义为表现凝血活性的最高稀释倍数的倒数。结果表明氨基酸序列为SEQ ID NO:1的多肽，其最高稀释倍数为9倍，即凝血活性为18。
3、对氨基酸序列为SEQ ID NO:1的多肽进行原子力显微镜(AFM)的检测：
将30μl的多肽溶液置于新解离的云母片上，室温下吸附10秒左右，用超纯水冲洗吸附后的云母片去除多余的溶液，用氮气轻轻吹扫确保云母片干燥。利用原子力显微镜观测组装体的形貌图，实验中采用TESP型探针(Veeco,Santa Barbara,CA)在轻敲模式下扫描云母片表面，扫描速率为1-1.5Hz，数字分辨率512×512，利用AFM自带软件(Nanoscope5.31r1)对图像进行检测。
对氨基酸序列为SEQ ID NO:1的多肽进行透射电子显微镜(TEM)的检测：
将30μl的多肽溶液滴在封口膜上，然后将300目的镀碳铜网贴放在样品上约30秒，用干净的镊子取出铜网，并用滤纸吸干铜网周围多余的液体静置晾干，然后将吸附有两亲性短肽的铜网一面贴附在2％的乙酸双氧铀溶液液滴(同样品溶液相同放置在封口膜上)上进行负染约1分钟，用镊子取出铜网将多余的液体用滤纸吸干静置晾干。采用JEOL JEM-2100UHR型电子显微镜观测形貌，加速电压为200k V。
其结果显示，通过原子力显微镜和透射电子显微镜观察到的多肽溶液都显示为纤维结构(图1和图2)。
实施例2：伤口清洗液的制备及抗菌性的检测
本发明所使用的羧甲基壳聚糖，其一种制备方法如下：
称取10g壳聚糖加入到250ml四颈瓶中，再加入计量的40％的NaOH溶液和75ml异丙醇，安装好搅拌装置，回流装置，然后在搅拌下水浴慢慢加热至40℃，并在此温度下碱化2h。碱化完成后，由滴液漏斗滴加入50％的氯乙酸水溶液，并控制滴加速度使四颈瓶中物料的温度不高于50℃。滴加完毕后升温，待结束反应，用10％的盐酸溶液调pH值使混合物的pH值在7左右，并对物料进行抽滤，滤渣用40ml 80％甲醇水溶液浸泡洗涤并抽滤，再用无水乙醇浸泡洗涤后用红外干燥至产品恒重，得到羧甲基化壳聚糖。
为了获得更好的杀菌效果，选用分子量为2000-10000的羧甲基壳聚糖(采用乌式粘度计粘度法测定分子量)，取代度60％或以上。
取4g分子量为6000-9000的羧甲基壳聚糖(取代度80％)，加入到90ml超纯水中，超声杀菌后，再在无菌状态下加入10ml浓度为100mg/ml的氨基酸序列为SEQ ID NO:1的多肽母液；再定容到100ml制成清洗液；其中羧甲基壳聚糖的浓度为4g/ml；多肽的浓度为10mg/ml。
对本实施例制备的清洗液对金黄色葡萄球菌与大肠杆菌这两种典型菌的抗菌性能，具体步骤如下：
釆用牛肉膏蛋白胨培养基，其中液体培养基的配置方法如下：用电子天平称取10g蛋白胨、10gNaCl、5g牛肉膏于1000ml烧杯中，加入去离子水至950ml刻度处，置于磁力搅拌器上搅拌让其完全溶解，用pH 1.0的NaOH溶液调节培养基的pH值到7.0，最后加去离子水将培养基定容到1000ml，分装到三口烧瓶中，封口，湿压灭菌锅灭菌，备用。
固定培养平板的制备方法如下：用电子天平称取10g蛋白胨、10gNaCl、5g牛肉膏、5g琼脂于烧杯中，加入去离子水至950ml刻度处，置于磁力搅拌器上边加热边搅拌让其完全溶解，用pH 1.0的NaOH溶液调节培养基的pH值到7.0，最后加去离子水将培养基定容到1000ml分装到三口烧瓶中，封口，湿压灭菌，倒板后制成固定培养基备用。
将本实施例制备的清洗液在无菌状态下涂布到固定培养平板上，每个平板涂布100μl作为实验组；而没有涂布清洗液的培养平板作为对照组。
