CN113181432A - 一种人工蚕丝瓣膜及其制备方法 - Google Patents

一种人工蚕丝瓣膜及其制备方法 Download PDF

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CN113181432A
CN113181432A CN202110404563.7A CN202110404563A CN113181432A CN 113181432 A CN113181432 A CN 113181432A CN 202110404563 A CN202110404563 A CN 202110404563A CN 113181432 A CN113181432 A CN 113181432A
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silk
valve
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CN113181432B (zh
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王文硕
王盛章
钟伯雄
王璐
刘顺
魏来
王春生
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Zhongshan Hospital Fudan University
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Abstract

本发明公开了一种人工蚕丝瓣膜及其制备方法。本发明利用转基因技术，将蜘蛛的牵引丝蛋白2基因植入到家蚕体内，从而让家蚕产出含有牵引丝蛋白2的蚕丝，其拉伸性及强度比普通蚕丝要强数百倍，同时利用静电纺织技术将蚕丝按照不同的纺织方式制成瓣膜，在受力较为集中的瓣膜根部和瓣膜边缘采用斜纹纺织或增加纺织层数方式进行加固，从而提高整体瓣膜的强度，在其他瓣叶部分为了保证瓣膜的柔韧性采用平行纹纺织；且本发明的蚕丝瓣膜具有相当光滑的表面，从而有效避免了瓣膜表面栓的形成，并且蚕丝的组织相容性也较好，成为理想的人工瓣膜。

