CN101259292A - A method for constructing tissue engineered blood vessels - Google Patents

Abstract

Translated fromChinese

本发明涉及一种组织工程血管的构建方法，属于组织工程领域。一种组织工程血管的构建方法，包括如下步骤：(1)将管状自体活组织基架材料在体外无菌条件下连接于持续灌流的生物反应器内，管腔外贮满培养液；(2)将体外培养获得的自体内皮细胞以M199悬浮，或将骨髓单个核细胞诱导生成的自体内皮样细胞以内皮祖细胞诱导培养液悬浮，按高密度种植法接种于管腔内表面；(3)待内皮细胞或者内皮样细胞黏附后接通管腔内循环和外循环，并模拟血液循环给予适当的压力刺激、剪切力刺激，于二氧化碳培养箱内培育5～7天。本发明的优点是：具有完美的细胞相容性和足够的力学强度，克服了异源性材料引起免疫排斥及炎症反应等缺陷，制备需时间较短。

The invention relates to a method for constructing tissue engineering blood vessels, belonging to the field of tissue engineering. A method for constructing tissue engineered blood vessels, comprising the steps of: (1) Connecting a tubular autologous living tissue matrix material to a continuous perfusion bioreactor under aseptic conditions in vitro, and storing a culture solution outside the lumen; (2 ) Suspend the autoendothelial cells obtained from in vitro culture with M199, or suspend the autoendothelial-like cells induced by bone marrow mononuclear cells with endothelial progenitor cell induction culture medium, and seed them on the inner surface of the lumen according to the high-density planting method; (3) After the endothelial cells or endothelial-like cells are adhered, the intraluminal circulation and external circulation are connected, and appropriate pressure stimulation and shear force stimulation are given to simulate blood circulation, and they are incubated in a carbon dioxide incubator for 5 to 7 days. The invention has the advantages of perfect cell compatibility and sufficient mechanical strength, overcomes defects such as immune rejection and inflammatory reaction caused by heterologous materials, and requires less time for preparation.

Description

Translated fromChinese

一种组织工程血管的构建方法A method for constructing tissue engineered blood vessels

技术领域technical field

本发明涉及一种组织工程血管的构建方法，更具体地说是一种自体活组织材料体外构建血管移植物的方法，属于组织工程领域。The invention relates to a method for constructing tissue engineering blood vessels, more specifically a method for constructing blood vessel grafts in vitro with autologous living tissue materials, belonging to the field of tissue engineering.

背景技术Background technique

目前，心血管疾病已经成为严重危害人类健康的疾病之一，特别是血管闭塞性疾病最终必须依靠外科手术的方法置换病变血管。目前常用的血管移植材料包括以不可降解的人工合成材料制作的人工血管、以及经过不同方法处理过的同种或异种血管和自体血管。人工血管材料的主要问题是存在血栓形成、凝血问题；同种或异种血管主要存在排异反应、钙化、耐久性差等问题。自体血管目前仍然是中小血管移植材料的主要来源，但自体血管来源受限，数量有限，特别是老年患者、肾脏疾病患者、糖尿病、高血压患者中，可移植的无病变血管更显稀少，尤其是在需要多支旁路或在手术病人中，更是如此。虽然经过多年的努力，但目前仍未获得突破性进展。At present, cardiovascular disease has become one of the diseases that seriously endanger human health, especially vascular occlusive disease, which ultimately requires surgery to replace diseased blood vessels. Currently commonly used vascular graft materials include artificial blood vessels made of non-degradable synthetic materials, homogeneous or heterogeneous blood vessels and autologous blood vessels that have been processed in different ways. The main problems of artificial blood vessel materials are thrombosis and coagulation problems; the same or heterogeneous blood vessels mainly have problems such as rejection, calcification, and poor durability. Autologous blood vessels are still the main source of materials for small and medium-sized blood vessel grafts, but the source of autologous blood vessels is limited and the number is limited. Especially in elderly patients, patients with kidney disease, diabetes, and hypertension, transplantable disease-free blood vessels are even rarer, especially This is especially true in patients requiring multivessel bypass or in surgical patients. Although after years of hard work, no breakthrough has been made yet.

近年来，组织工程学方法的提出，有望彻底的解决这些问题，其原理是应用细胞种植的方法，将病人的活细胞在体外培育成为有活性的组织，再植入到病人体内，活细胞分泌细胞外基质成分逐渐改造，最终实现活组织器官的再生。In recent years, the proposal of tissue engineering method is expected to completely solve these problems. The principle is to use the method of cell planting to cultivate the patient's living cells into active tissues in vitro, and then implant them into the patient's body. The extracellular matrix components are gradually remodeled, eventually achieving the regeneration of living tissue organs.

传统的组织工程技术以可降解的高分子材料为支架，但高分子材料存在力学性能差、难以塑形、降解产物在局部存在不同程度的毒性等缺点。因此，生物来源的材料越来越受到重视。以胶原基架为代表的重组细胞外基质材料在生物相容性上具传统材料无法比拟的优势，然而在力学性能、顺应性等方面存在的问题极大的制约了这类材料在组织工程中的广泛应用。异种或异体来源的脱细胞材料在力学强度、顺应性上存在着特有的优势，但同时存在传染疾病、抗原残留等问题。此外，组织钙化已经成为制约此类材料应用的重要原因。近年来，受体自身来源的组织材料以其独具的优势，引起了人们的注意。Traditional tissue engineering techniques use degradable polymer materials as scaffolds, but polymer materials have disadvantages such as poor mechanical properties, difficulty in shaping, and local toxicity of degradation products to varying degrees. Therefore, materials of biological origin are gaining more and more attention. Recombinant extracellular matrix materials represented by collagen matrix have incomparable advantages in biocompatibility with traditional materials. However, problems in mechanical properties and compliance have greatly restricted the use of such materials in tissue engineering. wide application. Acellular materials derived from xenogeneic or allogeneic sources have unique advantages in mechanical strength and compliance, but at the same time have problems such as infectious diseases and antigen residues. In addition, tissue calcification has become an important reason restricting the application of such materials. In recent years, the tissue material derived from the receptor itself has attracted people's attention due to its unique advantages.

发明内容Contents of the invention

本发明要解决的技术问题是提供一种以自体活组织材料构建组织工程血管移植材料的方法，在体外应用该方法可以构建适合于作为血管移植材料的组织工程血管移植物。The technical problem to be solved by the present invention is to provide a method for constructing a tissue engineering vascular graft material with autologous living tissue material, which can be used in vitro to construct a tissue engineering vascular graft suitable as a vascular graft material.

