CN115154662A - 一种兼具抗凝血和促进内皮化功能的人工血管及其制备方法 - Google Patents
一种兼具抗凝血和促进内皮化功能的人工血管及其制备方法 Download PDFInfo
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Abstract
本发明属于人工血管领域,一种兼具抗凝血和促进内皮化功能的人工血管,其特征在于,含有钠尿肽。其有益效果在于:本发明将钠尿肽负载到人工血管上,通过钠尿肽实现了抗凝与促内皮化双重功能,操作简便,体内植入效果良好。
Description
技术领域
本发明属于人工血管领域,具体涉及一种兼具抗凝血和促进血管组织再生功能的人工血管及其制备方法。
背景技术
全球范围内因心血管疾病而死亡的人数已经达到死亡构成比的30%左右,心血管疾病已经成为人类健康的头号杀手。心血管疾病的发生常由于血管狭窄或阻塞导致血流减少和营养物质缺乏,从而使组织或器官受损,通常表现为冠心病、脑血管病、外周动脉疾病。自体血管是最理想的血管替代品,但由于来源有限、供体部位感染风险以及尺寸不匹配等问题限制了自体血管的使用。因此,多数情况下需要使用人工血管替代材料进行血管移植。
对于大口径(口径≥6mm)血管,虽然临床上已有由聚苯二甲酸乙二醇酯膨体聚四氟乙烯(ePTFE,)等材料制备产品,但是目前产品不具有再生能力,无法实现内皮和平滑肌再生,始终存在凝血风险。临床上心脏冠脉搭桥、膝盖以下外周血管替换、肝/肾器官移植等手术均需要使用小口径(口径<6mm)血管,但是目前尚无可以医用的小口径人工血管产品。小口径人工血管植入体内后常会出现血栓、内膜增生、钙化等问题,进而导致移植失败。
目前的人工血管通过负载肝素、比伐泸定、等抗凝药物,能够实现人工血管的短期抗凝血,但他们不具备促进人工血管内皮化等组织再生的能力,而有些人工血管通过负载CD133、VEGF、TPS等能够促进内皮化的效果,但是这些分子不具备抗凝血的作用。
目前的观点认为小口径人工血管体内植入后既需要具有良好的抗凝血性能,也需要具有良好地促进内皮化的性能,两者性能的结合是解决上述问题和实现长期通畅的关键所在。
为解决上述问题,为了实现抗凝与促内皮化双重效果,往往需要对人工血管进行多步处理,以达到两种功能的复合,但是操作步骤繁琐,增加的制备时间和经济成本。
发明内容
本发明的目的在于克服现有人工血管的缺点,提供同时具有抗凝血和促进内皮化功能的人工血管。
为实现上述目的,本发明采用的技术方案为:
一种兼具抗凝血和促进内皮化功能的人工血管,含有钠尿肽。
进一步地,还包括基质材料,所述基质材料采用常见人造血管用高分子材料。
进一步地,以质量分数计,以质量分数计,所述基质材料为1份,所述钠尿肽为8.8×10-9-8.8×10-5g份。
进一步地,所述常见人造血管用高分子材料采所述常见人造血管用高分子材料采用聚己内酯、聚乙醇酸、聚乳酸-羟基乙酸共聚物、聚(3-羟基丁酸酯-co-4- 羟基丁酸酯)、聚乳酸、聚L-丙交酯-己内酯、聚对二氧六环己酮中至少一种或几种的任意比例混合物。
优选地,所述常见人造血管用高分子材料采用聚己内酯。
一种兼具抗凝血和促进内皮化功能的人工血管的制备方法,包括如下步骤:
将基质材料溶解于有机溶液中;待高分子材料完全溶解后形成基质溶液,向基质溶液中添加钠尿肽粉末制成混合溶液;再利用常规纺织手段将上述混合溶液制备成负载钠尿肽的人工血管或膜材料。
进一步地,有机溶剂采用氯仿、甲醇中一种或集中的任意比例混合物。
优选地,所述机溶剂采用氯仿、甲醇的混合物,氯仿与甲醇的体积比为 5:1。
优选地,所述基质溶液中,溶质的质量浓度为0.10-0.30mg/mL。
更优选地,所述基质溶液中,溶质的浓度为0.25mg/mL。
优选地,钠尿肽(CNP)溶解于混合有机溶液中的浓度为10-9-10-5mol/L。
更优选地,钠尿肽(CNP)溶解于混合有机溶液中的浓度为10-7mol/L。