Description

一种人工蚕丝瓣膜及其制备方法
技术领域
本发明涉及一种人工蚕丝瓣膜及其制备方法，属于医用材料技术领域。
背景技术
随着生活水平的不提高，人们对于身体的健康越来越重视。如今，心血管疾病已成为威胁人类健康的头号“杀手”。全国每年有数百万人因心血管疾病而死亡，其中有一部分是由于心脏瓣膜疾病而导致的。心脏瓣膜疾病大多是由于瓣膜结构异常引起瓣口的反流或者狭窄。目前主要的治疗方式是将人工瓣膜替换原先病变的瓣膜，人工瓣膜目前分为机械瓣膜和生物瓣膜，前者具有较长使用寿命的优点，但由于是金属材质，容易导致组织排斥性和血栓形成；后者具有良好的组织相容性，但由于牛心包或猪心包的抗拉伸性较差，导致生物瓣膜的寿命较短，患者需要在一段时间后再次行手术换瓣，这些都对患者身体健康造成不小的影响，如何设计一款即有良好组织相容性又具有很好的抗拉伸性的人工瓣膜成为关键。
发明内容
本发明所要解决的技术问题是：如何获得一款兼具良好的组织相容性和抗拉伸性的人工瓣膜。
为了解决上述技术问题，本发明提供了一种人工蚕丝瓣膜，利用静电纺丝技术将转基因桑蚕丝制成具有不同纺织方式的蚕丝瓣膜，所述蚕丝瓣膜的瓣叶边缘和瓣叶根部采用斜纹纺织或多层纺织、其他部位采用单层平行纹纺织，所述的转基因桑蚕丝含有黑寡妇蜘蛛牵引丝蛋白2。
本发明还提供了一种所述的人工蚕丝瓣膜的制备方法，包括如下步骤：
步骤1：利用转基因技术，制备含有黑寡妇蜘蛛牵引丝蛋白2的转基因桑蚕丝；
步骤2：将转基因桑蚕丝经过脱胶、干燥和酶溶解后制得蚕丝蛋白溶液，再将蚕丝蛋白溶液制成可纺性蚕丝极化液，将蚕丝极化液置于供液系统中；
步骤3：将高压电源的正极接到纺丝针头上，负极接在接收装置上，开启高压电源和供液系统进行静电纺丝，在静电场作用下，蚕丝极化液飞向接收装置过程中被拉伸，随着溶液的挥发，最终形成平行纹纺织纹路的瓣叶；
步骤4：瓣叶边缘和瓣叶根部采用斜纹纺织或多层纺织进行加固处理，得到人工蚕丝瓣膜。
优选地，所述步骤3中的静电纺丝的工艺条件为：电压10-15KV，温度22-28℃，供液系统的流速0.5-2mL/h，纺丝针头与接收装置的距离为10-20cm。
优选地，所述步骤4中的斜纹纺织为：改变高压电源的正负极位置，使瓣叶边缘和瓣叶根部的位置形成斜纹纺织纹路。
优选地，所述的步骤1～步骤4均在医用无菌、洁净要求的环境下进行。
与现有技术相比，本发明的有益效果在于：
本发明利用转基因技术，将蜘蛛的牵引丝蛋白2基因植入到家蚕体内，利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白2，其拉伸性及强度比普通蚕丝要强数百倍，同时利用静电纺织技术将蚕丝按照不同排列方式编织成瓣膜，在受力较为集中的瓣膜根部和瓣膜边缘采用斜纹纺织或增加纺织层数方式进行加固，从而提高整体瓣膜的强度，在其他瓣叶部分为了保证瓣膜的柔韧性采用平行纹纺织；此外，本发明的蚕丝瓣膜具有相当光滑的表面，从而有效避免了瓣膜表面血栓的形成，并且蚕丝的组织相容性也较好，能够有效降低组织排异性，因此本发明的人工蚕丝瓣膜是一种兼具良好的组织相容性和抗拉伸性的理想人工瓣膜。
附图说明
图1是实施例1制备的人工蚕丝瓣膜的实物图；
图2是本发明的不同纺丝方向的蚕丝瓣膜与牛心包材料制成的瓣膜的力学性能测试比较图；其中，a:牛心包轴向应力，b：斜纹纬向应力，c：斜纹经向应力，d：平纹纬向应力，e：平纹经向应力；
图3是不同纺丝方向的蚕丝瓣膜的水渗透性能测试图。
具体实施方式
为使本发明更明显易懂，兹以优选实施例，并配合附图作详细说明如下。
首先，本发明利用转基因技术将黑寡妇蜘蛛的牵引丝蛋白2表达基因转移到家蚕的体内，从而让家蚕吐出具有一定牵引丝蛋白2含量的蚕丝，这种蚕丝具有非常好的抗拉伸性和强度，具体工艺参考《利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白2的方法》专利(专利号：CN201510697644.5)。
其次，本发明通过静电纺丝工艺制备蚕丝瓣膜，静电纺丝是一种利用可纺性蚕丝极化液或熔体在强电场作用下形成喷射流进行纺丝加工工艺。其核心原理是带电荷的高分子或熔体在高压静电场中流动与变形，经溶剂蒸发或熔体冷却而固化得到纤维状物质；其中，静电纺丝装置主要有三部分：1、让聚合物溶液带电荷产生静电场的高压电源；2、压力泵组成供液系统；3、接收得到电纺纤维的接收器。本发明通过改变电极方向来实现不同方式的纺织(比如斜纹纺织或平行纹纺织等)。由于静电纺丝技术可以让蚕丝瓣膜表面洁净且光滑，在人体血液环境中不易聚集红细胞从而形成血栓，避免不必要的抗凝治疗。其次，设计的静电纺丝可根据不同要求对人工蚕丝瓣膜不同部位进行加固，在局部受力较强的部位采用斜纹纺织或多层纺织，以增加人工瓣膜的寿命。
实施例1
一种人工蚕丝瓣膜的制备，包括如下步骤：
步骤1：按照专利《利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白2的方法》中的方法制备转基因蚕丝：
(1)采用分子生物学方法构建pBac[3xP3-DsRed]-MaSp2质粒作为在家蚕丝腺中表达的黑寡妇蜘蛛牵引丝蛋白2基因载体，pBac[3xP3-DsRed]-MaSp2质粒以piggBac转座子为基础包含有作为外源基因的黑寡妇蜘蛛牵引丝蛋白2基因和作为标记基因的红色荧光DsRed基因表达框；
(2)采用显微注射转基因家蚕方法将pBac[3xP3-DsRed]-MaSp2质粒及能够提供piggyBac转座酶的辅助质粒pHA3PIG按浓度比1:1的比例导入家蚕产卵后6小时以内的受精卵内，利用pBac[3xP3-DsRed]-MaSp2质粒中的piggyBac转座子将黑寡妇蜘蛛牵引丝蛋白2基因插入到家蚕基因组内；
(3)蚕卵孵化后饲养至成虫，然后与非基因家蚕交配制种续代，此代为G1代，在G1代蚕卵的转青期，通过荧光体视显微镜观察筛选出单眼表达红色荧光DsRed标记基因的转基因家蚕，饲养至成虫再与非转基因家蚕交配制种续代成为G2代；
(4)G2代家蚕采用单蛾育，同蛾区表达红色荧光DsRed标记基因的蚕蛾相互交配，制成G4代；
(5)G3代家蚕采用单蛾育，同蛾区表达红色荧光DsRed标记基因的蚕蛾相互交配，制成G4代；
(6)从G4代开始并经连续3代采用红眼表型纯一的蛾区饲养、单蛾育和同蛾区蚕蛾交配的相同方法进行选择和交配，育成红眼基因和黑寡妇牵引丝蛋白2基因纯合、丝腺细胞能够合成分泌黑寡妇蜘蛛牵引丝蛋白2的转基因家蚕；
(7)通过家蚕丝腺细胞合成分泌黑寡妇蜘蛛牵引丝蛋白2，并随家蚕吐丝结茧行为得到蚕茧。
步骤2：采用静电纺丝技术制备人工蚕丝瓣膜(在符合医用无菌、洁净要求的环境下进行制备)：
(1)将步骤1得到的蚕茧经过脱胶、干燥、蛋白酶溶解后制得蚕丝蛋白溶液，再与高分子聚合物溶液混合，制成可纺性蚕丝极化液，供液系统将以0.5-2ml/h的流速对静电池内持续供液，其接收装置为一块规格为20*20cm附着有铝箔的钢板，钢板与纺丝针头的距离为10-20cm，纺丝环境温度为25℃左右；
(2)将高压电源的正极接到纺丝针头上，负极接在接收装置上，设置电纺电压为10-15KV；
(3)开启高压电源，然后启动供液装置，在静电场作用下，蚕丝极化液飞向接收装置过程中被拉伸，随着溶液的挥发，最终形成平行纹编织方式的瓣叶；
(4)蚕丝瓣膜局部加强处理：改变高压电源正负极的相对位置，使瓣叶边缘和瓣叶根部的位置形成斜纹纺织纹路，进行加固，从而得到人工蚕丝瓣膜，其实物图如图1所示，不同纺丝方向的蚕丝瓣膜与牛心包瓣膜的力学性能测试比较如图2所示，由图2可知，在相同应力的情况下，相比于牛心包瓣膜，蚕丝瓣膜具有良好的弹性和延展性，表现出较好的韧性，相应的瓣膜寿命更长，其次，在蚕丝瓣膜中，我们可以看到斜纹编织的瓣叶边缘和瓣叶根部部位相比于平行纹编织的其他部位，具有更好的弹性和延展性，而瓣叶边缘和瓣叶根据为瓣膜受力相对集中的部位，其采用了斜纹编织进行加固，使得相应的蚕丝瓣膜具有更长的使用寿命；不同纺丝方向的蚕丝瓣膜的水渗透性能测试图如图3所示，从图3可以看出斜纹编织相比于平纹编织具有更好地防水性，而蚕丝瓣膜的瓣叶边缘和瓣叶根据采用斜纹编织，可以有效降低瓣膜的渗血。
实施例2
一种人工蚕丝瓣膜的制备，包括如下步骤：
步骤1：同实施例1的步骤1；
步骤2：采用静电纺丝技术制备人工蚕丝瓣膜(在符合医用无菌、洁净要求的环境下进行制备)：
(1)将步骤1得到的蚕茧经过脱胶、干燥、蛋白酶溶解后制得蚕丝蛋白溶液，再与高分子聚合物溶液混合，制成可纺性蚕丝极化液，供液系统将以0.5-2ml/h的流速对静电池内持续供液，其接收装置为一块规格为20*20cm附着有铝箔的钢板，钢板与纺丝针头的距离为10-20cm，纺丝环境温度为25℃左右；
(2)将高压电源的正极接到纺丝针头上，负极接在接收装置上，设置电纺电压为10-15KV；
(3)开启高压电源，然后启动供液装置，在静电场作用下，蚕丝极化液飞向接收装置过程中被拉伸，随着溶液的挥发，最终形成平行纹纺织纹路的瓣叶；
(4)蚕丝瓣膜局部加强处理：在瓣叶边缘和瓣叶根部的位置进行多次纺织增加纺织层数，进行加固，从而得到人工蚕丝瓣膜。
上述实施例仅为本发明的优选实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。

