CN108939176A - 负载纳豆激酶的小口径人工血管及其制备方法 - Google Patents

负载纳豆激酶的小口径人工血管及其制备方法 Download PDF

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CN108939176A
CN108939176A CN201810830187.6A CN201810830187A CN108939176A CN 108939176 A CN108939176 A CN 108939176A CN 201810830187 A CN201810830187 A CN 201810830187A CN 108939176 A CN108939176 A CN 108939176A
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王淑芳
侯丁
史杰
张祥云
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Nankai University
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Abstract

本发明公开了一种负载纳豆激酶的小口径人工血管及其制备方法，其特点在于利用多巴胺在生物可降解脂肪族聚酯材料上自聚合形成具有超强黏性的聚多巴胺涂层，再将纳豆激酶通过聚多巴胺涂层负载到聚酯材料上，形成负载纳豆激酶的小口径人工血管。所构建的人工血管具有良好的亲水性和生物相容性，有利于细胞在材料上的黏附和增殖，从而有利于血管再生；纳豆激酶具有抗凝血和溶解血栓的作用，使得所构建的小口径人工血管具备良好的血液相容性，避免血栓的形成，具有良好抗凝血效果，能够保持血管畅通。

Description

负载纳豆激酶的小口径人工血管及其制备方法
技术领域
本发明涉及一种负载了抗凝血物质的小口径人工血管及其制备方法，具体说是在聚酯材料上负载纳豆激酶的小口径人工血管及其制备方法。属于生物材料及生物医学工程领域。
背景技术
随着人们生活水平的提高，心血管疾病已成为全球造成死亡的主要原因，它持续威胁着人类的健康，如何治疗心血管疾病成为一项非常重要的课题。在各类心血管疾病的诸多治疗方法中，血管移植是一种重要的治疗方法。血管移植是通过外科手术，在患者体内需要进行血管移植的部位植入血管代替物。现在临床上常用的血管代替物一般包括天然血管和人工血管。目前人工血管中大口径人工血管已有商品化的产品，而小口径人工血管还没有满意的替代物。小口径人工血管血栓及内膜增生的问题至今仍未能得到妥善解决，仍然是研究的热点和难点。所以构建新型小口径人工血管，使其减少血栓形成，具有良好的细胞相容性和血液相容性，并且有利于内皮细胞黏附，保持血管畅通，这对于小口径人工血管的研究与应用具有重要意义。
纳豆激酶是一种在纳豆发酵过程中产生的，能够有效溶解血栓的蛋白酶，纳豆激酶还具有将其负载于小口径人工血管上，以期解决血栓和内膜增生的问题。
纳豆激酶不仅具有良好的溶解血栓的作用，同时也具有抗凝血的效果。研究发现，纳豆激酶可以通过抑制内源性和外源性的凝血途径，从而抑制凝血活酶形成。纳豆激酶还可以显著延长凝血酶引起的纤维蛋白凝固时间。因此纳豆激酶具有抗凝血的效果，并且和肝素相比，作用更为缓和，不易引起出血。
发明内容
本发明针对现有的小口径人工血管材料存在的血栓以及内膜增生的问题，以及纳豆激酶抗凝血和溶解血栓的性能，制备负载纳豆激酶的小口径人工血管，使其能够抑制和溶解血栓，促进血管的再生。
本发明所述负载纳豆激酶的小口径人工血管，是利用多巴胺在生物可降解脂肪族聚酯材料上自聚合形成具有超强黏性的聚多巴胺涂层，再将纳豆激酶通过聚多巴胺涂层负载到聚酯材料上，形成负载纳豆激酶的小口径人工血管；其具体制备方法包括以下步骤：
1)配制静电纺丝溶液：首先将生物可降解脂肪族聚酯溶于氯仿/甲醇混合溶剂，其中氯仿与甲醇的体积比为3∶1-6∶1，聚酯的质量/体积浓度为10-25％，用来进行静电纺丝；静电纺丝条件为：流速1-8mL/h，电压为12-18kV，接收距离为15-20cm，以滚动不锈钢棒(直径2mm)作为接收器；收集得到管状纤维材料，每3-8min收集一根；将得到的聚酯纤维管置于真空干燥箱中干燥48h以去除有机溶剂；
2)配制多巴胺(2mg/mL)的10mmol/L Tris-HCl(pH＝8.5)溶液，用恒流泵将纤维管连接在管路中，恒流20-30h，取下纤维管，用蒸馏水冲洗内表面3次，室温干燥，得到了聚多巴胺纤维管；
3)配制纳豆激酶(1mg/ml)的PBS溶液，用恒流泵将聚多巴胺纤维管连接在管路中，恒流3-8h，用蒸馏水冲洗内表面，室温干燥，获得负载纳豆激酶的小口径人工血管。
