PCL-PTMC静电纺丝血管支架材料中PTMC含量变化对巨噬细胞表型和组织再生的影响

发布时间：2018-04-28 03:52

本文选题：巨噬细胞 + 生物可降解材料　；参考：《兰州大学》2017年硕士论文

【摘要】：周围血管性疾病是炎症性、阶段性和反复发作的慢性闭塞性疾病。该病常侵袭四肢中小动静脉,以下肢多见,多次发作后症状逐渐明显和加重,其治疗方法主要包括非手术疗法和手术疗法。非手术治疗即选用抗血小板聚集与扩血管药物、高压氧仓治疗和中医疗法;手术治疗的目的是重建动脉血流通道,增加肢体血供,改善缺血。局部血管发生严重病变,不能保证血液的正常供应且不适于保守治疗时,则需进行外科血管移植治疗。在实际生活中,血管损伤也是临床工作中经常遇到的疾病,受伤原因主要包括炸伤、枪伤、机器致伤、车祸伤及刀伤等,受伤类型主要有血管完全或部分断裂、创伤性动脉瘤、创伤性动静脉瘘等,因此也常常需要外科血管移植治疗。目前,临床上应用的血管移植物主要是自体血管,虽然自体血管手术效果较好,但常常因来源有限而面临无血管可用的问题。因此,人们不得不把目光集中到人工血管替代物上。目前临床上可得到的人工血管替代物多限于可膨性聚四氟乙烯(expanded polytetrafluoroethylene,e-PTFE)或聚对苯二甲酸乙二醇酯(polyethylene terephthalate,PET),即涤纶(Dacron)制成的人工合成血管。这些血管替代物用于中等直径(6-10 mm,ID)和大直径的(10 mm,ID)血管是比较成功的,但当用于小口径血管(6 mm ID),易造成血栓形成和内膜增生,其有效性受到严重限制,远不能满足临床需要。聚己内酯(polycaprolactone,PCL)被美国FDA批准作为植入人体的可降解材料。它具有出众的力学性能,易于组织工程支架的加工制作;具有良好的生物相容性,支持多种细胞的粘附生长;可以在体内缓慢降解,且降解产物能够被组织很快吸收。聚三亚甲基碳酸酯(poly(1,3-trimethylene carbonate),PTMC)与PCL相似,同属线性脂肪族聚酯。体内的生物相容性和毒性分析显示,PTMC对心、肝、肾等重要器官无影响,采用PTMC制备的多孔支架支持人脐静脉内皮细胞,平滑肌细胞和间充质干细胞等多种细胞的黏附与生长。PTMC较之PCL的主要区别在于PTMC具有更好的弹性,且能在体内快速降解,因此聚三亚甲基碳酸酯作为原位组织工程血管支架材料具有潜在优势。本实验以PCL-PTMC材料为研究对象,自行制备不同质量比PCL-PTMC(3:1、3:2、1:1)和PCL四组管型静电纺丝材料,通过小鼠皮下组织埋植实验,在预先设计的时间点(3天,1周,3周,5周,7周)取材,采用冰冻组织切片技术,以HE染色及巨噬细胞分类标记的结果为基础,研究不同质量比PCL-PTMC静电纺丝材料在动物体内降解速率变化情况,并观察不同质量比材料对巨噬细胞极化分型和组织再生的影响,初步揭示该材料降解速率、巨噬细胞行为与组织重塑三者之间的内在联系,评价其作为小口径血管替代物的可能性,为获得理想的血管移植物材料提供实验基础和理论依据。主要实验结果如下:一、PCL-PTMC静电纺丝物理特性静电纺丝拉伸实验表明,该纳米材料具有一定弹性和收缩特性,与自体中小型动脉血管物理特性相似。PTMC与PCL具有类似的化学结构,在PCL-PTMC纤维材料中,PCL、PTMC的特征峰不发生显著变化,两者具有相似相溶性的特点,经有机溶剂溶解后制备静电纺丝管型假体,材料外表光滑,管壁及管腔均匀,直径约2mm,长约5cm。为进一步明确机体细胞与静电纺丝相融特性,本研究对埋植材料进行冰冻切片行HE染色,观察细胞浸润情况。二、PCL-PTMC管型材料埋植后HE染色结果埋植材料经冰冻切片行HE染色,经观察发现早期材料表面及材料内部有大量的炎症细胞浸润,随埋植时间延长,材料周围细胞数量在1周左右时达到高峰,随后细胞数量减少,周围形成纤维结缔组织包膜,包膜与静电纺丝材料之间形成浸润细胞层。为明确静电纺丝材料表面细胞类型以及对巨噬细胞分型极化的影响,本研究进行了免疫组织细胞化学荧光染色。三、免疫组织化学荧光染色结果:埋植材料行免疫组织细胞化学荧光染色结果显示,分别由CD68标记未分型巨噬细胞、CD11b标记的炎症细胞、CCR7和CD206标记的M1、M2型巨噬细胞在材料植入后1周达到高峰,随时间推移,不同质量比的生物材料表面粘附细胞数量均开始减少,PCL-PTMC(3:1)和PCL-PTMC(3:2)组材料表面M1型巨噬细胞和M2型巨噬细胞分布较均匀,支架材料内形成比较平衡的生物降解和组织重塑现象。
[Abstract]:Peripheral vascular disease is an inflammatory, stage and recurrent chronic occlusive disease. The disease often invades the middle and small arteriovenous limbs of the extremities. The following limbs are often seen, and the symptoms are gradually obvious and aggravated after multiple episodes. The treatment methods mainly include non operative therapy and surgical treatment. Non operative treatment is the use of antiplatelet aggregation and vasodilator. The purpose of the operation is to reconstruct the arterial blood flow, increase the blood supply of the limbs, improve the blood supply of the limbs, improve the ischemia. The local blood vessels have serious lesions, and the normal supply of blood is not guaranteed and the surgical treatment is not suitable for conservative treatment. In the actual life, the vascular injury is also the clinical work. The main causes of the disease are injury, gunshot, machine injury, car accident and knife injury. The main types of injury include complete or partial rupture of blood vessels, traumatic aneurysm, traumatic arteriovenous fistula and so on. Therefore, surgical vascular transplantation is often needed. Autologous vascular surgery has a good effect, but it often