为实现上述目的，本发明采用以下技术方案(见图1)：To achieve the above object, the present invention adopts the following technical solutions (see Fig. 1):

一种组织工程血管的构建方法，包括如下步骤：A method for constructing tissue engineered blood vessels, comprising the steps of:

(1)将管状自体活组织基架材料在体外无菌条件下连接于持续灌流的生物反应器内，管腔外贮满培养液；血管培养生态模拟系统分内循环和外循环，分别由两台多档可调蠕动泵驱动。反应器参照文献设计(见图2)(参见Mall JW，Philipp AW，RademacherA，et al.Re-endothelialization of punctured ePTFE graft：an in vitro study under pulsedperfusion condition.Nephrol Dial Transplant，2004，19：61-67.)。内外循环分别充盈自行配置的内循环培养液和外循环培养液。图中(A)内循环的蓄液瓶；(B)内循环的蠕动泵，液流方向如箭头所示；(C)内循环的液容；(D)反应器的灌流室；(E)外循环的蠕动泵；(F)外循环的蓄液瓶。a1、a2分别为内循环和外循环的气体交换进气道，b1、b2分别为内循环和外循环的气体交换排气道，均接有孔径小于0.2μm的针式微孔滤器，过滤除菌。(1) The tubular autologous living tissue scaffold material is connected to a continuous perfusion bioreactor under sterile conditions in vitro, and the tube cavity is filled with culture solution; the vascular culture ecological simulation system is divided into internal circulation and external circulation, respectively. Driven by a multi-level adjustable peristaltic pump. The reactor was designed with reference to the literature (see Figure 2) (see Mall JW, Philipp AW, RademacherA, et al. Re-endothelialization of punctured ePTFE graft: an in vitro study under pulsed perfusion condition. Nephrol Dial Transplant, 2004, 19: 61-67 .). The inner and outer circulations are respectively filled with self-configured inner circulation culture fluid and outer circulation culture fluid. In the figure (A) the liquid storage bottle of the internal circulation; (B) the peristaltic pump of the internal circulation, the liquid flow direction is shown by the arrow; (C) the liquid capacity of the internal circulation; (D) the perfusion chamber of the reactor; (E) Peristaltic pump for external circulation; (F) liquid storage bottle for external circulation. a1 and a2 are the gas exchange inlets of the inner circulation and the outer circulation respectively, and b1 and b2 are the gas exchange exhaust passages of the inner circulation and the outer circulation respectively, both of which are connected with needle-type microporous filters with a pore size less than 0.2 μm. bacteria.

(2)将体外培养获得的自体内皮细胞以M199悬浮，或将骨髓单个核细胞诱导生成的自体内皮样细胞以内皮祖细胞诱导培养液悬浮，按高密度种植法接种于管腔内表面；(2) Suspend the autoendothelial cells obtained from in vitro culture with M199, or suspend the autoendothelial-like cells induced by bone marrow mononuclear cells with endothelial progenitor cell induction culture medium, and inoculate them on the inner surface of the lumen according to the high-density planting method;

(3)待内皮细胞或者内皮样细胞黏附后接通管腔内循环和外循环，并模拟血液循环逐渐给予适当的剪切力刺激(5～15dyn/cm2)，压力95/55mmHg，于37℃5％二氧化碳培养箱内培育5～7天，获得组织工程血管样复合物。(3) After the adhesion of endothelial cells or endothelial-like cells, connect the intraluminal circulation and external circulation, and simulate the blood circulation and gradually give appropriate shear force stimulation (5-15dyn/cm2), pressure 95/55mmHg, at 37°C Cultivate in a 5% carbon dioxide incubator for 5-7 days to obtain tissue-engineered blood vessel-like complexes.

所述自体活组织基架材料的构建方法：以微创小切口技术将适合长度的医用硅胶管埋植入实验动物腹腔或皮下，做为异物模型，2～3周后，以无菌手术的方法将自体活组织包裹形成的硅胶管荚囊完整取出。剪除封闭荚囊的两端后，抽出硅胶管后，形成管状自体活组织结构，作为组织工程血管构建的基架材料。The construction method of the autologous living tissue scaffold material: using minimally invasive small incision technology, implanting a suitable length of medical silicone tube into the abdominal cavity or subcutaneous of the experimental animal as a foreign body model. Methods The silicone tube pod formed by autologous living tissue was taken out completely. After the two ends of the closed pod are cut off, the silicone tube is pulled out to form a tubular autologous living tissue structure, which is used as the base frame material for tissue engineering blood vessel construction.

本发明首先以异物植入体内，诱导机体产生包裹反应，以构建可供组织工程应用的血管基架材料(一般来说肌性管道及囊性器官的构建都适宜用此方法)。同时在体外扩增培养自体血管内皮细胞或将骨髓来源的间充质细胞向内皮方向诱导，作为种子细胞。然后在静态或旋转条件下，将种子细胞接种于血管基架材料的内表面。接着在体外持续灌流情况下，给以脉动和搏动刺激，在保证基架材料存活的基础上，促进活细胞的成熟和分化。最终形成可供移植的血管样移植材料，达到应用组织工程技术构建血管移植材料的目的。In the present invention, a foreign body is firstly implanted into the body to induce a wrapping reaction in the body to construct a vascular scaffold material that can be used in tissue engineering (generally speaking, this method is suitable for the construction of muscular conduits and cystic organs). At the same time, autologous vascular endothelial cells are expanded and cultured in vitro or bone marrow-derived mesenchymal cells are induced towards the endothelium as seed cells. The seed cells are then seeded on the inner surface of the vascular scaffold material under static or rotating conditions. Then, under the condition of continuous perfusion in vitro, pulsation and pulsation stimulation are given to promote the maturation and differentiation of living cells on the basis of ensuring the survival of the matrix material. Finally, a vascular-like graft material that can be transplanted is formed, and the purpose of applying tissue engineering technology to construct a vascular graft material is achieved.

我们以自体活组织为基架构建的组织工程血管移植材料移植到细胞供体的股动脉，1个月后随访，无明显阻塞及血栓形成，组织学发现内膜无明显增生表现。We transplanted the tissue-engineered vascular graft material based on the autologous living tissue into the femoral artery of the cell donor. Follow-up 1 month later showed no obvious obstruction and thrombosis, and no obvious intimal hyperplasia was found in histology.