上述生物可降解脂肪族聚酯材料包括聚己内酯(PCL)，3-羟基丁酸-4-羟基丁酸共聚物(P3HB4HB)，聚丙交酯和聚己内酯共聚物(PLCL)，聚丙交酯和乙交酯的共聚物(PLGA)。
本发明与现有技术相比突出优点在于：
1)材料选取上，生物可降解脂肪族聚酯材料拥有较好的生物相容性和力学性能，使得材料符合小口径人工血管要求。
2)制备工艺上，利用静电纺丝技术制备纤维材料。静电纺丝工艺可制备多种不同材料，纺丝条件相对温和，参数易控制。由静电纺丝制备的材料，更加符合细胞外基质环境，有利于细胞的黏附、迁移与增殖。
3)功能上，静电纺丝制备的纤维材料有利于细胞浸润，聚多巴胺涂层和纳豆激酶改善了材料的亲水性，提高了材料的生物相容性，有利于细胞在材料上的黏附和增殖，从而有利于血管再生。纳豆激酶具有抗凝血和溶解血栓的作用，使得材料具备良好的血液相容性，减少小口径人工血管血栓的形成，具有良好抗凝血效果，能够保持血管畅通。
实施例1：
1)量取15mL三氯甲烷和3mL甲醇(混合液体积比5∶1)，接着称取3.6g聚己内酯(PCL)放入玻璃瓶中，溶液在磁力搅拌机上搅拌过夜，得到浓度为20％的充分溶解均一的聚己内酯纺丝溶液。
静电纺丝的条件为：流速6mL/h，电压12kV，收集距离15cm；以滚动不锈钢棒(直径2mm)作为接收器，收集得到管状纤维材料，每5min收集一根；收集到的静电纺丝纤维管材料在显微镜下初步观察，纤维取向无规则，纤维上无小液滴；将得到的纤维管置于真空干燥箱中干燥以去除有机溶剂。
2)配制多巴胺(2mg/mL)的10mmol/L Tris-HCl(pH＝8.5)溶液，用恒流泵将纤维管连接在管路中，恒流24h，取下纤维管，用蒸馏水冲洗内表面3次，室温干燥，得到了聚多巴胺纤维管；
3)配制纳豆激酶(1mg/ml)的PBS溶液，用恒流泵将聚多巴胺纤维管连接在管路中，恒流4h，用蒸馏水冲洗内表面，室温干燥，获得负载纳豆激酶的小口径人工血管。
实施例2：
1)量取15mL三氯甲烷和5mL甲醇(混合液体积比3∶1)，接着称取2g P3HB4HB(3-羟基丁酸酯-4羟基丁酸酯共聚物，PHA的一种)放入玻璃瓶中，溶液在磁力搅拌机上搅拌过夜，得到浓度为10％的充分溶解均一的P3HB4HB纺丝溶液；静电纺丝的条件为：流速1mL/h，电压18kV，收集距离20cm，以滚动不锈钢棒(直径2mm)作为接收器，收集得到管状纤维材料，每5min收集一根。在显微镜下初步观察，纤维取向无规则，纤维上无小液滴。将得到的纤维膜置于真空干燥箱中干燥以去除有机溶剂。
2)配制多巴胺(2mg/mL)的10mmol/L Tris-HCl(pH＝8.5)溶液，用恒流泵将纤维管连接在管路中，恒流28h，取下纤维管，用蒸馏水冲洗内表面3次，室温干燥，得到了聚多巴胺纤维管；
2)配制纳豆激酶(1mg/ml)的PBS溶液，用恒流泵将聚多巴胺纤维管连接在管路中，恒流5h，用蒸馏水冲洗内表面，室温干燥，获得负载纳豆激酶的小口径人工血管。
实施例3：
1)量取12mL三氯甲烷和2mL甲醇(混合液体积比6∶1)，接着称取3.5g的PLCL放入玻璃瓶中，溶液在磁力搅拌机上搅拌过夜，得到浓度为25％的充分溶解均一的PLCL纺丝溶液；静电纺丝的条件为：流速8mL/h，电压16kV，收集距离15cm；以滚动不锈钢棒(直径2mm)作为接收器，收集得到管状纤维材料，每5min收集一根。收集到的静电纺丝纤维膜在显微镜下初步观察，纤维取向无规则，纤维上无小液滴。将得到的纤维膜置于真空干燥箱中干燥以去除有机溶剂。
2)配制多巴胺(2mg/mL)的10mmol/L Tris-HCl(pH＝8.5)溶液，用恒流泵将纤维管连接在管路中，恒流26h，取下纤维管，用蒸馏水冲洗内表面3次，室温干燥，得到了聚多巴胺纤维管；
3)配制纳豆激酶(1mg/ml)的PBS溶液，用恒流泵将聚多巴胺纤维管连接在管路中，恒流6h，用蒸馏水冲洗内表面，室温干燥，获得负载纳豆激酶的小口径人工血管。
实施例4：
通过凝血时间实验，实施例1负载了纳豆激酶的PCL人工血管材料与没有负载纳豆激酶的PCL人工血管材料相比，APTT(30s/25s)和TT(23s/16s)的时间都有明显的延长，并且显著降低了纤维蛋白原的量(1.5g/L/2.8g/L)，说明材料良好的抗凝效果。通过血小板黏附实验看出，负载了纳豆激酶的人工血管材料具备抑制血小板黏附的功能。通过动静脉分流实验，负载了纳豆激酶的材料可以避免急性血栓形成，体外循环1h后，扫描电子显微镜下纤维形貌仍然清晰，证明了负载纳豆激酶的材料有良好的抗凝血效果。
实施例5：
进行大鼠体内原位移植，在4周进行取材，并对取出的材料进行了观察和染色。相比于纯PCL材料，实施例1所得到的负载了纳豆激酶的人工血管材料组内皮化程度更高，细胞浸润程度更好，胶原在材料中分布更多，显示出了良好的血管再生性能。

