CN114246986B - 一种基于原位调控免疫反应的心血管植入物及其制备方法 - Google Patents

一种基于原位调控免疫反应的心血管植入物及其制备方法 Download PDF

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CN114246986B
CN114246986B CN202111646804.5A CN202111646804A CN114246986B CN 114246986 B CN114246986 B CN 114246986B CN 202111646804 A CN202111646804 A CN 202111646804A CN 114246986 B CN114246986 B CN 114246986B
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dcas9
cardiovascular implant
cardiovascular
sustained
plasmid
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CN114246986A (zh
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曾文
李彦朝
汪艳宏
薛方超
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Third Military Medical University TMMU
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Abstract

本发明公开了一种基于原位调控免疫反应的心血管植入物及其制备方法，属于生物医药技术领域，其技术方案要点是：包括心血管植入物本体和修饰于心血管植入物本体上的H4000‑CD25/dcas9缓释纳米颗粒；H4000‑CD25/dcas9缓释纳米颗粒包括H4000质粒纳米载体(Engreen)、CD25抗体和dcas9质粒序列；心血管植入物的制备方法包括如下步骤：构建心血管植入物本体、制备H4000‑CD25纳米转染载体、制备H4000‑CD25/dcas9缓释纳米颗粒和将H4000‑CD25/dcas9缓释纳米颗粒偶联于心血管植入物本体上。本发明主要用于构建修饰有H4000‑CD25/dcas9缓释纳米颗粒的心血管植入物，可诱导神经纤维长入工程血管，并利用Treg细胞对免疫反应的调控能力，提高心血管植入物的抗血栓功能和促进心血管植入物的原位再生。

Description

一种基于原位调控免疫反应的心血管植入物及其制备方法
技术领域
本发明涉及生物医药技术领域，尤其涉及一种基于原位调控免疫反应的心血管植入物及其制备方法。
背景技术
心血管疾病是威胁人类健康的第一大疾病，全球每年有730万人死于缺血性心脏病，列各病之首，因此对心血管植入物的需求日益增大。其中，生物人工血管是冠状动脉旁路移植术、血液透析和外周血管闭塞治疗的血管植入物的发展方向。并且肝、肾、肺、胰岛等复杂组织器官的构建也均需血管化，导致对生物人工血管的需求量的进一步增加。人是一个整体，一个系统某个器官功能的发挥往往需要其他多个系统的相互作用和调控。特别是对于移植的组织和器官，需要尽快的融入受体，重建与受体各个系统的正常联系。
但目前器官移植的研究主要集中在同种异体排斥和功能的重建，却很少关注神经网络重建。神经特别是交感神经在免疫稳态维持中发挥着至关重要的作用。交感神经支配血管收缩和腺体分泌，交感神经分泌的Catecholamines(CAs),ATP,and adenosine被证明可以抑制interleukin(IL-12),tumor necrosis factor(TNF)-a,and interferon(IFN)-g的产生，但是可以促进IL-10的生成，保护组织和器官免受过度炎症反应的损伤。因此神经网络重建可能是维持renal allografts免疫豁免和功能长时辰维持的一个新的靶点。
炎症对于组织再生是一把“双刃剑”，低强度的炎症可以促进干细胞的动员和增殖，持续存在又会对干细胞功能产生损害；如何实现原位有效调控免疫反应，促进炎症消退，仍是领域内的重要难题。
为了解决上述问题，在现有技术的基础上提供了一种基于原位调控免疫反应的心血管植入物及其制备方法。
发明内容
本发明的目的是提供一种基于原位调控免疫反应的心血管植入物及其制备方法，本方法利用不同程度的炎症对于心血管植入物再生的影响和作用，构建了修饰有H4000-CD25/dcas9缓释纳米颗粒的心血管植入物，可诱导神经纤维长入工程血管，并利用Treg细胞对免疫反应的调控能力，提高了心血管植入物的抗血栓功能和促进了心血管植入物的原位再生；此外，采用本方法制备的心血管植入物进行血管移植治疗，缓释纳米颗粒可原位调控免疫反应并促进血管再生，有效克服血栓形成，促进其长期通畅。
本发明的上述技术目的是通过以下技术方案得以实现的：
一种基于原位调控免疫反应的心血管植入物，包括心血管植入物本体和修饰于心血管植入物本体上的H4000-CD25/dcas9缓释纳米颗粒；所述H4000-CD25/dcas9缓释纳米颗粒包括H4000质粒纳米载体(Engreen)、CD25抗体和dcas9质粒序列；所述H4000质粒纳米载体和CD25抗体为共价连接，且所述dcas9质粒序列用于增强去甲基化酶TET2表达。
进一步地：所述dcas9质粒序列为pZdonor_U6-sgRNA-EF1α-dSpCas9-NLS-VP64-2A-EGFP-2A-Puro。