本发明的优点是：首次尝试了以自体活组织作为组织工程基架材料构建组织工程器官，在保证自体活组织材料存活的基础上，在材料表面进行了细胞种植的研究。自体来源的活组织基架管具有完美的细胞相容性和足够的力学强度，且所构建的器官可完全排除异种、异体免疫排斥反应发生的风险，克服了异源性材料引起免疫排斥及炎症反应等缺陷。本发明所需时间较短，远远低于同类技术所需时间的3～6个月的周期。The invention has the advantages that: it is the first attempt to use autologous living tissue as tissue engineering base material to construct tissue engineered organs, and on the basis of ensuring the survival of autologous living tissue material, the research on cell planting is carried out on the surface of the material. The autologous living tissue scaffold tube has perfect cytocompatibility and sufficient mechanical strength, and the constructed organ can completely eliminate the risk of heterogeneous and allogeneic immune rejection, and overcome the immune rejection and inflammation caused by heterologous materials Response and other defects. The time required by the present invention is shorter, which is far lower than the 3-6 month period of time required by similar technologies.

下面结合具体实施方式对本发明作进一步说明，并非对本发明的限定，依照本领域公知的现有技术，本发明的实施方式并不限于此，因此凡依照本发明公开内容所作出的本领域的等同替换，均属于本发明的保护范围。The present invention will be further described below in conjunction with the specific embodiments, and the present invention is not limited. According to the prior art known in the art, the embodiments of the present invention are not limited thereto. Therefore, all equivalents in the field made according to the disclosure of the present invention Replacement all belong to the protection scope of the present invention.

附图说明Description of drawings

图1为本发明基本原理的示意图，将种植于腹腔内获得的自体活组织生物管连接于体外反应器，于管腔面种植内皮细胞，形成类似血管样结构。Fig. 1 is a schematic diagram of the basic principle of the present invention. The autologous living tissue biological tube obtained by planting in the abdominal cavity is connected to an in vitro reactor, and endothelial cells are planted on the lumen surface to form a blood vessel-like structure.

图2为本发明所使用的反应器及血管生物模拟系统的示意图。Fig. 2 is a schematic diagram of a reactor and a vascular biosimulation system used in the present invention.

图3为自体活组织生物管的HE染色400倍普通光镜照片。Figure 3 is a 400-fold ordinary light microscope photograph of HE staining of an autologous living tissue biological tube.

图4为经本发明构建的组织工程化血管复合物的HE染色400倍普通光学显微镜照片。Fig. 4 is a 400 times ordinary optical microscope photograph of HE staining of the tissue engineered blood vessel complex constructed by the present invention.

图5为种植细胞后的自体活组织生物管Masson染色的200倍普通光学显微镜照片。Figure 5 is a 200-fold ordinary optical microscope photo of Masson stained autologous living tissue biological tube after planting cells.

图6为天然血管的Masson染色的200倍普通光学显微镜照片。Fig. 6 is a 200 times normal light microscope photograph of Masson staining of natural blood vessels.

图7为经本发明方法构建的组织工程化血管复合物的地衣红法弹力纤维染色的200倍普通光学显微镜照片。Fig. 7 is a 200 times ordinary optical microscope photograph of elastic fibers stained with lichenin of the tissue engineered vascular complex constructed by the method of the present invention.

图8为天然动脉血管的弹力纤维染色的200倍普通光学显微镜照片。Fig. 8 is a 200 times normal optical microscope photograph of elastic fiber staining of natural arteries.

图9为未种植细胞前的新鲜自体活组织生物管表面结构的200倍扫描电镜照片。可见大量纤维结构及细胞外基质成分，偶见血细胞及成纤维细胞突起。Fig. 9 is a 200 times scanning electron micrograph of the surface structure of the fresh autologous living tissue biological tube before the cells are planted. A large number of fibrous structures and extracellular matrix components can be seen, with occasional protrusions of blood cells and fibroblasts.

图10为旋转高密度法接种内皮6小时后管腔内表面的200倍扫描电镜照片。Fig. 10 is a 200 times scanning electron micrograph of the inner surface of the lumen 6 hours after inoculation of the endothelium by the rotating high-density method.

图11为图10放大至1000倍的扫描电镜照片。可见管腔内表面大量细胞黏附，多数细胞仍呈圆形，部分细胞伸展黏附呈梭形。Fig. 11 is a scanning electron micrograph of Fig. 10 enlarged to 1000 times. It can be seen that a large number of cells adhere to the inner surface of the lumen, most of the cells are still round, and some cells are stretched and adhered in a spindle shape.

图12为静态种植24小时后管腔内表面的200倍扫描电镜照片。Fig. 12 is a 200 times scanning electron micrograph of the inner surface of the lumen after static implantation for 24 hours.

图13为图12放大至1000倍的扫描电镜照片。可见黏附细胞贴壁生长，伸展，局部有一定的极向性，但总体极性不明显。Fig. 13 is a scanning electron micrograph of Fig. 12 enlarged to 1000 times. It can be seen that the adherent cells adhere to the wall, grow and stretch, and have a certain polarity locally, but the overall polarity is not obvious.

图14为给予渐增的剪切力刺激后，种植72小时后管腔内表面的200倍扫描电镜照片。Fig. 14 is a 200-fold scanning electron micrograph of the inner surface of the lumen after implantation for 72 hours after being stimulated by increasing shear force.

图15为图14放大至1000倍的扫描电镜照片。可见细胞生长极向性明显，细胞长轴普遍沿液流方向排列，可见细胞间有汇合生长趋势。Fig. 15 is a scanning electron micrograph of Fig. 14 enlarged to 1000 times. It can be seen that the polarity of cell growth is obvious, and the long axis of cells is generally arranged along the direction of liquid flow. It can be seen that there is a confluent growth trend between cells.

图16为动态灌流培养达5天时，管腔内表面的200倍扫描电镜照片。Figure 16 is a 200-fold scanning electron micrograph of the inner surface of the lumen when the dynamic perfusion culture reaches 5 days.

图17为图16放大至1000倍的扫描电镜照片。可见细胞极向性明显，细胞排列紧密，管腔内表面光滑，细胞生长汇合。Fig. 17 is a scanning electron micrograph of Fig. 16 enlarged to 1000 times. It can be seen that the polarity of the cells is obvious, the cells are closely arranged, the inner surface of the lumen is smooth, and the cells grow confluent.

图18为天然犬股动脉血管内腔面200倍扫描电镜照片。Figure 18 is a 200-fold scanning electron micrograph of the lumen surface of the natural canine femoral artery.

图19为图18放大至1000倍的扫描电镜照片。可见细胞汇合生长，表面光滑，细胞排列极向性明显。Fig. 19 is a scanning electron micrograph of Fig. 18 enlarged to 1000 times. It can be seen that the cells grow confluently, the surface is smooth, and the polarity of cell arrangement is obvious.

图20为本方明构建的组织工程化血管样复合物的平滑肌特异性肌动蛋白(SM-α-actin)免疫组化染色的400倍普通光学显微镜照片。Figure 20 is a 400-fold ordinary optical microscope photograph of smooth muscle-specific actin (SM-α-actin) immunohistochemical staining of the tissue-engineered blood vessel-like complex constructed by Fang Ming.

