CN112118860A - 用于治疗人体皮肤溃疡、烧伤以及创面的胶原蛋白-层粘连蛋白基质的生产方法 - Google Patents
用于治疗人体皮肤溃疡、烧伤以及创面的胶原蛋白-层粘连蛋白基质的生产方法 Download PDFInfo
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Abstract
本发明涉及医学生物技术，更具体地涉及再生医学中的组织工程方法。该方法的特征在于，与保守治疗方法一起，提供了替代疗法，用于通过聚合物基质来治疗受损皮肤，该聚合物基质的组织结构类似于人体组织结构，并且包括来源细胞的生物活性剂，尤其是胶原蛋白和层粘连蛋白，可促进受损部位的结构形成活性。基于聚合物基质和由人类上皮细胞合成的产物的组合的可生物降解的创面愈合复合材料，尽管所述材料不含细胞成分，但其特征在于生产过程相对简单并且储存寿命长，并且例如在大面积烧伤、营养性溃疡等情况下，可以避免皮肤移植。
Description
本发明涉及医学生物技术，尤其涉及用于解决再生医学问题的细胞技术和组织工程方法。根据该方法，提出了与保守治疗方法一起，使用聚合物基质(matrice)(在组织学上类似于人体组织)和生物活性剂(细胞衍生物-胶原蛋白和层粘连蛋白)，其中胶原蛋白和层粘连蛋白有助于形成受损区域结构的功能，来治疗皮肤损伤的替代疗法。
细胞技术的使用是现代再生医学最重要且发展迅速的领域。胶原蛋白-层粘连蛋白基质的任务是将活细胞移植到缺损区域，完全恢复皮肤的结构和功能，还可以刺激再生过程并创造微环境，以实现自身组织和细胞的潜力。组织工程技术应用于解决此类问题[8]。组织工程旨在解决再生医学的许多问题，通过恢复器官和组织的丧失的结构和功能来实现个人生命周期的延长和生活质量的提高。使用基于细胞技术创造的新材料的使用可以成为治疗和恢复组织的前瞻性方法[2、3、5、6]。
目前正在开发基于亲皮的(dermotropic)生物医学细胞产品的方法，以治疗长期的创面(营养性溃疡、烧伤)并恢复或暂时闭合大面积的皮肤。刺激组织再生的细胞技术使用包括利用不同类型，(例如)生物基质，和以不同变体及其组合的类似组织的结构的细胞。
已知现有使用表皮自体移植(epidermal autografting)覆盖大面积烧伤的方法。然而，在同种异体(allogenic)自体移植的情况下，使用表皮层治疗烧伤患者在组织存活率方面因为具有免疫原性和可受性(affordability)而遭遇困境。各种其他类型的细胞-真皮成纤维细胞(dermal fibroblasts)、间充质骨髓干细胞(mesenchymal bone marrow stemcells)，诱导多能干细胞(induced pluripotent stem cells)-已成功地用于临床前和临床研究。但是，这种处理通常需要大量的细胞材料。在小创面的治疗中这不是主要问题，但在大伤口的治疗中可能会出现[8]。
一些有效利用亲皮成分以用于皮肤再生的方法(例如胶原蛋白凝胶、海绵以及胶原蛋白基)，是由天然和合成聚合物改性而来，但是再生过程由于稳定性和生物兼容性低而变得复杂[9]。
在闭合较小伤口方面取得了积极成果的临床试验表明，已使用表皮和真皮等效物，包括牛胶原蛋白与人类新生儿成纤维细胞(Apligraf)、新生儿包皮的异基因成纤维细胞在合成基体上(Dermagraft)，然而，在这些变体中，具有成纤维细胞的三维胶原蛋白凝胶在组织工程结构的感染、免疫原性、储存以及运输方面遭遇难题。[8,10].
所要求保护的方法如下执行。I型胶原蛋白是从大鼠尾部肌腱获得的。这要求在实验室饲养条件(排除无感染、寄生虫等)下体重等于或大于300克的成年Wistar品系大鼠(或类似动物)。动物安乐死后的大鼠尾部可以在-20℃下冷冻保存数年。在分离胶原蛋白之前，将尾部用标准洗涤剂洗涤，并在提取胶原蛋白之前用70％乙醇灭菌24小时。所有操作均在无菌条件下进行(在生物安全等级为第一级或第二级的层流柜中)。用手术刀和镊子从尾部上去除皮肤，将尾部切成2-3厘米的小块。将所得的尾部片段再次在70％乙醇中孵育15分钟。然后用镊子从尾部除去肌腱，将肌腱分裂成细纤维，并放入装有0.1％冰醋酸无菌溶液的容器中。胶原蛋白几乎立即开始被提取到溶液中，溶液变得粘稠，呈蓝色乳浊雾状。为了获得正确的浓度和溶液浓度，应将一整根尾部的肌腱溶解在100-150ml的溶液中。然后将溶液在+4℃下孵育一个月。如果可能，应每天取出装有溶液的瓶子，并搅拌沉淀物。结果获得粘稠的液体，底部有不溶的沉淀物。然后将胶原蛋白溶液离心(在室温下以1500g～4000rpm的转速旋转1小时，转子半径为10cm)以沉淀未溶解的胶原蛋白纤维。将上清液排入无菌储存容器中。溶液的浓度可通过在干燥空气恒温器中蒸发水分并称重所得沉淀物来计算。胶原蛋白浓度在不低于1mg/ml(通常3-5mg/ml)的浓度下可以被认为是可接受的胶原蛋白溶液。