进一步地,所述兼具抗凝血和促进内皮化功能的人工血管的应用,可以改变形状用做血管补片、带瓣血管、心脏瓣膜。
与现有技术相比,本发明的有益效果在于:
本发明将钠尿肽负载到人工血管上,通过钠尿肽实现了抗凝与促内皮化双重功能,操作简便,体内植入效果良好。
附图说明
图1负载CNP与未负载CNP静电纺丝PCL人工血管的结构表征图;
图2负载CNP与未负载CNP静电纺丝PCL人工血管的力学表征图;
图3负载CNP静电纺丝PCL人工血管的CNP释放特性曲线图;
图4负载CNP与未负载CNP静电纺丝PCL人工血管的抗凝血分析图;
图5人主动脉平滑肌细胞(HASMCs)在负载CNP与未负载CNP静电纺丝PCL膜材料上的生长情况图;
图6CNP与未负载CNP静电纺丝PCL膜材料对人脐静脉内皮细胞 (HUVECs)行为的调控作用分析图;
图7对比例1与实施例2电纺血管植入大鼠腹主动脉1个月后的内皮化分析图。
具体实施方式
下面结合实施例对本发明的具体实施方式作进一步描述,以下实施例仅用于更加清楚地说明本发明的技术实施例,而不能以此来限制本发明的保护范围。
本发明实施例所用材料如下:
实施例1
以聚己内酯(PCL)为原材料,溶解PCL于氯仿和甲醇的混合有机溶液中,氯仿与甲醇的体积比为5:1,待PCL完全溶解后添加钠尿肽粉末,PCL溶解于上述混合有机溶液中的终浓度为0.25mg/mL,钠尿肽(CNP)溶解于上述混合有机溶液中的终浓度为10-5mol/L,PCL与CNP的质量比为1:8.8×10-5。以上述溶液进行静电纺丝,制备负载CNP的电纺PCL人工血管或电纺膜,命名为 PCL-CNP-10-5,紫外过夜灭菌后备用。
实施例2
制备方法同实施例1,但是钠尿肽(CNP)溶解于混合有机溶液中的终浓度为10- 7mol/L,PCL与CNP的质量比为1:8.8×10-7。所制备负载CNP的电纺 PCL人工血管或电纺膜,命名为PCL-CNP-10-7。
实施例3
制备方法同实施例1,但是钠尿肽(CNP)溶解于混合有机溶液中的终浓度为10- 9mol/L,PCL与CNP的质量比为1:8.8×10-9。所制备负载CNP的电纺 PCL人工血管或电纺膜,命名为PCL-CNP-10-9。
对比例1
以聚己内酯(PCL)为原材料,溶解PCL于氯仿和甲醇的混合有机溶液中,氯仿与甲醇的体积比为5:1,PCL溶解于上述混合有机溶液中的终浓度为 0.25mg/mL。以上述PCL溶液进行静电纺丝,制备PCL电纺人工血管或PCL 电纺膜,紫外过夜灭菌后备用。
为了进一步说明本发明的有益效果,特对进行如下测试:
1、结构表征:
将实施例1-3及对比例1中所制备的人工血管进行结构表征。结果如附图 1所示。
图1中(A)血管材料的横截面扫描电子显微镜(SEM)观察图片;(B) 血管内径统计数据;(C)血管壁厚统计数据;(D)血管材料内腔面SEM观察图片;(E)血管材料纤维直径统计数据(F)血管材料孔径统计数据。
扫描电子显微镜(SEM)观察显示出四组血管材料的具有相同的管体结构 (图1A),各组血管支架的内径和壁厚没有统计差异(图1B-C)。血管材料内腔面SEM显示4种人工血管的纤维形貌良好(图1D),基本一致,在纤维直径和孔径上也没有统计差异(图1E-F)。这些结果表明CNP的添加没有影响电纺纤维过程。
2、力学表征:
将实施例1-3及对比例1中所制备的人工血管进行力学表征。结果如附图 2所示。
图2中(A)血管材料的爆破压检测统计数据;(B)血管材料的缝合强度统计数据;(C)各组血管材料的拉伸应力-应变曲线;(D)血管材料杨氏模量统计数据;(E)血管材料拉伸应力的统计数据(F)血管材料的断裂伸长率统计数据。
4种人工血管在爆破压(>1600mmHg,图2A)和缝合强度(1.5N左右,图2B)上没有差异,均满足作为人工使用的需求。4种人工血管也展示了相似的应力-应变曲线(图2C),在杨式模量(图2D)、拉伸应力(图2E)、断裂伸长率(图2F)这三个检测项目上也没有差别。这些结果表明CNP的负载没有影响PCL人工血管的力学强度。