进一步地：所述心血管植入物为管状，口径为1-4mm，长度为0.5-20cm。
本发明还提供了一种基于原位调控免疫反应的心血管植入物的制备方法，包括如下步骤：
(1)构建心血管植入物本体；
(2)制备H4000-CD25纳米转染载体；
(3)通过H4000-CD25纳米转染载体和Crisper/dcas9系统质粒，制备得到H4000-CD25/dcas9缓释纳米颗粒；
(4)将H4000-CD25/dcas9缓释纳米颗粒偶联于心血管植入物本体上：用所述H4000-CD25/dcas9缓释纳米颗粒和胶原共孵育所述心血管植入物本体，获得心血管植入物。
进一步地：步骤(1)所述的构建心血管植入物本体的方法是：先脱去离体血管中的细胞，然后脱去所述离体血管中的核酸和脂肪，获得血管基质材料；并在所述血管基质材料表面覆盖胶原，获得心血管植入物本体。
进一步地：步骤(3)所述的H4000-CD25/dcas9缓释纳米颗粒的制备方法为：将所述Crisper/dcas9系统质粒包载到H4000-CD25纳米转染载体上；且所述Crisper/dcas9系统质粒与H4000-CD25纳米转染载体的孵育比例为0.4ug质粒/uL转染载体。
进一步地：步骤(4)将所述H4000-CD25/dcas9缓释纳米颗粒偶联于心血管植入物本体上的方法为：先用H4000-CD25/dcas9缓释纳米颗粒孵育所述心血管植入物本体10min，再用胶原孵育所述心血管植入物本体10min；再重复2次上述孵育过程，从而获得自组装修饰有H4000-CD25/dcas9缓释纳米颗粒的心血管植入物。
技术原理：心血管植入物本体由于具有一定的免疫原性，所以在植入体内后会引起机体炎症反应，尤其在血管和心血管植入物的吻合口处。并且，血管发生急性炎症还会通过促进白细胞和血浆蛋白渗透而传播慢性炎症，而急慢性炎症反应是引起血管植入物血栓和内膜再生困难或异常增生的重要诱因。
首先，为避免机体强烈的免疫排斥，先脱去离体血管中的细胞，然后脱去离体血管中的核酸和脂肪，获得血管基质材料；将本方案的心血管植入物制作成小口径心血管植入物(即小口径组织工程血管，也称为小口径TEBV，口径为1-4mm)，可解决小口径血管移植后失败率高的临床问题。一般小口径组织工程血管的长度在0.5-20cm，可满足血管移植等临床应用的需求。
在本技术方案中，转染试剂H4000为阳离子聚合物，携带大量氨基，与CD25PE抗体上的羧基进行偶联成H4000-CD25PE特异性纳米载体，再与dcas9质粒形成H4000-CD25/dcas9缓释纳米颗粒修饰到心血管植入物本体上；血管移植后，由于炎症反应，导致Treg细胞浸润到血管外，此包载后的纳米材料得以转染浸润到血管外的Treg细胞，通过在纳米转染材料上连接CD25抗体提高Treg细胞的在体靶向转染效果，原位调控免疫反应并促进血管神经再生，有效克服血栓形成，促进血管的长期通畅。
Crisper/dcas9系统转染进Treg细胞后，增强了TET2基因启动子的表达促使TET2蛋白表达量得到了提高，从而调控T-reg细胞相关细胞因子分泌，进而促进了crisper/dcas9修饰工程血管的神经重建，可以促进移植的人工血管更快的植入神经，融入到机体的稳态当中，防止人工血管的钙化和堵塞。
综上所述，本发明具有以下有益效果：
1.本发明利用了不同程度的炎症对于心血管植入物再生的影响和作用，构建了修饰有H4000-CD25/dcas9缓释纳米颗粒的心血管植入物，可诱导神经纤维长入工程血管。
2.本发明制备得到了修饰有H4000-CD25/dcas9的缓释纳米系统的心血管植入物。利用该心血管植入物进行血管移植治疗，缓释纳米颗粒可原位调控免疫反应并促进血管再生，有效克服血栓形成，促进其长期通畅。
3.通过本方法构建的心血管植入物能够高效识别聚集到的Treg细胞并激活和增强其炎症抑制功能，同时对趋附而来的巨噬细胞实现原位调控，从而促进炎症转归、营造良好的局部微环境促进血管神经再生；提高了心血管植入物的抗血栓功能和促进了心血管植入物的原位再生。
4.本方法所使用的胶原安全性和生物相容性均较高。利用胶原纳米颗粒机械强度大、承压能力强和生物相容性好的特点，通过层层自主装的方式将转染纳米颗粒包裹于胶原中并交联于心血管植入物本体表面，可实现H4000-CD25/dcas9纳米粒子的缓释，延长心血管植入物通畅的有效期。
附图说明
图1为本发明实施例1中的Crisper/dcas9系统质粒序列示意图；
图2为本发明实施例1的H4000-CD25的空间分布和H4000-CD25/dcas9转染质粒的制作过程示意图；
图3为测试H4000和H4000-CD25PE的水动力尺寸；
图4为H4000-CD25/dcas9血管缓释系统的制备和移植示意图；
图5为用H4000-Cy3/dcas9和活体荧光体外和在体检测血管缓释荧光维持效果图；
图6为本发明实施例1的纳米复合物尺寸和表面形貌(生物组织内和生物组织外，扫描电镜观察)；
图7为H4000-CD25/dcas9系统对Treg细胞的转染率流式图(包含对照组和阳性结果)；
图8为本发明转染后对巨噬细胞炎症因子表达的影响(荧光显微镜观察)；
图9为心血管植入物对Treg抑炎因子分泌的影响(ELISA检测)；
图10为检测血管植入后的血管形态和细胞聚集情况的HE染色、小动物CT和超声测血流通畅情况图；
图11为质粒PCR扩增观察质粒缓释情况；
图12为神经长入情况和血管的神经3D重建情况。
具体实施方式
下面结合附图和实施方式对本发明作进一步的详细说明：
实施例：一种基于原位调控免疫反应的心血管植入物的制备方法，如图1、图2、图3所示，包括如下步骤：
