【作者】 刘昭；

【导师】 丁义涛；

【作者基本信息】 南京大学 ， 外科学， 2011， 博士

【摘要】 血管疾病发病率有逐年增高的趋势,而血管移植手术仍是临床治疗这类疾病的最常用手段之一。目前临床常用人工血管有Dacron和ePTFE等,由于顺应性较差,血管表面没有内皮细胞覆盖,内膜过度增生,易形成血栓等原因,在小口径小血管(<5mm)移植时,长期通畅率低,是血管外科领域有待解决的难题。近年来,血管组织工程学方面的研究为解决上述难题提供了新的途径。组织工程血管有两方面关键内容：仿生学细胞外基质支架的构建和种子细胞的种植方法。电纺丝技术可以模拟天然细胞外基质形态结构构建纳米纤维支架,且技术成熟,应用越来越广。外周血内皮祖细胞取材方便,具有强大的增殖潜能,具有稳定的表型和功能,是组织工程血管理想的种子细胞。目前在种子细胞的种植技术领域已有许多研究,但存在细胞粘附率低、技术复杂、耗时长等缺点。生物电场作用广泛存在于血管组织的发育和修复,研究发现人造电场可以诱导内皮细胞的血管生成反应,影响细胞形状、运动和组织结构,引起明显的细胞延伸和定向迁移。本研究经兔外周血分离获取内皮祖细胞作为种子细胞,使用电纺丝技术制备聚己内酯纳米纤维血管支架,通过研制的反应器,利用脉冲电场刺激诱导方法,将内皮祖细胞和聚己内酯纳米纤维血管支架复合培养构建成组织工程血管补片,再移植到兔颈动脉和股动脉进行动物实验,评估其各项性能,尝试创建一种全新、高效的组织工程材料种子细胞种植方法。全文分为四个部分：第一部分外周血内皮祖细胞的分离、培养及鉴定目的：建立由外周血分离内皮祖细胞(EPCs)的方法,并进行体外培养和鉴定,选择合适的EPCs作为种子细胞用于体外构建组织工程补片。方法：经兔心脏穿刺获取外周血,使用密度梯度离心法,淋巴分离液分离外周血,得到外周血单核细胞悬液,采用EGM-2培养液置于预先铺板Fn的培养瓶中,在体外进行诱导培养获取人和兔内皮祖细胞。对其生长增殖、细胞表型、蛋白表达、形成血管能力、分泌生长因子情况和一氧化氮释放进行鉴定,以确认内皮祖细胞适用于用于构建组织工程血管补片。结果：内皮祖细胞可来源于单核细胞。EGM-2培养基培养贴壁单核细胞10-14天后,细胞呈集落状生长,在培养的2-3周后出现大量生长迅速的卵圆形外观细胞。其具有强大的增殖能力,细胞表型类似于内皮细胞,能摄取Dil-ad-LDL和UEA-I。CD31、CD34和VEGFR-2内皮细胞表面抗原标记强阳性,但不表达CD133。内皮祖细胞可在Matrigel上形成毛细血管网状结构。此外,内皮祖细胞还可以释放一氧化氮。结论：密度梯度离心法结合贴壁分离培养法,可以有效分离外周血单核细胞,有效分离和纯化外周血来源的EPCs,分离培养的细胞具有内皮祖细胞的基本表型和特征,且体外增殖活跃。可以用来构建组织工程血管补片。第二部分聚己内酯纤维组织工程血管支架的制备和其性能研究目的：应用电纺丝技术制备聚己内酯纳米纤维组织工程血管支架,为构建组织工程血管提供理想的支架材料。方法：按质量比为7：3分别准确称取PCL和PEO,将两者溶于氯仿中配置均匀纺丝液。应用电纺丝技术构建膜状聚己内酯纳米纤维组织工程血管支架。通过倒置显微镜及扫描电镜研究此支架结构及形态,并对其力学性能、亲水性等性能进行研究。为了检测此组织工程血管支架的生物相容性,将内皮祖细胞种植于支架表面,研究了细胞在支架上分布、粘附、生长、增殖及功能情况。结果：应用电纺丝技术成功构建了聚己内酯纳米纤维支架,其纳米纤维直径平均约700±35mm,呈无序排列,具有与天然血管类似的结构。聚己内酯纳米纤维支架具有优秀的生物力学性能,其缝合撕裂强度为(4.4±0.6),超过天然动脉和脱细胞支架；通过计算细胞粘附率、MTT法及一氧化氮释放分析结果表明聚己内酯支架与内皮祖细胞有生物相容性良好。结论：聚己内酯纳米纤维具有优异的生物力学性能和良好的生物相容性,表明其可作为组织工程血管支架材料用于进一步研究。第三部分应用脉冲电场刺激构建组织工程血管材料的实验研究目的：探讨应用脉冲电场刺激构建内皮祖细胞复合聚己内酯纳米纤维支架组织工程血管的可行性。方法：应用第一部分方法构建组织工程血管膜状支架备用,同时尝试构建具良好导电性的大面积柔性自支撑聚苯胺纳米结构薄膜也用于此实验。将两种支架置于自制的脉冲电场刺激器中,消毒备用。使用培养的第3代的兔内皮祖细胞胰酶消化,CM-DiI标记后制成细胞悬液,将细胞悬液加入反应器孔中,之后打开电刺激器,按照电场强度(OV,1V/cm,2V/cm,4V/cm)和刺激时间(1h和2h)的反应条件将其分为多组。2小时后,取出复合有内皮祖细胞的支架,行光镜、电镜观察和比较在不同电场刺激条件下,内皮祖细胞在聚己内酯纳米纤维管状支架和聚苯胺纳米结构薄膜上的粘附、生长及分布情况；荧光显微镜下观查CM-DiI染色阳性细胞的粘附及生长情况。使用MTT法和NO试剂盒测定脉冲电场刺激对细胞活力及细胞功能等影响。同时利用生物力学测定仪检测电场刺激培养后组织工程血管补片的力学性能。结果：在脉冲电场刺激下培养后,内皮祖细胞与组织工程支架构建结合。组织学、电子显微镜分析表明,适合的电场强度是2V/cm,在此条件下细胞在支架表面粘附最多,形态最好,连接最为紧密,2小时即可形成完整稳定的内皮单层。但是4V/cm的电场强度不利于细胞的粘附和生长,反而对细胞有害。电刺激培养后,聚乙内酯纳米纤维补片保持了良好的力学性能。荧光染色分析也证实晚期内皮祖细胞与支架材料结合紧密,生长良好。MTT和NO测试也显示2V/cm的电场强度不影响细胞活力,同时可能有助于增强内皮细胞功能。聚苯胺纳米结构薄膜由于电导率良好,更有利于电场下细胞在支架表面的粘附,但是其物理性质脆弱。结论：应用脉冲电场刺激体外培养构建组织工程血管补片,可明显促进细胞在支架表面的粘附,缩短构建时间,对细胞活性及功能无不利影响。成功于体外在脉冲电场刺激下构建了兔外周血内皮祖细胞复合聚己内酯纳米纤维支架组织工程血管补片。第四部分组织工程血管补片动物体内移植实验研究目的：建立兔颈动脉和股动脉血管补片模型,观察经体外脉冲电场刺激构建的组织工程血管补片在移植后短期的组织形态学变化、内皮细胞覆盖情况及通畅率情况。方法：于体外在脉冲电场刺激下将CM-DiI标记的兔内皮祖细胞与聚己内酯纳米纤维支架复合体外构建成组织工程血管补片。以新西兰大白兔为研究对象,将构建的组织工程血管补片移植于兔颈动脉和股动脉,并将静态种植组和脱细胞组作为对照。移植后每周使用多普勒超声随访检查,观察对比组织工程血管补片在动物体内通畅率及管腔结构变化。移植后3周,取出移植段动脉标本,行大体观察,HE染色,在光镜和扫描电镜下观察组织工程血管结构变化及内皮化情况。应用荧光显微镜观察CM-DiI和FITC-vWF荧光染色阳性细胞来检测补片管腔内壁内皮化情况及内皮细胞来源。同时,对移植后组织工程血管的生物力学性能变化情况也进行了检测。结果：移植后,所有静态培养的血管补片在3周内均发生了血管闭塞(0/7),后经解剖证实为血栓形成。脉冲电场刺激构建的血管补片,仅有1例在3周内发生了血管闭塞(6/7),余下血管在3周过程中均保持通畅,形态结构无明显退行性变。脱细胞血管补片2例发生感染,2例在三周内发生闭塞(3/7)。电场刺激组短期内通畅率最高,为85.6%(6/7)。3周后取出组织工程血管补片,通畅的组织工程血管补片结构完整,无狭窄及血栓形成。而在闭塞的血管中,血栓形成明显可见。HE染色显示通畅的组织工程血管补片与周围组织相融合,结构完整,内壁覆盖有内皮细胞；扫描电镜也证实组织工程血管补片内壁已完成内皮化过程。荧光染色分析示,补片内壁存在CM-DiI和FITC-vWF荧光双染色细胞,表明种植内皮祖细胞可分化为内皮细胞参与内皮化过程。此外生物力学性能检测发现补片的机械性能略有降低。结论：本研究构建的组织工程血管补片在动物体内移植后,具有良好的生物相容性,近期通畅率高。本研究方法可提高种子细胞的种植效率,加快内皮化过程,可能为构建组织工程血管提供一种高效的新技术。更多还原