3、CNP释放检测:
酶联免疫吸附测定试剂盒(Elisa kIt)检测显示,实施例1-3中制备的CNP 负载的PCL人工血管均能实现缓释30天以上(图3),且缓释效率随着随着 CNP与PCL的质量比的增加而增加。
4、抗凝血性能检测
将实施例1-3及对比例1中所制备的人工血管进行血液相容性表征。结果如附图4所示。
图4中(A)各组血管材料对凝血酶活性的抑制作用统计分析;(B)各组血管材料对血小板粘附的抑制作用统计分析;(C)各组血管材料对血小板激活的抑制作用统计分析;(D)各组血管材料的溶血率统计分析。
由图中可知相比于PCL人工血管,三种负载CNP的PCL人工血管均显著减少了凝血酶的激活(图4A)、血小板的粘附(图4B)和血小板的激活(图 4C)。这些结果表明CNP的负载能够增强PCL人工血管的抗凝血特性。但是,三种负载CNP的PCL人工血管与单纯的PCL人工血管相比,在溶血率上没有差别,表明CNP的负载不会引起红细胞的破裂。此外,我们也进行了兔子颈/ 静动脉的AV-Shunt实验,结果显示PCL-CNP-10-5和PCL-CNP-10-7血管内腔几乎没有血栓形成,而PCL-CNP-10-9可见部分血栓形成,而PCL人工血管则可见明显血栓附着。这些结果表明PCL-CNP-10-7血管中的CNP负载量已经满足抗凝血需求。
5、平滑肌细胞增殖抑制作用
将人主动脉平滑肌细胞(HASMCs)分别接种于对比例1及实施例1-3,培养5天。从第3天开始,三种负载CNP的PCL膜材料就显示出了对HASMCs 生长的抑制作用,其中实施例2制备的PCL-CNP-10-7膜材料具有最强的抑制 (图5),这对体内植入后抑制内膜过度增生具有重要作用。
6、内皮细胞功能调控
将人脐静脉内皮细胞(HUVECs)分别接种于对比例1及实施例1-3所制备的电纺膜材料上,分析各组膜材料对HUVECs的调控作用。结果如附图6 所示。
图6中(A)HUVECs在各组膜材料上的生长情况;(B)HUVECs在各组材料上培养5天后的死活染色分析;(C)HUVECs在各组材料上培养3天后的分泌VEGF能力的分析;(D)HUVECs在各组材料浸泡液中的迁移分析;(E) HUVECs迁移的统计分析;(F)DAF-FM探针检测HUVECs在各组材料调控下生产NO的情况;(D)DAF-FM探针相对荧光强度统计分析,PCL膜材料培养组HUVECs的DAF-FM荧光强度被定义为100%。DAF-FM为NO的检测探针,DAF-FM进入细胞内,与NO结合会产生荧光,因此可以通过DAF-FM 探针的荧光强度来反应细胞产生NO的能力。
HUVECs分别接种于对比例1及实施例1-3所制备的电纺膜材料上,培养 5天。从第3天开始,三种负载CNP的PCL膜材料就显示出了对HUVECs的生长的促进作用,其中实施例2制备的膜材料具有最强的促进HUVECs增殖的作用(图6A),而且培养5天后,所有膜材料上未见红色的死细胞,细胞生长状态良好(图6B)。
但是从绿色活细胞的密度上也能看出实施例2具有最高的HUVECs密度 (图6B)。HUVECs在各组膜材料上培养三天后,收集上清,Elisa检测显示三种负载CNP的PCL膜材料均促进了HUVECs分泌VEGF,其中实施例2制备的膜材料具有最强的促进HUVECs分泌VEGF的能力(图6C)。
划痕实验显示出三种负载CNP的PCL膜材料的浸泡液均促进了HUVECs 迁移,其中实施例2制备的膜材料的浸泡液具有最强的促进HUVECs迁移的能力(图6D,E)。
通过transwell孔板对HUVECs产生一氧化氮(NO)的能力进行了检测。膜材料置于transwell孔板的上室,HUVECs种植于下室,培养3天后,采用 NO荧光探针(DAF-FM)对HUVECs释放NO的能力进行检测,结果显示三种负载CNP的PCL膜材料均促进了HUVECs产生NO,其中实施例2制备的膜材料具有最强的促进HUVECs产生NO的能力(图6F,G)。这些结果表明实施例2具有最强的促进HUVECs功能的能力,这对体内植入后快速的功能内皮再生具有重要作用。