(1)构建心血管植入物本体：
1)在无菌条件下，从250-300g SD大鼠中提取颈总动脉，生理盐水冲洗血液，分离取出颈总动脉的外层结缔组织，然后剪成长0.5-1cm的小段血管(一般长度为0.5-20cm的组织工程血管可满足血管移植等临床应用的需求，制成的心血管植入物的移植对象为大鼠，所以以长0.5-1cm的小段血管为宜)。
用M199培养基稀释胰酶为0.05％浓度并在37℃的条件下消化处理血管30min去除细胞，之后用RNase、DNase及脂肪去除核酸和脂肪从而获得只有胶原和弹力纤维的血管基质材料。其中，血管基质材料的口径为1-4mm，以保证制备获得口径符合要求的小口径心血管植入物(TEBV)。
2)使用4mg/ml的胶原溶液孵育血管基质材料24h，获得心血管植入物本体。
(2)制备H4000-CD25纳米转染载体：转染试剂H4000偶联CD25-PE抗体(12-0390-82，eBioscience)成H4000-CD25转染纳米颗粒，具体步骤如下：
1)H4000测水动力尺寸，取20uL稀释到1mL。
2)H4000测Zeta电位，分别取20uL稀释到1mL，测试，结果pH7.4为3.5mV，pH4.7为3.5mV，无明显电位变化。
3)取CD25-PE 40uL 0.2mg/mL即8ug，加入10×MES pH 5.5缓冲液15uL。
4)取H4000100uL，混匀避光摇床25℃，孵育1小时，加入EDC 1mg/mL MES稀释，8uL，即8ug，避光摇床孵育过夜。
5)取混合物，使用纯水，100KDa超滤三次，定容至100uL。
产物测水动力和Zeta电位判断偶联效果，取用量20uL，动力尺寸由H4000的193nm增加到306nm，说明偶联成功。扫描电镜观测纳米粒子的粒径。
(3)将Crisper/dcas9系统质粒包载到H4000-CD25纳米转染载体上形成H4000-CD25/dcas9缓释纳米颗粒，具体步骤如下：
1)将0.8μg的dcas9质粒用25μl无血清稀释液稀释，充分混匀，制成dcas9稀释液。
无血清稀释液建议采用OPTI-MEM、无血清DMEM或1640。
2)2μl的EntransterTM-H4000/H4000-CD25PE用25μl无血清稀释液体稀释，充分混匀，制成EntransterTM-H4000/H4000-CD25PE稀释液，室温静置5min。
3)将EntransterTM-H4000稀释液分别加入到dcas9稀释液中，充分混匀，室温静置15min(可用振荡器振荡或用加样器吹吸10次以上)，H4000-CD25/dcas9缓释纳米颗粒制备完成。Crisper/dcas9系统质粒与H4000-CD25纳米转染载体的孵育比例为0.4ug质粒/uL转染载体。
(4)将H4000-CD25/dcas9缓释纳米颗粒偶联于心血管植入物本体上，具体方法如下：
1)将H4000-CD25/dcas9缓释纳米颗粒加入到含细胞和完全培养基的培养容器上，轻柔混匀。
2)将心血管植入物本体浸泡在含有H4000-CD25PE/dcas9缓释纳米颗粒的工作液浓度中10min，然后PBS浸泡2min；再将心血管植入物本体浸泡于可溶性胶原PBS溶液(1g/L)中10min，然后PBS浸泡2min,再重复孵育洗脱步骤2次，得到自组装修饰有H4000-CD25/dcas9缓释纳米颗粒的心血管植入物。
本发明还做了一系列质量控制与表征的试验，结果如下：
(1)观测形貌和各项指标
将心血管植入物取出，观测形貌和各项指标，如图6所示。本实验设置了对照组，对照组为心血管植入物本体。
扫描电镜检测纳米粒径：扫描电镜共做了5组，其中包含血管3组(H4000组，H4000-CD25组和H4000-CD25/dcas9组)，血管孵育2组(纯脱细胞血管组，H4000-CD25/dcas9血管孵育组)。不含血管组滴一滴到云母片上晾干，血管孵育组扫描前需戊二醛孵育过夜，酒精和叔丁醇梯度脱水，各组同时固定到载物托上镀金拍照。
(1)检测缓释效果：
1)心血管植入物纳米颗粒残留检测，如图4、图5所示；
制备H4000-Cy3纳米颗粒：
S1、H4000与Cy3反应：取活化的Cy3(5mg/mL)10uL，200uL的H4000和200u L的NaHCO3(0.1M,PH7.8)，三者混匀，4℃避光反应过夜。
S2、透析(透析袋规格3500)：先用50％乙醇煮沸半小时活化,两端以夹子封口，放入去离子水浴锅中，搅拌，每2h换一次水,最后一次过夜，收集。
S3、将上述H4000-Cy3心血管植入物置于1.5％琼脂糖生理盐水凝胶中,分别于1/3/7/15/30d拍照比较Cy3荧光残留情况，并分别于移植后的1/3/7/15/30d用活体成像仪观察在体DIR荧光残留情况。
2)心血管植入物质粒释放检测：如图11所示，将上述H4000-CD25PE/dcas9工程血管置于1.5％琼脂糖生理盐水溶液中。分别于1/3/7/15/30d取质粒PCR扩增观察质粒缓释情况。
(2)检测转染率和对细胞炎症因子释放的影响：如图7和图8所示，体外转染Treg细胞后24-48h,孵育兔GFP(bs-0844r，bioss)和小鼠FOXP3一抗(ab22510,abcam)，兔488和小鼠568二抗(thermofisher),流式细胞仪(C6,BD)检测转染率。Elisa试剂盒(信誉公司)检测IL10,TGF,IL35的表达变化。将转染2天后的H4000-CD25PE和H4000-CD25 PE/dcas9组Treg细胞取上清，按1:1比例添加到上述巨噬细胞培养基里。继续培养2天，用小鼠CD68(TA318150，ORIGENE)和兔CCR2(GB11326,servicebio)一抗孵育，孵育小鼠488和兔568二抗，荧光拍照。