【Abstract】 The incidence of vascular disease has tended to increase year by year. The bypass graft surgery is still one of the most commonly used methods in clinical means of treating such diseases. Currently, the commonly used clinical prosthetic vascular graft materials are Dacron and expanded polytetrafluoroethylene (ePTFE). However, when applied to small-diameter (<5mm) vascular revascularization, prosthetic vascular graft materials have a low patency rate by reasons of poor compliance, vascular surface not covered by endothelial cells, chronic intimal hyperplasia, easy to cause thrombosis, etc. Thus, this is a difficult problem remaining to be solved in fields of vascular surgery.In recent years, researches on tissue engineered blood vessels have provided new ways to solve these problems. The construction of bionics extracellular matrix scaffold and cultivation methods of cell resources are the two key elements. Electrospinning technology can fabricate nanofiber scaffolds with morphology of natural extracellular matrix, and this technology has been more and more mature and widely used. Endothelial progenitor cells (EPCs) from peripheral blood are ideal cell resources in the field of tissue engineered blood vessels owing to their easy obtainment, strong proliferation potential, and stable phenotype and function. There are many studies on fields of cell resources planting technology at present, but shortcomings such as low cell adhesion, technical complexity and time-consuming still occur. Bioelectric field exists widely in vascular tissue development and regeneration. Studies have found that artificial electric field can induce angiogenic response of endothelial cells, influence cell shapes, movements and structures, and cause significant cell extension and directed migration. In this study, the EPCs from rabbit peripheral blood were isolated to be cell resources. Electrospinning technology was used to fabricate polycaprolactone (PCL) nanofibrous scaffolds. Using home-made reactor and pulsed electric field stimulation and induction, the EPCs and PCL nanofibrous scaffolds are co-cultured and constructed tissue engineered blood vessel patches. They were then transplanted into the rabbit’s carotid artery and femoral artery for vivo animal experiments. The performance was evaluated, and a new high-efficiency method of cultivating cell resources for tissue engineered materials was attempted to create. This research is divided into four parts:Part Ⅰ. Isolation, culture and identification of endothelial progenitor cells from peripheral bloodObjective To establish a method for the isolation of EPCs from peripheral blood, to culture and identificate in vitro, and to choose proper EPCs as cell resources for constructing tissue engineered blood vessel patches in vitro.Methods Peripheral blood was obtained by cardiac puncture from healthy rabbits. Using density gradient centrifugation, Lymphocytes separation Medium separated peripheral blood, and peripheral blood mononuclear cells suspension were obtained. EGM-2Medium is placed in culture plates with pre-plank Fn. Mononuclear cells were cultured in vitro on these