CN107198793A - 一种智能化组织工程血管 - Google Patents

一种智能化组织工程血管 Download PDF

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CN107198793A
CN107198793A CN201610150254.0A CN201610150254A CN107198793A CN 107198793 A CN107198793 A CN 107198793A CN 201610150254 A CN201610150254 A CN 201610150254A CN 107198793 A CN107198793 A CN 107198793A
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spheres
sustained
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李温斌
马小龙
辛志飞
李殿坤
万居易
周子凡
张燕
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Abstract

一种智能化组织工程血管，心血管疾病已成为危害人类健康的主要疾病，最常用的治疗手段为血管移植术，临床上需要大量的人工血管替代物。通过截取绵羊的双侧颈动脉采用采用TritonX‑100＋DNA/RNA酶消化脱细胞去抗原方法；使用高分子物质（PLGA）、聚乙烯醇及VEGF等通过乳化挥发法制备载有VEGF的缓释微球；将脱细胞羊颈动脉支架和缓释微球通过冷冻干燥法紧密结合，制出具有定向诱导内皮细胞吸附分化增殖作用的载VEGF缓释微球组织工程血管，有望解决临床上血管移植遇到的难题：血管移植来源有限问题、单纯异种血管移植排斥反应问题以及现有组织工程血管移植远期通畅效果差问题等，做为治疗人类心血管疾病的血管移植替代物。

Description

一种智能化组织工程血管
技术领域
基础医学 临床医学 免疫学 组织工程学 动物学。
背景技术
心血管疾病已成为危害人类健康的主要疾病，最常用的治疗手段为血管移植术。但是由于自体血管移植来源有限，而且也受到自身血管条件的限制，临床上需要大量的人工血管替代物。人们尝试了各种异体血管移植的方法，但其远期效果差，血栓形成，血管变性、增生，血管狭窄和闭塞以及出现一些并发症。近年来兴起的组织工程为这一临床难题提供了新的思路，其以细胞生物学、材料科学和工程学的基础理论和方法，在体外将组织脱细胞去除抗原性，植入体内来替代宿主因疾病、外伤等原因所造成组织细胞结构缺损、功能丧失的组织或器官以达到对宿主组织结构的修复和功能重建。
其中包含研究制备小口径人工血管（直径《6 mm》）替代自身血管移植。但截至目前，尚无小口径人工血管移植完全适应于临床的报道。其主要原因是材料血液相容性和抗凝性能不佳，当与血液接触后激活体内的凝血系统，引起血小板和纤维蛋白聚集形成血栓，造成管腔狭窄及闭塞。血液相容性差。当其移置入体内后，容易导致血栓形成，严重影响移植血管的再通率，远期还易出现内膜增生。解决这些问题的关键在于促进人工血管腔的内皮化，形成完整的内皮层抑制血管狭窄及堵塞的发生。为解决这一难题我们在进一步改进脱细胞去抗原性方法的基础上，制备出具有定向诱导内皮细胞吸附分化增殖作用的载VEGF缓释微球，将两者通过一定的方法结合后制成智能化组织工程血管，具有抗排异反应、促进内皮细胞吸附、抗血栓且能大量生产制备的优点。
发明内容
一种智能化组织工程血管，可以作为血管移植的理想替代物。
附图说明 图1：载VEGF缓释微球的组织工程血管结构示意图。
图2：智能化组织工程血管的制备流程示意图。
具体实施方式 截取绵羊的双侧颈动脉采用采用TritonX-100＋DNA/RNA酶消化脱细胞去抗原方法；使用高分子物质（PLGA）、聚乙烯醇及VEGF等通过乳化挥发法制备载有VEGF的缓释微球；将脱细胞羊颈动脉支架和缓释微球通过冷冻干燥法紧密结合，制出具有定向诱导内皮细胞吸附分化增殖作用的载VEGF缓释微球组织工程血管。
基本步骤（图:2）：
一、组织工程血管的制备：
2.1修剪、清洗血管：在无菌操作台中把新鲜的羊的颈动脉在容器中进行适当的清洗及修剪，去除血管外膜附着物及管腔内血栓，保留血管内膜、中层、外膜的完整结构，反复用漂洗液清洗。
2.2脱细胞阶段：将血管分量，置入含有脱细胞终液的无菌Fisher（250ml）瓶中，在恒温振荡器中匀速震荡24h后，进行漂洗。
2.3核酶消化阶段：将漂洗后的血管置入装有DNA酶+RNA酶的Fisher（250ml）瓶中，进行24h的核酶液消化处理，进行漂洗处理。漂洗结束后将一部分血管置入无菌培养皿中在-80℃超低温冰箱进行冻存处理备用。
二、载VEGFl65缓释微球的制备
采用有机乳化溶剂高速搅拌挥发法制备载有VEGF的缓释微球。称取6mg的VEGF冻干粉溶于3ml的蒸馏水中，制成液体①（内水相），称取200mg的PLGA溶于20ml的二氯甲烷，制成液体②（有机相）。将液体①+液体②混合于Fisher（100ml）瓶中密封，在磁力搅拌器下搅拌（10min，2000r/min）,形成乳化液③（乳相）。再向液体③中加入20ml聚乙烯醇溶液（1％）（外水相），密封好Fisher瓶后搅拌（10min，2000r/min）形成复乳④。去除密封状态后，保持室内空气流通，继续搅拌3小时以挥发有机溶剂，固化缓释微球。最后用高速离心机离心（12000r/min，10min）,收集沉淀微球，用蒸馏水洗涤、离心3遍后，将沉淀物在冷冻干燥机干燥36h后在-80℃冻存备用。
三、含有VEGF的组织工程血管缓释模型的构建
将已经过脱细胞处理的羊颈动脉均与摆放在无菌玻璃培养皿中，置于-80℃冰箱冻存2小时后取出，放进冷冻式真空干燥机中冷冻干燥6小时。冷冻结束后取出血管浸泡于装有缓释微球悬浊液的Fisher瓶中，静置6小时后，再次取出血管后并均匀摆放在无菌培养皿中，再次进行冷冻干燥处理，然后再浸泡于缓释微球悬浊液中6小时。浸泡结束后取出血管放入-80℃冰箱中冻存备用。