图21为天然股动脉的SM-α-actin免疫组化染色的400倍普通光学显微镜照片。Figure 21 is a 400-fold ordinary light microscope photograph of SM-α-actin immunohistochemical staining of natural femoral artery.

图22为本方明构建的组织工程化血管样复合物的第VIII因子相关抗原免疫组化染色的400倍普通光学显微镜照片。Figure 22 is a 400-fold ordinary optical microscope photograph of the factor VIII-related antigen immunohistochemical staining of the tissue-engineered blood vessel-like complex constructed by Fang Ming.

图23为天然动脉的第VIII因子相关抗原免疫组化染色的400倍普通光学显微镜照片。Fig. 23 is a 400-fold ordinary light microscope photograph of the immunohistochemical staining of the factor VIII-related antigen of the natural artery.

图24为以PKH-26标记的内皮细胞种植后，血管内腔面的激光共聚焦显微镜200倍照片。Fig. 24 is a laser confocal microscope image at 200 times of the endothelial cells labeled with PKH-26 after implantation.

图25为图24标本的激光共聚焦显微镜400倍照片。Fig. 25 is a 400-fold photograph of the specimen in Fig. 24 under a laser confocal microscope.

图26为图24标本的横切面激光共聚焦显微镜200倍照片。Fig. 26 is a 200X photo of the cross-section laser confocal microscope of the specimen in Fig. 24 .

图27为图24标本的横切面激光共聚焦显微镜的400倍照片。可见荧光标记的细胞种植并铺展生长于管腔内表面，呈单层排列。Fig. 27 is a 400X photograph of the cross-section laser confocal microscope of the specimen in Fig. 24. It can be seen that the fluorescently labeled cells planted and spread on the inner surface of the lumen, arranged in a single layer.

图28为未种植细胞的自体活组织生物管与血小板作用后的内表面的1000倍扫描电镜照片。未种植内皮细胞的组织表面可见明显的血小板黏附聚集，呈团状，难于分辨单个血小板的形态。Fig. 28 is a 1000 times scanning electron micrograph of the inner surface of the autologous living tissue biological tube without cells planted with platelets. On the surface of the tissue without endothelial cells, obvious platelet adhesion and aggregation can be seen, in the form of agglomerates, and it is difficult to distinguish the shape of a single platelet.

图29为种植内皮细胞后的组织工程复合物与血小板作用后的内表面1000倍扫描电镜照片。种植内皮细胞的组织工程复合物表面几乎无明显血小板黏附，表面依然保持光滑。Fig. 29 is a 1000 times scanning electron micrograph of the inner surface of the tissue engineering compound after planting endothelial cells interacting with platelets. There was almost no obvious platelet adhesion on the surface of the tissue engineering compound planted with endothelial cells, and the surface remained smooth.

图30为本发明构建的组织工程化血管复合物内表面的6000倍透射电镜照片。可见内皮细胞间有紧密连接形成，细胞表面有绒毛样结构。Fig. 30 is a 6000 times transmission electron micrograph of the inner surface of the tissue engineered blood vessel complex constructed in the present invention. It can be seen that there are tight junctions formed between endothelial cells, and there are villi-like structures on the cell surface.

图31为本发明构建的组织工程化血管复合物管壁细胞的20,000倍透射电镜照片。Figure 31 is a 20,000-fold transmission electron micrograph of the wall cells of the tissue-engineered vascular complex constructed in the present invention.

图32为本发明构建的组织工程化血管复合物管壁细胞的30,000倍透射电镜照片。可见细胞膜表面可见多个致密斑、细胞内可见成束的细丝样结构及吞饮小泡、细胞表面被覆蛋白多糖等细胞外基质。Fig. 32 is a 30,000-fold transmission electron micrograph of the wall cells of the tissue-engineered vascular complex constructed in the present invention. It can be seen that multiple dense spots can be seen on the surface of the cell membrane, bundled filament-like structures, pinocytic vesicles, and extracellular matrix such as proteoglycans can be seen on the cell surface.

具体实施方式Detailed ways

实施例1.Example 1.

一.试剂和材料1. Reagents and materials

1.磷酸盐缓冲液(PBS液)：市售PBS粉末(Gibco)一小包，溶于1000ml蒸馏水中，调整pH 7.2，过滤除菌后，分装，置4℃备用。1. Phosphate buffer saline (PBS solution): a small packet of commercially available PBS powder (Gibco), dissolved in 1000ml distilled water, adjusted to pH 7.2, filtered and sterilized, aliquoted, and placed at 4°C for later use.

2.M199培养液：市售M199粉末(Gibco)一小袋，加碳酸氢钠1.2g，1.5mol/LHEPES 10ml，三蒸水加至1L，调节pH至7.2，过滤除菌，按250ml分装，置4℃贮存备用。2. M199 culture medium: a small bag of commercially available M199 powder (Gibco), add 1.2g sodium bicarbonate, 1.5mol/LHEPES 10ml, add three-distilled water to 1L, adjust the pH to 7.2, filter and sterilize, and pack in 250ml, Store at 4°C for later use.

3.DMEM培养液：市售DMEM粉末(Gibco)一小袋，加碳酸氢钠1.2g，1.5mol/LHEPES 10ml，三蒸水加至1L，调节pH至7.2，过滤除菌，按250ml分装，置4℃贮存备用。3. DMEM culture medium: a small bag of commercially available DMEM powder (Gibco), add 1.2g of sodium bicarbonate, 1.5mol/LHEPES 10ml, add triple distilled water to 1L, adjust the pH to 7.2, filter and sterilize, and pack in 250ml, Store at 4°C for later use.

4.内皮细胞及内循环培养液：含15～20％FBS和0.1mg/ml的内皮细胞生长补充物(ECGS，购自中日友好医院临床医学研究所)的M199培养液。4. Endothelial cells and internal circulation culture medium: M199 culture medium containing 15-20% FBS and 0.1 mg/ml endothelial cell growth supplement (ECGS, purchased from Institute of Clinical Medicine, China-Japan Friendship Hospital).

5.外循环培养液：含20％胎牛血清(FBS，Gibco)、2ng/ml碱性成纤维细胞生长因子(b-FGF，PeproTech)、50ng/ml血小板衍生生长因子-BB(PDGF-BB，PeproTech)、10ng/ml胰岛素样生长因子(IGF，PeproTech)、0.5ng/ml表皮细胞生长因子(EGF，PeproTech)的DMEM溶液。5. External circulation culture medium: containing 20% fetal bovine serum (FBS, Gibco), 2ng/ml basic fibroblast growth factor (b-FGF, PeproTech), 50ng/ml platelet-derived growth factor-BB (PDGF-BB , PeproTech), 10 ng/ml insulin-like growth factor (IGF, PeproTech), 0.5 ng/ml epidermal growth factor (EGF, PeproTech) in DMEM.