culture plates to get human and rabbit EPCs. Their proliferation, cell phenotype, protein expression, capacity for blood vessel formation, secretion of growth factors and nitric oxide release conditions were identified to confirm that EPCs are suitable for constructing tissue engineered blood vessel patches.Results EPCs can be derived from monocytes. Within10-14days of culturing adherent monocytes in EGM-2Medium, cells grew in a colony-like shape. After2-3weeks of culture, a large number of fast-growing cells with oval appearances occurred. They have strong proliferation, and the cell phenotypes are similar to endothelial cells. They can uptake Dil-ad-LDL and UEA-I. CD31, CD34and VEGFR-2endothelial cell surface antigen marked strongly positive, but this do not express CD133. EPCs in Matrigel formed a capillary network structure. In addition, EPCs also can release nitric oxide.Conclusion Combining density gradient centrifugation with isolated adherent culture can effectively isolate mononuclear cells from peripheral blood, and can effectively separate and purify EPCs derived from peripheral blood. Isolated and cultured cells have the phenotypes and characteristics of EPCs, with an active proliferation in vitro. They can be used to fabricate tissue engineered blood vessel patches.Part II. Preparation and properties of polycaprolactone nanofibrous scaffolds for vascular tissue engineeringObjective To fabricate PCL nanofibrous scaffold by using electrospinning technology, and to provide ideal stent materials for tissue engineered blood vessels.Methods With the mass ratio of7:3, accurately weighed PCL and polyethylene oxide (PEO) and dissolved the two in chloroform to make homogeneous spinning solution. Using electrospinning technology to fabricate membrane PCL nanofibrous scaffolds. By using inverted microscope and scanning electron microscopy, the structure and morphology of the scaffolds were investigated, and their mechanical properties, hydrophilicity and other properties were studied. To test the biocompatibility of the scaffolds for vascular tissue engineering, EPCs were planted on the scaffold surface to study the distribution, adhesion, growth, proliferation and function of the cells on the scaffolds.Results PCL nanofibrous scaffolds were successfully fabricated with electrospinning techniques. Their average diameter of nano-fiber is700±35nm, showing a disordered arrangement. They have a similar structure to natural blood vessels. PCL nanofibrous scaffolds have excellent mechanical properties. Their stitch tear strength is (4.4±0.6), exceeding the natural artery and decellularized scaffolds. By calculating cell adhesion rate, MTT method and nitric oxide release, the results showed that PCL scaffolds have well biocompatibility with EPCs.Conclusion PCL nanofibers have excellent biomechanical properties and biocompatibility, which shows that they can be used as materials for tissue engineered blood vessel scaffolds for further studies.Part Ⅲ. Experimental study of tissue engineered blood vessel materials under pulsed electrical field stimulationObjective To investigate the feasibility of fabricating tissue engineered blood vessel materials of EPCs compounding PCL nanofibrous scaffolds under pulsed