将选育好的金黄色葡萄球菌与大肠杆菌分别用牛肉膏蛋白胨液体培养基稀释50倍后，用移液枪吸取50μl稀释的受试菌分别涂布到实验组和对照组的平板上，用涂布棒将菌液涂布均匀，将然后将平板置于37℃的恒温培养箱中培养过夜，其中实验组和对照组，金黄色葡萄球菌与大肠杆菌分别做三个平行。
结果表明，本发明制备的清洗液对金黄色葡萄球菌和大肠杆菌均有明显的抑制作用(图3)。
为了研究清洗液中羧甲基壳聚糖和多肽添加浓度对清洗液抑菌效果的影响，将清洗液中的羧甲基壳聚糖的浓度设定为2g/ml、3g/ml、4g/ml；多肽的含量为10mg/ml、20mg/ml、40mg/ml；在实施例3中制备的培养平板(没有涂布清洗液)上分别涂布金黄色葡萄球菌和大肠杆菌；然后在涂布了菌株的平板上放置无菌牛津杯，在杯内分别注入含有不同浓度的羧甲基壳聚糖、多肽的清洗液；以超纯水作为对照；在37℃的恒温培养箱中培养48h后，之后取出测量抑菌圈直径大小；其中不同浓度羧甲基壳聚糖的抑菌效果如表1所示。
表1：不同浓度的羧甲基壳聚糖的抗菌效果
其中，－代表没有抑菌圈；+为抑菌圈直径为6.0mm～8.0mm；++为抑菌圈直径为8.0mm～10.0mm；+++为抑菌圈直径为10.0mm～12.0mm；++++为抑菌圈直径为14.0mm以上。
而对多肽抑菌圈的分析，其20mg/ml浓度的抑菌圈要大于10mg的，但20mg/ml与40mg/ml的抑菌圈大小没有显著的区别。
研究表明本发明的清洗液对表皮葡萄球菌、绿脓菌和白色念珠菌等常见的感染菌也有明显的抑制效果。
对实施例1制备的清洗液进行原子力显微镜(AFM)的检测，将20μl的多肽溶液置于新解离的云母片上，室温下吸附30秒左右，用超纯水冲洗吸附后的云母片去除多余的溶液，用氮气轻轻吹扫确保云母片干燥。利用原子力显微镜观测组装体的形貌图，实验中采用TESP型探针(Veeco,Santa Barbara,CA)在轻敲模式下扫描云母片表面，扫描速率为1-1.5Hz，数字分辨率512×512，利用AFM自带软件(Nanoscope 5.31r1)对图像进行检测。
结果表明多肽在底部形成了纤维网状结构，而羧甲基壳聚糖分布其中(图4)，上述结构表明制备的清洗液会形成凝胶状结果，从而有效的保护伤口。
将SD大鼠腿部的肌肉暴露出来，找出大鼠腿部静脉，用注射器针头扎破静脉，制造割伤点和出血点；然后用本发明实施例1制备的清洗液进行清洗后，用无菌纱布进行包扎处理；并每天用清洗液清洗。观察结果表明，在小鼠伤口愈合期间，并没有发生细菌感染的症状。而且，由于多肽的存在，在小鼠伤口处形成了膜状结构，产生明显的止血效果。
序列表
<110> 青岛市妇女儿童医院
<120> 一种伤口清洗液
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
Asp Glu Leu Lys Val Cys Cys Cys Ile Ile Cys Cys Cys Glu Leu Lys
1 5 10 15
Val Arg
Claims (5)
1.一种伤口清洗液，其特征在于，所述的清洗液为包含有羧甲基壳聚糖和多肽的溶液，所述的多肽的氨基酸序列为SEQ ID NO:1；
所述的羧甲基壳聚糖的浓度为2-4g/ml；
所述的清洗液中多肽的浓度为10-50mg/ml。
2.如权利要求1所述的清洗液，其特征在于，所述的羧甲基壳聚糖，其分子量为2000-10000。
3.如权利要求2所述的清洗液，其特征在于，所述的羧甲基壳聚糖，其分子量为6000-9000。
4.如权利要求1所述的清洗液，其特征在于，所述的羧甲基壳聚糖取代度为60%或以上。
5.如权利要求4所述的清洗液，其特征在于，所述的羧甲基壳聚糖取代度为80%。
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