6.内皮祖细胞诱导培养液：将购自美国Canbrex bio science valkersville公司的EBM-2加EGM-2MV SingleQuots无菌条件下按说明书混合，分装后置4℃保存。6. Endothelial progenitor cell induction culture medium: EBM-2 plus EGM-2MV SingleQuots purchased from Canbrex bio science valkersville, USA, were mixed under sterile conditions according to the instructions, and stored at 4°C after aliquoting.

7.0.05％胰蛋白酶-0.53mmol/L EDTA.4Na溶液：胰蛋白酶(1∶250)(Gibco)0.5g，EDTA.4Na 0.2g，加无Ca²⁺、Mg²⁺BSS加至1L，过滤除菌，分装小瓶，-20℃保存。7. 0.05% trypsin-0.53mmol/L EDTA.4Na solution: trypsin (1:250) (Gibco) 0.5g, EDTA.4Na 0.2g, add Ca²⁺ , Mg²⁺ BSS to 1L, filter Sterilize, divide into vials, and store at -20°C.

8.医用硅胶管选择直径4mm的医用中空硅胶管，分别剪裁为长度2cm、4cm、6cm的小段，密封包装后环氧乙烷消毒灭菌，室温空气中放置1周后备用。8. For the medical silicone tube, choose a medical hollow silicone tube with a diameter of 4 mm, cut it into small sections with a length of 2 cm, 4 cm, and 6 cm, respectively, seal the package, sterilize it with ethylene oxide, and place it in the air at room temperature for 1 week before use.

9.青霉素/链霉素溶液(100倍浓度)青霉素1.0106IU，链霉素1g，加0.9％生理盐水至100ml，过滤除菌，分装小瓶，置-20℃保存。9. Penicillin/streptomycin solution (100 times concentration) penicillin 1.0106IU, streptomycin 1g, add 0.9% normal saline to 100ml, filter and sterilize, divide into vials, and store at -20°C.

10.0.1％胶原酶溶液：市售IV型胶原酶(Sigma)100mg，加PBS溶液至100ml，过滤除菌，分装置-20℃保存。10. 0.1% collagenase solution: 100 mg of commercially available type IV collagenase (Sigma), add PBS solution to 100 ml, filter and sterilize, store in separate devices at -20°C.

二.方法：2. Method:

(一)内皮细胞体外培养(1) In vitro culture of endothelial cells

无菌手术条件下，切取实验动物颈外静脉血管组织约10cm，以0.1％的胶原酶腔内灌注消化后收集细胞，离心洗涤后，以1×10⁶个/ml细胞密度接种于预先以明胶铺底的培养瓶，加入20％胎牛血清、0.1mg/ml血管内皮生长补充物(ECGS)、200U/ml青霉素、0.2ug/ml链霉素的M199培养液，37℃5％CO2培养箱内帖壁培养，取第二到六代细胞用于实验。实验细胞留取样本进行VIII因子、CD31免疫组织化学染色鉴定。Under sterile surgical conditions, excise about 10 cm of the external jugular vein tissue of the experimental animal, digest it with 0.1% collagenase in the cavity, collect the cells, centrifuge and wash them, and inoculate them at a density of 1×10⁶ cells/ml on gelatin-prepared Put the culture bottle on the bottom, add 20% fetal bovine serum, 0.1mg/ml vascular endothelial growth supplement (ECGS), 200U/ml penicillin, 0.2ug/ml streptomycin M199 culture medium, 37 ℃ 5% CO2 incubator Adhesive culture, the second to sixth generation cells were used for experiments. Samples of experimental cells were collected for identification of factor VIII and CD31 immunohistochemical staining.

骨髓间充质细胞向内皮的诱导培养与鉴别：Induction culture and differentiation of bone marrow mesenchymal cells into endothelium:

试验动物麻醉后，无菌条件下抽取骨髓10ml。以PBS 1∶1稀释后，铺于密度为1.073的淋巴细胞分离液表面，室温下2000转离心20分钟，吸取单个核细胞层，以PBS液稀释后1500转离心洗涤5～10分钟，以EBM-2EGM-2V培养液悬浮，计数并调整细胞密度，按5×10⁵/cm²接种于预先以纤维连接蛋白(Fn)铺底的培养瓶内，置于37℃5％CO₂饱和湿度的培养箱内培养，诱导骨髓基质干细胞定向分化为内皮细胞。接种培养后每2～3天换液一次，相差显微镜下观察细胞形态，待细胞长满瓶底80％时用0.05％胰蛋白酶-0.53mmol/L EDTA.4Na(Gibco)溶液消化传代。第二代细胞种植于盖玻片上，进行VIII因子、CD31、FLK-1免疫组化染色鉴定。After the experimental animals were anesthetized, 10 ml of bone marrow was extracted under aseptic conditions. Dilute with PBS 1:1, spread on the surface of lymphocyte separation medium with a density of 1.073, centrifuge at 2000 rpm for 20 minutes at room temperature, absorb the mononuclear cell layer, dilute with PBS solution, wash by centrifugation at 1500 rpm for 5-10 minutes, and use EBM - Suspend in 2EGM-2V culture medium, count and adjust the cell density, inoculate at 5×10⁵ /cm² in culture flasks pre-coated with fibronectin (Fn), and culture at 37°C with 5% CO₂ saturated humidity Cultured in the box to induce bone marrow stromal stem cells to differentiate into endothelial cells. After inoculation, the medium was changed every 2-3 days, and the cell morphology was observed under a phase-contrast microscope. When the cells covered 80% of the bottom of the bottle, they were digested and passaged with 0.05% trypsin-0.53mmol/L EDTA.4Na (Gibco) solution. The second-generation cells were planted on coverslips, and identified by immunohistochemical staining for factor VIII, CD31, and FLK-1.

(二)自体活组织生物材料的获得(2) Obtaining biomaterials from autologous living tissue

实验动物麻醉后，无菌条件下，作一长1～2cm切口，将适合长度的医用硅胶管埋植入实验动物腹腔内或背部皮下。2～3周后，同样以微创的方法将腹膜或皮下组织包裹的硅胶管荚囊完整取出。于无菌条件下适当修整后，剪除封闭荚囊的两端后，抽出硅胶管后，形成管状结构。After the experimental animals were anesthetized, under aseptic conditions, a 1-2 cm long incision was made, and a suitable length of medical silicone tube was implanted into the abdominal cavity or subcutaneously on the back of the experimental animal. After 2 to 3 weeks, the silicone tube pods wrapped in the peritoneum or subcutaneous tissue were completely removed by the same minimally invasive method. After proper trimming under aseptic conditions, the two ends of the closed pods are cut off, and the silicone tube is pulled out to form a tubular structure.