(3)系统在体调控炎症反应：如图9所示，体外转染Treg细胞后24-48h，孵育兔GFP(bs-0844r,bioss)和小鼠FOXP3一抗(ab22510,abcam),兔488和小鼠568二抗(thermofisher),流式细胞仪(C6,BD)检测转染率。Elisa试剂盒(信誉公司)检测IL10,TGF,IL35的表达变化。具体操作步骤如下：
1)分别于3/7/15/30d取上述移植的仅脱细胞心血管植入物本体和正常未移植血管，2min内生理盐水冲刷血管凝血后，转入4％甲醛溶液中孵育3h，30％蔗糖过夜，冰冻切片，分别孵育cd3和foxp3(abcam)一抗,cd68和CCR2(abcam)一抗,孵育小鼠488和兔568二抗和DAPI,共聚焦拍照,以在体检测未转染情况下移植血管免疫细胞随时间变化情况。
2)分别于3/7/15d取上述3组(H4000-CD25PE，H4000/dcas9，H4000-CD25PE/dcas9)移植的工程血管，2min内生理盐水冲刷血管凝血后，转入4％甲醛溶液中孵育3h，30％蔗糖过夜，冰冻切片，分别孵育GFP(servicebio)和foxp3(abcam)一抗,cd68(ORIGENE)和CCR2(abcam)一抗，孵育小鼠488和兔568二抗和DAPI,共聚焦拍照,以在体检测转染情况下移植血管免疫细胞随时间变化和转染情况。
(4)观测血流通畅情况：如图10所示，将构建好的功能分子修饰的组织工程血管(心血管植入物)和对照组通过端端吻合移植到同种或异种的颈总动脉，用外科缝合线在显微镜下行端端吻合术，每组10例。
手术后1个月，用多普勒血流仪测量移植组织工程血管的颈总动脉以及对侧健康颈总动脉的血流量。静脉注射碘海醇进行MicroCT扫描造影，从而判断血管形态和连通性差异。生物人工血管移植到大鼠颈动脉，1月后通过本发明制备的H4000-CD25/dcas9缓释纳米颗粒生物工程血管保持通畅，平均血流量显著高于对照组；形态学拍照和冰冻切片HE染色可见血管无血栓形成和内膜增生，对照组血栓形成明显。
(5)观测血管神经三维重建情况：如图12所示，分别于30/60/90d取上述移植的H4000-CD25PE/dcas9组转染移植工程血管，2min内生理盐水冲刷血管凝血后，转入4％甲醛溶液中孵育3h，30％蔗糖过夜，撕外膜铺片，分别孵育PGP9.5(abcam)和TUBB3(BIOSS)一抗,以检测在体神经生长情况；孵育小鼠488和兔568二抗,共聚焦拍照拼图,用3Dmax软件根据血管神经模型进行血管神经三维重建，检测比较在体神经生长情况。
在本发明的上述实施例中，本发明利用了不同程度的炎症对于心血管植入物再生的影响和作用，构建了修饰有H4000-CD25/dcas9缓释纳米颗粒的心血管植入物，可诱导神经纤维长入工程血管。
本发明制备得到了修饰有H4000-CD25/dcas9的缓释纳米系统的心血管植入物。采用本方案制备的心血管植入物进行血管移植治疗，缓释纳米颗粒可原位调控免疫反应并促进血管再生，有效克服血栓形成，促进其长期通畅。
通过本方法构建的心血管植入物能够高效识别聚集到的Treg细胞并激活和增强其炎症抑制功能，同时对趋附而来的巨噬细胞实现原位调控，从而促进炎症转归、营造良好的局部微环境促进血管神经再生，利用Treg细胞对免疫反应的调控能力，提高了心血管植入物的抗血栓功能和促进了心血管植入物的原位再生。
胶原是生物医药领域常用物质，安全性和生物相容性均较高。利用胶原纳米颗粒机械强度大、承压能力强和生物相容性好的特点，通过层层自主装的方式将转染纳米颗粒包裹于胶原中并交联于心血管植入物本体表面，可实现H4000-CD25/dcas9纳米粒子的缓释，延长心血管植入物通畅的有效期。
dcas9质粒序列为SEQ ID NO：1：
ATGGACAAGAAGTACTCCATTGGCCTCGCCATCGGAACAAATAGCGTGGGCTGGGCTGTCATCACAGATGAGTACAAGGTGCCTAGCAAGAAATTTAAGGTGCTGGGAAATACAGACAGACATAGCATCAAGAAGAACCTCATTGGCGCTCTCCTGTTTGACTCCGGCGAAACAGCCGAAGCTACCAGACTCAAGAGAACCGCTAGGAGAAGGTACACCAGAAGGAAAAACAGGATTTGCTACCTGCAGGAAATTTTTTCCAACGAGATGGCCAAGGTGGACGATTCCTTCTTCCATAGGCTGGAAGAGAGCTTCCTCGTGGAGGAAGACAAGAAACACGAGAGGCATCCTATTTTTGGCAATATTGTGGATGAGGTCGCCTACCATGAGAAGTATCCCACAATCTATCATCTGAGAAAAAAACTGGTGGATAGCACCGACAAGGCCGATCTCAGGCTCATTTATCTCGCTCTGGCTCACATGATCAAGTTTAGGGGCCACTTCCTGATCGAAGGCGACCTGAATCCCGACAACTCCGACGTGGACAAACTGTTCATCCAGCTCGTCCAGACCTACAATCAACTCTTCGAGGAGAACCCCATCAATGCTTCCGGCGTGGATGCCAAGGCCATCCTGAGCGCTAGGCTCTCCAAGTCCAGGAGGCTGGAAAATCTGATCGCCCAACTCCCTGGAGAGAAGAAGAACGGCCTGTTTGGCAATCTGATTGCCCTGAGCCTCGGACTCACCCCCAACTTCAAGAGCAACTTCGATCTCGCCGAAGACGCCAAACTCCAACTGAGCAAGGATACCTACGACGACGATCTCGATAATCTCCTCGCCCAGATCGGCGATCAATATGCCGACCTCTTTCTGGCCGCCAAAAACCTGAGCGACGCTATTCTGCTCAGCGACATTCTCAGGGTGAATACAGAAATCACAA