Claims (4)

1.负载纳豆激酶的小口径人工血管，是利用多巴胺在生物可降解脂肪族聚酯材料上自聚合形成具有超强黏性的聚多巴胺涂层，再将纳豆激酶通过聚多巴胺涂层负载到聚酯材料上，形成负载纳豆激酶的小口径人工血管。
2.负载纳豆激酶的小口径人工血管的制备方法，包括以下步骤：
1)配制静电纺丝溶液：首先将生物可降解脂肪族聚酯溶于氯仿/甲醇混合溶剂，其中氯仿与甲醇的体积比为3∶1-6∶1，聚酯的质量/体积浓度为10-25％，用来进行静电纺丝；静电纺丝条件为：流速1-8mL/h，电压为12-18kV，接收距离为15-20cm，以滚动不锈钢棒(直径2mm)作为接收器；收集得到管状纤维材料，每3-8min收集一根；将得到的聚酯纤维管置于真空干燥箱中干燥48h以去除有机溶剂；
2)配制多巴胺(2mg/mL)的10mmol/L Tris-HCl(pH＝8.5)溶液，用恒流泵将纤维管连接在管路中，恒流20-30h，取下纤维管，用蒸馏水冲洗内表面3次，室温干燥，得到了聚多巴胺纤维管；
3)配制纳豆激酶(1mg/ml)的PBS溶液，用恒流泵将聚多巴胺纤维管连接在管路中，恒流3-8h，用蒸馏水冲洗内表面，室温干燥，获得负载纳豆激酶的小口径人工血管。
3.根据权利要求1所述的负载纳豆激酶的小口径人工血管，其生物可降解脂肪族聚酯材料包括聚己内酯(PCL)，3-羟基丁酸-4-羟基丁酸共聚物(P3HB4HB)，聚丙交酯和聚己内酯共聚物(PLCL)，聚丙交酯和乙交酯的共聚物(PLGA)。
4.根据权利要求1所述的负载纳豆激酶的小口径人工血管的用途，其特征在于提高人工血管的抗凝血性能，所制备的负载纳豆激酶的人工血管内皮化速度快，可促进血管的再生。
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CN117462770A (zh) * 2023-10-31 2024-01-30 西南交通大学 一种抗凝血促内皮的血流导向装置及表面改性方法
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