(三)自体活组织基架管的体外培养及细胞种植(3) In vitro culture and cell planting of autologous living tissue scaffold tube

将获得的自体活组织生物材料管以3-0丝线无菌条件下连接于体外持续灌流的血管生物反应器内，管腔外腔注满外循环液，采用高密度种植法将体外培养获得的自体颈静脉内皮细胞以内循环培养液悬浮，按5×10⁶/ml浓度接种于试验管腔内，于二氧化碳温箱内(5％CO2 37℃)以手工翻转或匀速缓慢旋转孵育2～4小时，以使细胞充分黏附，然后接通内循环和外循环，首先静态培养24小时，然后模拟血液循环，调节灌流速度，3天内逐渐由增加到3～9ml/s，剪切力刺激(5～15dyn/cm²)，压力95/55mmHg。于二氧化碳培养箱内继续培育至5天，即得组织工程血管。The obtained autologous living tissue biomaterial tube was connected to an in vitro continuous perfusion vascular bioreactor under 3-0 silk aseptic conditions, and the outer cavity of the tube was filled with external circulation fluid, and the high-density planting method was used to culture the obtained in vitro Autologous jugular vein endothelial cells were suspended in circulating culture medium, inoculated into the test tube at a concentration of 5×10⁶ /ml, and incubated in a carbon dioxide incubator (5% CO2 37°C) for 2 to 4 hours by manual turning or slow rotation at a uniform speed , so that the cells can fully adhere, and then connect the internal circulation and external circulation. First, culture statically for 24 hours, then simulate blood circulation, adjust the perfusion speed, and gradually increase it to 3-9ml/s within 3 days. 15dyn/cm² ), pressure 95/55mmHg. Continue culturing in a carbon dioxide incubator for up to 5 days to obtain tissue-engineered blood vessels.

实施例2.结果评价Example 2. Results Evaluation

对实施例1涉及的新鲜自体活组织生物管及组织工程血管分别留取标本，依下法进行力学测定和生物学检测。Specimens were collected from the fresh autologous living tissue biological tubes and tissue engineered blood vessels involved in Example 1, and mechanical measurements and biological tests were carried out according to the following methods.

一.方法1. Method

(一)力学检测(1) Mechanical testing

1.断裂拉伸强度的测定：将标本裁成板材状，在拉力机上将其拉断，所测的最大拉力除以实验材料的横截面积，即为标本的材料拉伸强度，表示自体活组织材料的抗拉伸能力。1. Determination of tensile strength at break: Cut the specimen into a plate shape and break it on a tensile machine. The measured maximum tensile force is divided by the cross-sectional area of the experimental material, which is the material tensile strength of the specimen, which indicates the self-activity. Stretch resistance of tissue materials.

2.断裂伸长率的测定：将标本裁成板材状，在拉力机上将其拉断，记录样品从被拉伸到被拉断的长度变化，除以样品的原始长度，所的百分比即为标本材料的断裂伸长率，表示材料的变形能力。2. Determination of elongation at break: Cut the specimen into a plate shape, break it on a tensile machine, record the change in length of the sample from being stretched to being broken, and divide it by the original length of the sample, the resulting percentage is The elongation at break of the specimen material indicates the deformability of the material.

3.标本的爆破强度测定：将自体活组织管标本连接于爆破压测量表，游离端封闭，装置内充盈PBS液，调节加压螺栓压缩腔内液体，以压力表记录管道系统内增加的压力。爆破压被定义为管型材料破裂前所达到的最高压力，表示材料对压力变化的耐受能力。3. Determination of the burst strength of the specimen: connect the autologous biopsy tube specimen to the burst pressure measuring gauge, close the free end, fill the device with PBS liquid, adjust the pressure bolt to compress the liquid in the cavity, and record the increased pressure in the pipeline system with a pressure gauge . Burst pressure is defined as the highest pressure reached before the tubular material ruptures, indicating the material's resistance to pressure changes.

4.标本的缝合强度测定：实验标本一端夹在拉力机上，另一端以6-0的尼龙缝线连接于拉力机的另一个夹具上。然后以1mm/min的速度匀速牵拉，记录完全断裂时的拉力。表示材料的可缝合性。4. Determination of the suture strength of the specimen: one end of the experimental specimen is clamped on the tensile machine, and the other end is connected to another clamp of the tensile machine with a 6-0 nylon suture. Then pull it at a constant speed of 1mm/min, and record the pulling force when it breaks completely. Indicates the seamability of the material.

(二)生物学检测：(2) Biological testing:

1.标本的基本结构：用10％的中性福尔马林固定标本，石蜡包埋，切成4微米厚的薄片，经二甲苯脱蜡、系列酒精脱水、苏木素-伊红染色，观察标本的大致细胞组成和排列分布。1. The basic structure of the specimen: fix the specimen with 10% neutral formalin, embed in paraffin, cut into 4 micron thick slices, dewax with xylene, dehydrate with serial alcohol, stain with hematoxylin-eosin, and observe the specimen The approximate cell composition and arrangement distribution.

2.标本的纤维结构：用10％的中性福尔马林固定标本，石蜡包埋，切成4微米厚的薄片，经二甲苯脱蜡、系列酒精脱水、Masson染色及地衣红法弹力纤维染色，观察标本的纤维排列和组成。2. The fiber structure of the specimen: fix the specimen with 10% neutral formalin, embed in paraffin, cut into thin slices with a thickness of 4 microns, dewax with xylene, dehydrate with serial alcohol, Masson stain and lichen red elastic fiber Stain and observe the fiber arrangement and composition of the specimen.

3.标本的表面结构：标本用2.5％戊二醛和1％锇酸双重固定，乙醇逐级脱水，二氧化碳临界点干燥，真空喷金，扫描电镜(SEM)观察生物管内腔面的超微结构。3. The surface structure of the specimen: the specimen was double-fixed with 2.5% glutaraldehyde and 1% osmic acid, dehydrated step by step with ethanol, dried at the critical point of carbon dioxide, sprayed gold in vacuum, and observed the ultrastructure of the inner surface of the biological tube with a scanning electron microscope (SEM) .

4.标本的免疫组化染色检查：用中性福尔马林固定标本，石蜡包埋，切成4微米厚的薄片，经二甲苯脱蜡、系列酒精脱水，抗原修复，按二步法依次加一抗及二抗后，滴加显色剂显色，苏木素复染，观察特定VIII因子、α-actin的表达情况。4. Immunohistochemical staining of specimens: fix the specimens with neutral formalin, embed in paraffin, cut into thin slices with a thickness of 4 microns, dewax with xylene, dehydrate with serial alcohol, and restore antigens in two steps. After adding the primary and secondary antibodies, the chromogen was added dropwise to develop the color, and counterstained with hematoxylin to observe the expression of specific factor VIII and α-actin.