AAGCCCCCCTGTCCGCCAGCATGATCAAAAGGTACGATGAACACCATCAGGACCTCACCCTGCTGAAGGCTCTGGTCAGGCAGCAACTCCCCGAAAAGTACAAGGAGATTTTCTTTGATCAGTCCAAGAATGGATATGCTGGCTATATTGATGGAGGCGCCTCCCAGGAGGAATTTTATAAATTCATCAAGCCCATTCTCGAAAAGATGGACGGAACCGAAGAGCTGCTGGTCAAACTCAATAGGGAGGATCTGCTGAGGAAGCAAAGGACCTTCGACAATGGCAGCATCCCCCACCAGATCCACCTCGGCGAACTCCACGCTATCCTCAGGAGGCAGGAAGACTTCTACCCTTTCCTGAAGGATAACAGGGAGAAAATCGAGAAAATCCTGACCTTCAGAATCCCCTACTACGTCGGACCTCTCGCCAGGGGCAATTCCAGATTCGCCTGGATGACAAGGAAGAGCGAGGAAACAATCACACCATGGAACTTCGAAGAAGTGGTCGATAAGGGCGCCAGCGCCCAGAGCTTCATTGAAAGGATGACCAACTTTGATAAGAACCTGCCCAATGAGAAGGTGCTGCCTAAGCACTCCCTGCTGTATGAGTATTTCACCGTGTATAATGAGCTGACCAAGGTCAAGTACGTCACCGAGGGAATGAGAAAGCCTGCTTTTCTCTCCGGCGAGCAGAAAAAAGCCATCGTGGACCTGCTGTTCAAGACCAACAGGAAGGTGACCGTCAAGCAACTCAAGGAGGACTACTTTAAGAAGATTGAGTGCTTTGATAGCGTGGAAATTAGCGGAGTCGAGGACAGGTTCAATGCCTCCCTCGGAACATATCACGACCTGCTGAAAATCATCAAAGACAAAGATTTTCTGGATAACGAGGAGAATGAAGACATTCTGGAGGACATTGTCCTCACCCTGACCCTGTTTGAGGACAGAGAGATGATTGAAGAGAGGCTGAAAACCTATGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTCAAAAGAAGGAGATATACCGGCTGGGGCAGACTGTCCAGGAAGCTGATCAACGGCATTAGGGACAAGCAGAGCGGCAAGACCATTCTCGACTTTCTCAAGTCCGACGGATTCGCCAACAGAAACTTTATGCAACTGATCCACGATGACAGCCTCACCTTTAAGGAAGATATTCAGAAGGCTCAGGTCAGCGGCCAAGGCGATTCCCTCCATGAGCACATCGCTAATCTGGCTGGCTCCCCTGCTATCAAAAAGGGCATCCTCCAGACAGTCAAAGTCGTCGATGAG CTGGTCAAGGTGATGGGCAGGCATAAACCCGAGAACATTGTGATTGAGATGGCTAGGGAGAACCAGACCACCCAGAAAGGCCAGAAAAACAGCAGAGAAAGAATGAAGAGGATCGAGGAGGGCATCAAAGAACTGGGCAGCCAAATCCTCAAGGAGCACCCCGTCGAAAATACACAACTCCAGAACGAAAAACTCTACCTCTACTATCTGCAGAACGGCAGAGACATGTACGTGGACCAGGAACTGGACATCAACAGGCTCTCCGATTACGATGTGGACGCCATCGTCCCTCAGTCCTTTCTGAAAGATGATAGCATCGACAACAAGGTGCTGACCAGGTCCGACAAGAATAGGGGCAAGAGCGATAATGTGCCCTCCGAGGAGGTCGTCAAAAAAATGAAAAACTACTGGAGACAACTCCTCAACGCTAAGCTCATCACCCAAAGAAAGTTCGACAATCTGACCAAAGCCGAGAGGGGCGGCCTCTCCGAACTGGACAAGGCCGGCTTCATCAAAAGGCAATTGGTGGAAACCAGGCAGATTACAAAGCATGTCGCTCAAATTCTCGATAGCAGGATGAATACCAAATATGACGAGAACGACAAGCTGATCAGAGAGGTCAAGGTCATCACACTCAAGTCCAAGCTCGTGAGCGACTTCAGAAAAGATTTCCAATTTTATAAAGTCAGGGAGATCAACAATTACCACCACGCTCACGACGCTTATCTCAACGCTGTCGTGGGAACCGCCCTGATCAAAAAATACCCCAAGCTGGAAAGCGAGTTCGTGTATGGCGATTATAAAGTGTACGACGTGAGGAAGATGATCGCTAAAAGCGAGCAGGAAATCGGCAAGGCTACAGCCAAGTACTTTTTCTACTCCAACATTATGAACTTCTTCAAGACCGAGATTACCCTCGCCAACGGCGAAATTAGGAAGAGGCCCCTGATTGAAACAAATGGAGAAACAGGCGAAATCGTCTGGGACAAGGGCAGGGACTTCGCCACAGTCAGAAAAGTGCTGTCCATGCCTCAAGTCAACATCGTCAAAAAGACCGAGGTGCAGACCGGCGGCTTTAGCAAAGAAAGCATCCTGCCCAAGAGAAACTCCGACAAGCTCATCGCTAGGAAGAAGGACTGGGACCCTAAGAAATACGGAGGATTTGACTCCCCTACCGTCGCCTATTCCGTCCTCGTCGTCGCTAAGGTGGAGAAGGGCAAGAGCAAGAAGCTCAAGAGCGTCAAGGAGCTGCTGGGAATCACCATCATGGAGAGGAGCTCCTTCGAAAAAAACCCTAT TGATTTCCTGGAGGCCAAGGGCTACAAGGAGGTCAAGAAGGACCTCATCATCAAGCTGCCCAAATACAGCCTCTTCGAACTGGAAAATGGCAGGAAGAGAATGCTCGCTAGCGCCGGCGAGCTCCAGAAAGGAAATGAGCTGGCTCTGCCCAGCAAGTACGTCAACTTCCTCTATCTCGCCAGCCACTATGAAAAGCTCAAGGGCAGCCCCGAAGACAATGAGCAGAAGCAGCTCTTCGTCGAGCAGCACAAGCACTACCTCGATGAAATCATCGAGCAAATCAGCGAGTTTTCCAAAAGGGTGATTCTCGCCGACGCTAACCTCGATAAGGTCCTCTCCGCTTACAACAAGCATAGAGACAAGCCCATCAGAGAACAGGCCGAGAACATCATCCACCTGTTTACACTCACAAACCTCGGAGCCCCTGCCGCTTTTAAATACTTCGATACAACCATTGATAGGAAGAGGTACACCTCCACCAAGGAGGTGCTGGATGCTACCCTGATTCATCAATCCATCACAGGACTCTACGAAACAAGGATTGACCTGTCCCAACTGGGAGGCGAC
本具体实施例仅仅是对本发明的解释，其并不是对本发明的限制，本领域技术人员在阅读完本说明书后可以根据需要对本实施例做出没有创造性贡献的修改，但只要在本发明的权利要求范围内都受到专利法的保护。
序列表
<110> 中国人民解放军陆军军医大学
<120> 一种基于原位调控免疫反应的心血管植入物及其制备方法
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4104
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggacaaga agtactccat tggcctcgcc atcggaacaa atagcgtggg ctgggctgtc 60
atcacagatg agtacaaggt gcctagcaag aaatttaagg tgctgggaaa tacagacaga 120
catagcatca agaagaacct cattggcgct ctcctgtttg actccggcga aacagccgaa 180
gctaccagac tcaagagaac cgctaggaga aggtacacca gaaggaaaaa caggatttgc 240
tacctgcagg aaattttttc caacgagatg gccaaggtgg acgattcctt cttccatagg 300
ctggaagaga gcttcctcgt ggaggaagac aagaaacacg agaggcatcc tatttttggc 360
aatattgtgg atgaggtcgc ctaccatgag aagtatccca caatctatca tctgagaaaa 420
aaactggtgg atagcaccga caaggccgat ctcaggctca tttatctcgc tctggctcac 480
atgatcaagt ttaggggcca cttcctgatc gaaggcgacc tgaatcccga caactccgac 540
gtggacaaac tgttcatcca gctcgtccag acctacaatc aactcttcga ggagaacccc 600
atcaatgctt ccggcgtgga tgccaaggcc atcctgagcg ctaggctctc caagtccagg 660
aggctggaaa atctgatcgc ccaactccct ggagagaaga agaacggcct gtttggcaat 720
ctgattgccc tgagcctcgg actcaccccc aacttcaaga gcaacttcga tctcgccgaa 780
gacgccaaac tccaactgag caaggatacc tacgacgacg atctcgataa tctcctcgcc 840
cagatcggcg atcaatatgc cgacctcttt ctggccgcca aaaacctgag cgacgctatt 900
ctgctcagcg acattctcag ggtgaataca gaaatcacaa aagcccccct gtccgccagc 960
atgatcaaaa ggtacgatga acaccatcag gacctcaccc tgctgaaggc tctggtcagg 1020