Claims (3)

1.该项申请的智能化组织工程血管，是将异种动物（羊）颈动脉组织通过脱细胞去抗原化处理后，与制成的的载有VEGF的缓释微球结合，形成具有定向诱导内皮细胞吸附生长的人工血管生物支架，制作流程简易，但是该智能化的人工血管支架将来可作为心外科血管移植的替代物，解决其移植来源短缺这一问题，应用价值较大，故要求以下专利权：1.其他各种动物动脉组织经过脱细胞去抗原化处理后得到不同口径和长度的组织工程血管都将视为本专利的范围。
2.运用本专利内使用的方法，包括各种高分子材料（PLGA)、VEGF等物质制成的载VEGF的缓释微球,将视为本专利范围。
3.将组织工程血管与缓释微球通过冷冻干燥法结合制成载VEGF缓释微球的组织工程血管，将视为本专利范围。
CN201610150254.0A 2016-03-16 2016-03-16 一种智能化组织工程血管 Pending CN107198793A (zh)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109498839A (zh) * 2018-11-13 2019-03-22 南开大学 一种生物复合人工血管和应用
CN114984330A (zh) * 2022-06-07 2022-09-02 新乡医学院 一种兼具抗凝与抗钙化的脱细胞血管支架及其制备方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109498839A (zh) * 2018-11-13 2019-03-22 南开大学 一种生物复合人工血管和应用
CN114984330A (zh) * 2022-06-07 2022-09-02 新乡医学院 一种兼具抗凝与抗钙化的脱细胞血管支架及其制备方法

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Application publication date: 20170926