5.内皮细胞的荧光标记示踪5. Fluorescence-labeled Tracing of Endothelial Cells

用PKH26-GL预标记内皮细胞，按3×10^-6M剂量标记1×10⁷个细胞。按前述方法将标记的内皮细胞种植于自体活组织血管基架材料的表面。所得组织管行2.5％的戊二醛固定。将管样结构修剪为片状，内腔面朝下，置于荧光共聚焦显微镜下观察，并摄像。同时取部分标本，以OCT胶包埋，液氮冰冻15秒，冰冻切片机内-25℃切片，厚度为8μm，载玻片上甘油指甲油封片后，置共聚焦显微镜下观察。Endothelial cells were pre-labeled with PKH26-GL, and 1×10⁷ cells were labeled at a dose of 3×10^-6 M. The labeled endothelial cells were planted on the surface of the autologous living tissue blood vessel base material according to the aforementioned method. The resulting tissue tubes were fixed with 2.5% glutaraldehyde. The tube-like structure was trimmed into sheets, with the lumen facing down, observed under a fluorescent confocal microscope, and photographed. At the same time, some specimens were taken, embedded in OCT glue, frozen in liquid nitrogen for 15 seconds, sliced at -25°C in a cryostat, with a thickness of 8 μm, sealed with glycerin nail polish on glass slides, and observed under a confocal microscope.

6.血小板黏附试验6. Platelet Adhesion Assay

以置入反应器经相同条件处理而未种植内皮细胞的基架管为对照，将血小板悬液与种植内皮后的血管样类似物静态孵育，2小时后取出，以PBS冲洗管腔面3遍，2.5％戊二醛固定后，行SEM观察。With the scaffold tube placed in the reactor treated under the same conditions but not planted with endothelial cells as a control, the platelet suspension was statically incubated with the blood vessel-like analog after planted with endothelial cells, removed after 2 hours, and the lumen surface of the tube was washed 3 times with PBS , 2.5% glutaraldehyde fixation, line SEM observation.

7.组织工程血管的透射电镜观察7. Transmission electron microscope observation of tissue engineered blood vessels

将2.5％戊二醛固定过夜的标本，PBS冲洗3次后，1％的四氧化锇4℃媒染固定30min，丙酮系列脱水，环氧树脂包埋，半薄切片制备，甲苯胺蓝染色，选择兴趣区域，切成8纳米的超薄切片，电镜下观察。本组试验标本均送首都医科大学附属天坛医院电子显微镜室完成。Specimens fixed overnight with 2.5% glutaraldehyde, rinsed with PBS for 3 times, fixed with 1% osmium tetroxide at 4°C for 30 min, dehydrated with acetone series, embedded with epoxy resin, prepared for semi-thin sections, stained with toluidine blue, selected The region of interest was cut into ultra-thin sections of 8 nm and observed under an electron microscope. All test specimens in this group were sent to the electron microscope room of Tiantan Hospital Affiliated to Capital Medical University.

三.实验结果3. Experimental results

(一)力学检测结果(1) Mechanical test results

1.断裂拉伸强度：新鲜自体活组织管拉伸强度4.7±2.3MPa，培养后血管类似物为4.5±1.8MPa，统计学无显著差异。1. Tensile strength at break: the tensile strength of the fresh autologous biopsy tube was 4.7±2.3MPa, and that of the cultured blood vessel analogue was 4.5±1.8MPa, with no statistically significant difference.

2.断裂伸长率：新鲜自体活组织管的断裂伸长率为34.2±8.3％，培养后血管类似物为33.1±10.2％，统计学无明显差异。2. Elongation at break: the elongation at break of the fresh autologous biopsy tube was 34.2±8.3%, and that of the cultured blood vessel analog was 33.1±10.2%, with no statistically significant difference.

3.爆破强度：新鲜自体活组织管的爆破强度1100±187mmHg，种植细胞后的组织工程血管复合物为1070±115mmHg，统计学无显著差异。3. Bursting strength: the bursting strength of fresh autologous living tissue tube is 1100±187mmHg, and the tissue engineered blood vessel composite after planting cells is 1070±115mmHg, there is no statistically significant difference.

4.缝合耐受强度：新鲜活组生物管的缝合耐受强度为2.5±0.3N，种植细胞后的组织工程血管复合物为2.4±0.7N，统计学无显著差异。4. Suture resistance strength: The suture resistance strength of the biological tube in the fresh living group was 2.5±0.3N, and that of the tissue engineered blood vessel composite after planting cells was 2.4±0.7N, and there was no statistically significant difference.

(二)生物学检测结果：(2) Biological test results:

1.基本结构：新鲜自体活组织生物管的普通HE染色染色显示由大量呈同心圆状排列的梭形细胞构成，细胞呈长梭形，内层较外层细胞稀薄，管壁内可见少量炎性细胞浸润(见图3)。种植后的组织工程复合物可见管腔内表面单层扁平细胞覆盖，管壁亦由大量同心圆状排列的梭形细胞组成，而炎性细胞明显减少，仅偶见(见图4)。1. Basic structure: Common HE staining of fresh autologous living tissue biological tubes shows that it is composed of a large number of spindle cells arranged in concentric circles. The cells are long spindle-shaped, and the inner layer is thinner than the outer layer of cells. Sex cell infiltration (see Figure 3). After implantation of the tissue engineering composite, the inner surface of the lumen was covered with a single layer of flat cells, and the wall of the tube was also composed of a large number of spindle cells arranged in concentric circles, and the inflammatory cells were significantly reduced, only occasionally (see Figure 4).