cagcaactcc ccgaaaagta caaggagatt ttctttgatc agtccaagaa tggatatgct 1080
ggctatattg atggaggcgc ctcccaggag gaattttata aattcatcaa gcccattctc 1140
gaaaagatgg acggaaccga agagctgctg gtcaaactca atagggagga tctgctgagg 1200
aagcaaagga ccttcgacaa tggcagcatc ccccaccaga tccacctcgg cgaactccac 1260
gctatcctca ggaggcagga agacttctac cctttcctga aggataacag ggagaaaatc 1320
gagaaaatcc tgaccttcag aatcccctac tacgtcggac ctctcgccag gggcaattcc 1380
agattcgcct ggatgacaag gaagagcgag gaaacaatca caccatggaa cttcgaagaa 1440
gtggtcgata agggcgccag cgcccagagc ttcattgaaa ggatgaccaa ctttgataag 1500
aacctgccca atgagaaggt gctgcctaag cactccctgc tgtatgagta tttcaccgtg 1560
tataatgagc tgaccaaggt caagtacgtc accgagggaa tgagaaagcc tgcttttctc 1620
tccggcgagc agaaaaaagc catcgtggac ctgctgttca agaccaacag gaaggtgacc 1680
gtcaagcaac tcaaggagga ctactttaag aagattgagt gctttgatag cgtggaaatt 1740
agcggagtcg aggacaggtt caatgcctcc ctcggaacat atcacgacct gctgaaaatc 1800
atcaaagaca aagattttct ggataacgag gagaatgaag acattctgga ggacattgtc 1860
ctcaccctga ccctgtttga ggacagagag atgattgaag agaggctgaa aacctatgcc 1920
cacctgttcg acgacaaggt gatgaagcag ctcaaaagaa ggagatatac cggctggggc 1980
agactgtcca ggaagctgat caacggcatt agggacaagc agagcggcaa gaccattctc 2040
gactttctca agtccgacgg attcgccaac agaaacttta tgcaactgat ccacgatgac 2100
agcctcacct ttaaggaaga tattcagaag gctcaggtca gcggccaagg cgattccctc 2160
catgagcaca tcgctaatct ggctggctcc cctgctatca aaaagggcat cctccagaca 2220
gtcaaagtcg tcgatgagct ggtcaaggtg atgggcaggc ataaacccga gaacattgtg 2280
attgagatgg ctagggagaa ccagaccacc cagaaaggcc agaaaaacag cagagaaaga 2340
atgaagagga tcgaggaggg catcaaagaa ctgggcagcc aaatcctcaa ggagcacccc 2400
gtcgaaaata cacaactcca gaacgaaaaa ctctacctct actatctgca gaacggcaga 2460
gacatgtacg tggaccagga actggacatc aacaggctct ccgattacga tgtggacgcc 2520
atcgtccctc agtcctttct gaaagatgat agcatcgaca acaaggtgct gaccaggtcc 2580
gacaagaata ggggcaagag cgataatgtg ccctccgagg aggtcgtcaa aaaaatgaaa 2640
aactactgga gacaactcct caacgctaag ctcatcaccc aaagaaagtt cgacaatctg 2700
accaaagccg agaggggcgg cctctccgaa ctggacaagg ccggcttcat caaaaggcaa 2760
ttggtggaaa ccaggcagat tacaaagcat gtcgctcaaa ttctcgatag caggatgaat 2820
accaaatatg acgagaacga caagctgatc agagaggtca aggtcatcac actcaagtcc 2880
aagctcgtga gcgacttcag aaaagatttc caattttata aagtcaggga gatcaacaat 2940
taccaccacg ctcacgacgc ttatctcaac gctgtcgtgg gaaccgccct gatcaaaaaa 3000
taccccaagc tggaaagcga gttcgtgtat ggcgattata aagtgtacga cgtgaggaag 3060