electrical field stimulation.Methods Membranous tissue engineered blood vessel scaffolds were fabricated according to the methods mentioned in Part I. At the meantime, try to build a large flexible self-supporting nano-structured polyaniline membrane with good electrical conductivity, which was also used for this experiment. Place the two stents in a home-made pulsed electric field stimulator, and sterilized. The trypsinized rabbit EPCs of third passage were marked with CM-DiI and made into cell suspensions. The cell suspensions were added into the reactor, and then the electrical stimulation device wad turned on. According to the reaction conditions including the electric field strength (0V,1V/cm,2V/cm,4V/cm) and stimulation time (1h and2h), the cell suspensions were divided into multiple groups.2hours later, take the scaffolds composited with EPCs. By using light and electron microscopy, under different electrical field stimulations, the adhesion, growth and distribution of EPCs on PCL nanofibrous scaffold tubes and on nano-structured polyaniline membranes were compared. Using the fluorescence microscope to check the adhesion and growth of CM-DiI staining cells. Using the MTT method and NO kits to test the influences on cell viability and cell function under pulse electrical field stimulation. Also, biomechanical analyzer was used to test the mechanical properties of tissue engineered blood vessel patches after pulse electrical field stimulation.Results After the culture of pulsed electric field stimulation, EPCs were combined with tissue engineered scaffolds. Histology research and the electron microscopy showed that the suitable electric field strength is2V/cm. Under this condition, cells in the scaffold surface adhered the most, formed the best, and were most closely connected. A complete stable endothelial monolayer could be formed after2hours.4V/cm is not conducive to cell adhesion and growth, but harmful to the cells. After the culture of pulsed electric field stimulation, PCL nanofibrous patches maintained good mechanical properties. Staining analysis also confirmed that the endothelial outgrowth cells linked closely together with scaffold materials and grew well. MTT and NO tests also showed that the electric field strength of2V/cm does not affect cell viability, and may contribute to enhancing endothelial functions. Nano-structured polyaniline membrane is more conducive to the adhesion of cells under the electric field, for it has good electrical conductivity. Conductivity of polyaniline nano-structured films as well, in the scaffolds, but it has a physical nature of vulnerability.Conclusion Under pulsed electrical field stimulation, the time for fabricating tissue engineered blood vessel patches in vitro can be shortened, and the adhesion of cells to the surface of the stent can be promoted obviously. This has no adversely effects on cell activity and function. Under pulsed electrical field stimulation in vitro, tissue engineered blood vessel patches of EPCs compounding PCL nanofibrous scaffolds from rabbit peripheral blood is successfully fabricated.Part IV. Experimental research on tissue engineered blood vessel patches in rabbitsObjective To develop an experimental model of blood vessel patches