Claims (5)

1.一种人工蚕丝瓣膜，其特征在于，利用静电纺丝技术将转基因桑蚕丝制成具有不同纺织方式的蚕丝瓣膜，所述蚕丝瓣膜的瓣叶边缘和瓣叶根部采用斜纹纺织或多层纺织、其他部位采用单层平行纹纺织，所述的转基因桑蚕丝含有黑寡妇蜘蛛牵引丝蛋白2。
2.权利要求1所述的人工蚕丝瓣膜的制备方法，包括如下步骤：
步骤1：利用转基因技术，制备含有黑寡妇蜘蛛牵引丝蛋白2的转基因桑蚕丝；
步骤2：将转基因桑蚕丝经过脱胶、干燥和酶溶解后制得蚕丝蛋白溶液，再将蚕丝蛋白溶液制成可纺性蚕丝极化液，将蚕丝极化液置于供液系统中；
步骤3：将高压电源的正极接到纺丝针头上，负极接在接收装置上，开启高压电源和供液系统进行静电纺丝，在静电场作用下，蚕丝极化液飞向接收装置过程中被拉伸，随着溶液的挥发，最终形成平行纹纺织纹路的瓣叶；
步骤4：瓣叶边缘和瓣叶根部采用斜纹纺织或多层纺织进行加固处理，得到人工蚕丝瓣膜。
3.如权利要求2所述的人工蚕丝瓣膜的制备方法，其特征在于，所述步骤3中的静电纺丝的工艺条件为：电压10-15KV，环境温度22-28℃，供液系统的流速0.5-2mL/h，所述纺丝针头与接收装置的距离为10-20cm。
4.如权利要求2所述的人工蚕丝瓣膜，其特征在于，所述步骤4中的斜纹纺织为：改变高压电源的正负极位置，使瓣叶边缘和瓣叶根部的位置形成斜纹纺织纹路。
5.如权利要求2所述的人工蚕丝瓣膜，其特征在于，所述的步骤1～步骤4均在医用无菌、洁净要求的环境下进行。
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