对于胶原蛋白聚合，应中和乙酸的酸性溶液。为此，将胶原蛋白溶液倒入无菌培养皿中(如有必要，培养皿半径为3cm、6cm或10cm)，并在搅拌下添加适量的0.1M NaOH溶液混合以形成的pH约为7.0至7.2的溶液。该溶液的酸度可以通过添加含有酚红染料的Ml 99配方来很容易地确定，该染料的颜色取决于pH值。溶液的所需颜色为深红色。通过改变胶原蛋白溶液的量，可以产生不同深度的基质。胶原蛋白的聚合在3-5分钟内发生，因此所有步骤均必须迅速进行，最好在冰上进行。加入所有必要的成分并混合后，将带有胶原蛋白凝胶的培养皿放在+37℃的培养箱中放置数小时(一天为最佳)。
第二天，将HaCat系细胞以每平方厘米10000-40000个细胞的密度植入在胶原蛋白凝胶上。HaCat系细胞是永生化的具有良好的增殖能力的人类角质形成细胞，，并广泛用于科学研究。从上方添加所需量的HaCaT细胞的完全生长培养基，并将该组合物进一步温育几天，直到达到致密和超密的细胞单层。在这段时间内，HaCat细胞在凝胶表面上合成了基底膜的层粘连蛋白层。
在达到所需的细胞密度后，将所得基质在4℃下用4％多聚甲醛固定1天。因此，组合物中的所有蛋白质融合在一起形成不溶的单一复合物，细胞失活。固定后，用无菌磷酸盐缓冲盐水(phosphate-salt buffer，PBS)的无菌溶液从甲醛中洗去移植物，在定轨摇床上振荡3次，每次持续15分钟。然后，基质在SDS洗涤剂和0.1％Triton-X-100洗涤剂中，在1天内于+4℃进行脱细胞阶段。在此过程中，脂质、脂质和蛋白质之间的键断裂，所有细胞内成分被释放并洗去，DNA和RNA细胞发生降解。脱细胞后，用无菌磷酸盐缓冲盐水(PBS)将移植物从洗涤剂上洗去，在定轨摇床上振荡6次，每次1小时。最后的洗涤阶段在+4℃下进行1天。然后可以将基质在PBS溶液中于+4℃下保存一个月，或在-20℃的冷冻冰箱中干燥两年。
所得的基质是由两种主要成分组成的蛋白质组合物，I型胶原蛋白和基底膜层粘连蛋白，它们在组织结构上与人类皮肤的结构完全相似。胶原蛋白形成移植物的基质，层粘连蛋白以基底膜的形式覆盖基质。该基质是无菌的、具有免疫耐受性的(因为胶原蛋白为具有高度保守性、免疫耐受性的蛋白质，并且层粘连蛋白为人源性的)，没有任何细胞成分，这使其成为医疗产品并大大简化了注册程序。
可以将各种添加剂(抗生素、生长因子、细胞因子)添加到最终基质组合物中，使其成为具有巨大开发潜力的便捷基础模型。HaCat细胞系可以合并到其他永生化的上皮细胞系中，产生其他类型的层粘连蛋白，从而让使用基质来治疗其他上皮-间质缺陷(输尿管尿道上皮、喉上皮、肠上皮等)成为可能。
借助细胞技术以及生物聚合基体的应用在体外进行的研究，使得有可能获得关于基于以凝胶膜形式提取的、具有脱细胞层粘连蛋白表面的胶原蛋白基质的稳定组织工程化组织型组合物的可能性数据。免疫组织化学荧光分析鉴定了复合蛋白(如I型胶原蛋白和层粘连蛋白)以及上皮细胞标记物泛细胞角蛋白(pan-cytokeratin)的表达。
所提出的三维基质的变体是一种蛋白质组合物，由两种主要成分组成，即第一类胶原和基底膜层粘连蛋白，它们与人类皮肤的结构具有完全的组织学相似性。胶原蛋白和层粘连蛋白是人类皮肤蛋白的两种最常见类型。胶原蛋白是皮肤真皮间质成分的基础，具有机械强度和弹性。层粘连蛋白是器官的任何基底膜的基础，包括皮肤的基底膜[1、7]。
在雄性Wistar大鼠的拼凑肌皮创面模型上获得的21天模拟创面过程动态的对照研究结果表明，在第7、14天使用胶原蛋白-层粘连蛋白基质的组中，呈现出伤口缺损的面积加速减少。从创面的底部观察到肉芽组织的生长，并且在结缔组织的肿瘤中观察到血管化。
关于皮肤完整性的恢复获得的数据表明，由于维持血管化过程的再生组织的定向生长，所产生的修复组合物具有高创面愈合效率。基于聚合物基体和人类角质形成细胞合成产物的组合而开发的生物兼容性胶原蛋白-层粘连蛋白创面愈合材料，尽管不含细胞成分，但其特点是易于制造且保质期长，因此可以在实践中推广本设计。
参考文献
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Claims (1)
1.一种用于治疗人体皮肤溃疡、烧伤以及创面的胶原蛋白-层粘连蛋白基质的生产方法，其特征在于，包括使用以下方法的组合获得组织工程化的创面组合物：从大鼠尾部肌腱中提取胶原蛋白，形成胶原蛋白基质，在所述胶原蛋白基质上培养HaCaT系皮肤的永生化角质形成细胞，借助洗涤剂SDS(最高至癸基硫酸钠)和Triton-X-100(辛基苯基聚氧乙烯)溶液，将形成的胶原蛋白膜去除细胞膜(从最终产物中去除细胞)，并在其表面保留层粘连蛋白(基底膜的主要蛋白质)，所述层粘连蛋白由HaCaT系细胞合成。
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