7、大鼠体内植入
将对比例1及实施例1-3中所制备的血管材料分别植入大鼠腹主动脉。移植一个月后,4组血管全部通畅。
图7中(A)SEM观察PCL与实施例2血管在缝合端(anastomotic)、1/4 处(quarter)、中段(midportion)内腔的内皮覆盖情况;(B)对比例1与实施例2血管纵切的CD31抗体的免疫荧光染色以及CD31+内皮细胞覆盖的统计分析。
SEM观察显示PCL仅缝合端有内皮覆盖,但是在血管1/4处和中部均无内皮覆盖,且呈现纤维裸露。虽然SEM显示实施例1和实施例3血管的缝合断和1/4处均呈现了内皮细胞,但是中部也没有完成内皮覆盖。只用实施例2 实现了3个位点的内皮全覆盖(图7A)。进一步的纵切片CD31抗体荧光染色结果显示出CD31+内皮细胞的覆盖情况和SEM观察到的情况基本一致,基于CD31抗体染色的内皮覆盖率统计结果显示,对比例的内皮覆盖率为38.64 ±2.21%(图7B),实施例1为63.47±5.64%,实施例2为90.50±2.55%(图 7B),实施例3为68.24±7.25%。
8、兔子体内植入
相比于大鼠,兔子更容易产生凝血反应,因此,我们将对比例1及实施例 1-3中所制备的血管材料分别植入了兔子颈动脉,进行更为严苛的凝血测试。移植1个月后的结果显示,PCL全部堵塞,实施例1-2的通畅率为100%,实施例3的通畅率为50%。这一结果表明,实施例2中制备的CNP负载的人工血管已经具有足够良好的抗凝血性能。
综合以上体内/外结果,本发明制备的CNP负载的人工血管兼具抗凝血和促内皮再生性能。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (11)
1.一种兼具抗凝血和促进内皮化功能的人工血管,其特征在于,含有钠尿肽。
2.根据权利要求1所述的兼具抗凝血和促进内皮化功能的人工血管,其特征在于,还包括基质材料,所述基质材料采用常见人造血管用高分子材料。
3.根据权利要求2所述的兼具抗凝血和促进内皮化功能的人工血管,其特征在于,以质量分数计,所述基质材料为1份,所述钠尿肽为8.8×10-9-8.8×10-5份。
4.根据权利要求2所述的兼具抗凝血和促进内皮化功能的人工血管,其特征在于,所述常见人造血管用高分子材料采用聚己内酯、聚乙醇酸、聚乳酸-羟基乙酸共聚物、聚(3-羟基丁酸酯-co-4-羟基丁酸酯)、聚乳酸、聚L-丙交酯-己内酯、聚对二氧六环己酮中至少一种或几种的任意比例混合物。
5.根据权利要求2所述的兼具抗凝血和促进内皮化功能的人工血管,其特征在于,所述常见人造血管用高分子材料采用聚己内酯。
6.一种兼具抗凝血和促进内皮化功能的人工血管的制备方法,包括如下步骤:
将基质材料溶解于有机溶液中;待高分子材料完全溶解后形成基质溶液,向基质溶液中添加钠尿肽粉末制成混合溶液;再利用常规纺织手段将上述混合溶液制备成负载钠尿肽的人工血管或膜材料。
7.根据权利要求6所述的兼具抗凝血和促进内皮化功能的人工血管的制备方法,其特征在于,有机溶剂采用氯仿、甲醇中一种或几种的任意比例混合物。
8.根据权利要求6所述的兼具抗凝血和促进内皮化功能的人工血管的制备方法,其特征在于,所述机溶剂采用氯仿、甲醇的混合物,氯仿与甲醇的体积比为5:1。
9.根据权利要求6所述的兼具抗凝血和促进内皮化功能的人工血管的制备方法,其特征在于,所述基质溶液中,溶质的质量浓度为0.10-0.30mg/mL。
10.根据权利要求6所述的兼具抗凝血和促进内皮化功能的人工血管的制备方法,其特征在于,钠尿肽溶解于混合有机溶液中的浓度为10-9-10-5mol/L。
11.根据权利要求1-5任意一项所述的兼具抗凝血和促进内皮化功能的人工血管的应用,其特征在于,可以改变形状用做血管补片、带瓣血管、心脏瓣膜。
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