atgatcgcta aaagcgagca ggaaatcggc aaggctacag ccaagtactt tttctactcc 3120
aacattatga acttcttcaa gaccgagatt accctcgcca acggcgaaat taggaagagg 3180
cccctgattg aaacaaatgg agaaacaggc gaaatcgtct gggacaaggg cagggacttc 3240
gccacagtca gaaaagtgct gtccatgcct caagtcaaca tcgtcaaaaa gaccgaggtg 3300
cagaccggcg gctttagcaa agaaagcatc ctgcccaaga gaaactccga caagctcatc 3360
gctaggaaga aggactggga ccctaagaaa tacggaggat ttgactcccc taccgtcgcc 3420
tattccgtcc tcgtcgtcgc taaggtggag aagggcaaga gcaagaagct caagagcgtc 3480
aaggagctgc tgggaatcac catcatggag aggagctcct tcgaaaaaaa ccctattgat 3540
ttcctggagg ccaagggcta caaggaggtc aagaaggacc tcatcatcaa gctgcccaaa 3600
tacagcctct tcgaactgga aaatggcagg aagagaatgc tcgctagcgc cggcgagctc 3660
cagaaaggaa atgagctggc tctgcccagc aagtacgtca acttcctcta tctcgccagc 3720
cactatgaaa agctcaaggg cagccccgaa gacaatgagc agaagcagct cttcgtcgag 3780
cagcacaagc actacctcga tgaaatcatc gagcaaatca gcgagttttc caaaagggtg 3840
attctcgccg acgctaacct cgataaggtc ctctccgctt acaacaagca tagagacaag 3900
cccatcagag aacaggccga gaacatcatc cacctgttta cactcacaaa cctcggagcc 3960
cctgccgctt ttaaatactt cgatacaacc attgatagga agaggtacac ctccaccaag 4020
gaggtgctgg atgctaccct gattcatcaa tccatcacag gactctacga aacaaggatt 4080
gacctgtccc aactgggagg cgac 4104

Claims (7)

1.一种基于原位调控免疫反应的心血管植入物，其特征是：包括心血管植入物本体和修饰于心血管植入物本体上的H4000-CD25/dcas9缓释纳米颗粒；所述H4000-CD25/dcas9缓释纳米颗粒包括H4000质粒纳米载体、CD25抗体和dcas9质粒序列；所述H4000质粒纳米载体和CD25抗体为共价连接，且所述dcas9质粒序列用于增强去甲基化酶TET2表达。
2.根据权利要求1所述的一种基于原位调控免疫反应的心血管植入物，其特征是：所述dcas9质粒序列为pZdonor_U6-sgRNA-EF1α-dSpCas9-NLS-VP64-2A-EGFP-2A-Puro。
3.根据权利要求1所述的一种基于原位调控免疫反应的心血管植入物，其特征是：所述心血管植入物为管状，口径为1-4mm，长度为0.5-20cm。
4.根据权利要求1所述的一种基于原位调控免疫反应的心血管植入物的制备方法，其特征是，包括如下步骤：
（1）构建心血管植入物本体；
（2）制备H4000-CD25纳米转染载体；
（3）通过H4000-CD25纳米转染载体和Crisper/dcas9系统质粒，制备得到H4000-CD25/dcas9缓释纳米颗粒；
（4）将H4000-CD25/dcas9缓释纳米颗粒偶联于心血管植入物本体上：用所述H4000-CD25/dcas9缓释纳米颗粒和胶原共孵育所述心血管植入物本体，获得心血管植入物。
5.根据权利要求4所述的一种基于原位调控免疫反应的心血管植入物的制备方法，其特征是，步骤（1）所述的构建心血管植入物本体的方法是：先脱去离体血管中的细胞，然后脱去所述离体血管中的核酸和脂肪，获得血管基质材料；并在所述血管基质材料表面覆盖胶原，获得心血管植入物本体。
6.根据权利要求4所述的一种基于原位调控免疫反应的心血管植入物的制备方法，其特征是，步骤（3）所述的H4000-CD25/dcas9缓释纳米颗粒的制备方法为：将所述Crisper/dcas9系统质粒包载到H4000-CD25纳米转染载体上；且所述Crisper/dcas9系统质粒与H4000-CD25纳米转染载体的孵育比例为0.4ug质粒/uL转染载体。
7.根据权利要求4所述的一种基于原位调控免疫反应的心血管植入物的制备方法，其特征是，步骤（4）将所述H4000-CD25/dcas9缓释纳米颗粒偶联于心血管植入物本体上的方法为：先用H4000-CD25/dcas9缓释纳米颗粒孵育所述心血管植入物本体10min，再用胶原孵育所述心血管植入物本体10min；再重复2次上述孵育过程，从而获得自组装修饰有H4000-CD25/dcas9缓释纳米颗粒的心血管植入物。
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