of rabbit carotid artery and femoral artery. After transplanting the tissue engineered blood vessel patches fabricated under pulsed electric field stimulation, examine the short-term histological and morphological changes, endothelial cell coverage and patency rate.Methods Under pulsed electrical field stimulation in vitro, fabricate tissue engineered blood vessel patches of CM-DiI staining rabbit EPCs compounding PCL nanofibrous scaffolds. The research objects are New Zealand white rabbits. Transplant the tissue engineered blood vessel patches fabricated into rabbit’s carotid artery and femoral artery. The static group and de-growing cells were as control. After transplantation, Doppler ultrasound was used every week to do follow-up examinations. The patency rate and lumen structural changes of the tissue engineered blood vessel patches in vivo were observed. After3weeks of transplantation, remove the transplanted artery specimens, follow with gross observation and HE staining. Light microscopy and scanning electron microscopy were used to examine the structural and endothelium changes of tissue engineered vessels. Fluorescence microscope was used to check CM-DiI and FITC-vWF staining positive cells, in order to detect endothelium changes in luminal wall of the patches and the source of endothelial cells. Meanwhile, the biomechanical properties of tissue engineered blood vessels after transplantation have also been detected.Results After transplantation, all static cultured vessel patches occluded in3weeks (0/7). The dissection showed that thrombosis occurred. For the vessel patches fabricated under pulsed electric field stimulation, only one case show vascular occlusion within3weeks, and the other vessels maintained patency in the three weeks, with no obvious morphological degeneration (6/7). For decellarized vessel patches,2had infection, and2cases of occlusion occurred in three weeks (3/7). The electric stimulation group had the highest rate of short-term patency, which is85.6%(6/7). After3weeks, tissue engineered blood vessel patches were taken out. The smooth patches had a complete structure without stenosis or thrombosis, but thrombosis occurred clearly in the occluded vessels. HE staining showed that the smooth patches blended with the surrounding tissue in a complete structure, and the inner wall was covered with endothelial cells. The scanning electron microscopy also confirmed that tissue engineered blood vessel patches had completed their endothelial lining process. The staining analysis indicated that CM-DiI and FITC-vWF fluorescence double-staining cells exist in patch wall. This demonstrates that the cultivation of EPCs can differentiate into endothelial cells and involve in the process. Biomechanical test also found that the mechanical properties of the patches became slightly lower.Conclusion After transplanted into the rabbits, the tissue engineered blood vessel patches constructed in this study showed good biocompatibility and a high short-term patency rate. This method can make the cultivation of cell resources more efficient, and it can also speed up the process of endothelial. This may provide a highly efficient new technology for the fabrication of tissue engineered blood vessels.更多还原