2.纤维结构：种植细胞后的组织工程复合物经Masson染色可见胶原纤维呈波浪状排列，类同心圆样结构，其间可见平滑肌样细胞，与天然血管结构相似(见图5、6)。两者相比较可以看出，两者的细胞外基质成分均有大量的胶原纤维构成，呈同心圆样排列，胶原纤维成波浪状排列，图中染为蓝色；纤维间可见的细胞结构，平滑肌细胞染成发紫红色；但图6中表层细胞下可见明显的连续的基底膜结构形成，图中呈粉红色线状，而图5中无此结构。地衣红法弹力纤维染色可见组织工程复合管中无明显弹力板样结构(见图7、8)。两者相比较可以看出，图7中无明显的弹力纤维板样结构形成，而图8中可见明显的弹力纤维板样结构。2. Fibrous structure: Masson staining of the tissue engineering composite after planting cells shows that the collagen fibers are arranged in a wavy, concentric circle-like structure, with smooth muscle-like cells in between, similar to the natural blood vessel structure (see Figures 5 and 6). Comparing the two, it can be seen that the extracellular matrix components of the two are composed of a large number of collagen fibers, which are arranged in concentric circles, and the collagen fibers are arranged in waves, which are dyed blue in the figure; the cell structure visible between the fibers, Smooth muscle cells were stained purple; however, in Figure 6, a continuous basement membrane structure can be seen under the superficial cells, which is pink in the figure, but there is no such structure in Figure 5. Elastic fiber staining by lichen red method showed that there was no obvious elastic plate-like structure in the tissue engineering composite tube (see Figures 7 and 8). Comparing the two, it can be seen that there is no obvious elastic fiber plate-like structure in Figure 7, but obvious elastic fiber plate-like structure can be seen in Figure 8.

3.表面结构：新鲜自体活组织生物管表面可见纤维状结构，及大量的细胞外基质成分，偶见血细胞成分及成纤维细胞突起(见图9)。种植细胞后的组织工程复合物表面由内皮细胞覆盖，且细胞随液流方向规律排列，且随时间延长而渐趋汇合，表面光滑，与天然血管类似(见图10、11、12、13、14、15、16、17、18、19)。3. Surface structure: Fibrous structures and a large amount of extracellular matrix components can be seen on the surface of fresh autologous living tissue biological tubes, and occasionally blood cell components and fibroblast protrusions can be seen (see Figure 9). The surface of the tissue engineering compound after planting cells is covered by endothelial cells, and the cells are arranged regularly with the direction of liquid flow, and gradually converge with time, and the surface is smooth, similar to natural blood vessels (see Figures 10, 11, 12, 13, 14, 15, 16, 17, 18, 19).

4.免疫组化染色：种植细胞后的组织工程血管复合物具有与天然血管类似的结构，管壁均含有大量的α-actin阳性细胞，内腔面被覆单层细胞，VIII因子染色阳性(见图20、21、22、23)。可以看出，本发明构建的组织工程样血管复合物与天然血管类似，内腔面被覆的单层细胞染色阳性，呈棕黄色。4. Immunohistochemical staining: the tissue-engineered vascular complex after planting cells has a structure similar to that of natural blood vessels, and the walls of the tubes all contain a large number of α-actin-positive cells. Figures 20, 21, 22, 23). It can be seen that the tissue engineering-like blood vessel complex constructed by the present invention is similar to the natural blood vessel, and the monolayer cells coated on the inner cavity surface are stained positively, showing brownish yellow.

5.内皮细胞的荧光标记示踪显示：PKH26示踪免疫荧光阳性的细胞被覆于组织工程血管复合物内腔面，共聚焦显微镜显示内腔面被覆细胞彼此汇合，几乎覆盖全部内腔面，横切面上可见荧光标记阳性的细胞呈单层状，局限被覆于管腔内表面(见图24、25、26、27)。5. Fluorescent labeling of endothelial cells showed that: PKH26 tracer immunofluorescence-positive cells covered the inner surface of the tissue engineered blood vessel complex. On the cut surface, the fluorescent marker-positive cells can be seen in a single layer, limited to the inner surface of the lumen (see Figures 24, 25, 26, and 27).

6.血小板黏附实验：种植内皮细胞后的组织工程复合物表面几乎无明显血小板黏附，而在反应器内经同样条件处理的未种植细胞的自体活组织生物管表面，则可见明显的血小板黏附聚集成团，难于分辨单个血小板的形态(见图28、29)。6. Platelet adhesion test: There is almost no obvious platelet adhesion on the surface of the tissue engineering compound after planting endothelial cells, but obvious platelet adhesion and aggregation can be seen on the surface of the autologous living tissue biological tube that has not been planted with cells treated under the same conditions in the reactor. Groups, it is difficult to distinguish the shape of a single platelet (see Figure 28, 29).

7.透射电镜检测：种植的内皮细胞呈单层排列于管腔表面，细胞间有紧密联结，可见细胞表面有微绒毛样结构(见图30)。内皮下管壁中层细胞具有明显的平滑肌样结构，如成束的细丝状结构、吞饮小泡、细胞表面被覆的蛋白多糖等细胞外基质成分(见图31、32)。7. Detection by transmission electron microscope: the planted endothelial cells were arranged in a single layer on the surface of the lumen, and there were tight junctions between the cells. It can be seen that there were microvilli-like structures on the cell surface (see Figure 30). The cells in the middle layer of the subendothelial tube wall have obvious smooth muscle-like structures, such as bundled filamentous structures, pinocytic vesicles, proteoglycans coated on the cell surface and other extracellular matrix components (see Figures 31 and 32).

Claims (5)

1. the construction method of an engineering blood vessel comprises the steps:

(1) tubulose is connected under external aseptic condition in the bioreactor of continous perfusion from body living tissue pedestal material, the full culture fluid of the outer storage of tube chamber, blood vessel is cultivated the ecological simulation system and is divided internal recycle and outer circulation;

What (2) In vitro culture is obtained suspends with M199 from the body endotheliocyte, or BMNC is induced suspending with endothelial progenitor cells inducing culture liquid from the body endothelioid cells of generation, is inoculated in the tube chamber inner surface by the high-density planting method;

(3) treat that endotheliocyte or endothelioid cells stick back on-tube intracavity circulation and outer circulation, and the simulate blood circulation, regulate perfusion rate, give suitable Pressure stimulation, shearing force stimulation, in CO2 gas incubator, cultivated 5～7 days, and obtained engineering blood vessel sample complex.

2. the construction method of a kind of engineering blood vessel according to claim 1, it is characterized in that: described construction method from body living tissue pedestal material is: laboratory animal abdominal cavity or subcutaneous is gone in the medical silicone tube heeling-in that will be fit to length with the mini-invasive incision technology, as the foreign body model, after 2～3 weeks, the complete taking-up of silica gel tube pod capsule that will be wrapped to form from the body living tissue with the method for aseptic operation; After wiping out the two ends of sealing pod capsule, behind the extraction silica gel tube, form tubulose from body living tissue structure, as the pedestal material of engineering blood vessel structure.

3. the construction method of a kind of engineering blood vessel according to claim 1, it is characterized in that: described pressure is 95/55mmHg.

4. the construction method of a kind of engineering blood vessel according to claim 1 is characterized in that: it is 5～15dyn/cm that described shearing force stimulates²

5. according to the construction method of the described a kind of engineering blood vessel of claim 1, it is characterized in that: described adjusting perfusion rate is to be increased to 3～9ml/s in 3 days gradually.

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