faces the problem of vascular availability because of its limited source. Therefore, people have to focus their attention on artificial vascular substitutes. Currently, the available artificial vascular replacement is limited to expanded polytetrafluoroethylene (e-PTFE), or polyterephthalic acid (PTA). The synthetic blood vessels made of polyethylene terephthalate (PET), that is, polyester (Dacron). These vascular substitutes are used for medium diameter (6-10 mm, ID) and large diameter (10 mm, ID) vessels that are relatively successful, but when used in small caliber vessels (6 mm ID), thrombosis and intimal hyperplasia are easily caused, and their effectiveness is severely limited. Polycaprolactone (PCL) has been approved by American FDA as a biodegradable material for the implantation of human body. It has outstanding mechanical properties and is easy to organize the fabrication of engineering scaffolds; it has good biocompatibility and supports the adhesion and growth of many cells; it can be degraded slowly in the body, and the degradation products can be used. The tissue is absorbed quickly. Poly Sanya methyl carbonate (poly (1,3-trimethylene carbonate), PTMC) is similar to PCL and belongs to linear aliphatic polyester. Biocompatibility and toxicity analysis in the body shows that PTMC has no influence on important organs such as heart, liver and kidney. The porous scaffold prepared by PTMC supports human umbilical vein endothelial cells, smooth muscle cells and intersections. The main difference between the adhesion and growth of a variety of cells such as mesenchymal stem cells and growth.PTMC is that PTMC has better elasticity and can be degraded rapidly in the body. Therefore, poly Sanya methyl carbonate has a potential advantage as an in situ tissue engineering vascular scaffold material. This experiment is based on PCL-PTMC material as the research object, to prepare different mass ratio PC to PC. L-PTMC (3:1,3:2,1:1) and PCL four groups of tube type electrospun materials were planted in mice by subcutaneous tissue implantation, and were harvested at a predetermined time point (3 days, 1 weeks, 3 weeks, 5 weeks, 7 weeks). Frozen tissue section technique was used to study the different mass ratio of PCL-PTMC electrospun materials in animals based on the results of HE staining and macrophage classification. In vivo degradation rate changes, and observe the effects of different mass ratio materials on the polarization and tissue regeneration of macrophages, preliminarily reveal the intrinsic relationship between the material degradation rate, macrophage behavior and tissue remodeling between the three, evaluate the possibility of as a small caliber vascular substitute, in order to obtain the ideal vascular graft material The experimental basis and theoretical basis are provided. The main experimental results are as follows: 1. The physical properties of PCL-PTMC electrospun electrospun electrospun tensile tests show that the nanomaterial has certain elastic and contractile properties, similar to the physical characteristics of the small and medium size arterial blood vessels,.PTMC and PCL have similar chemical structure, in PCL-PTMC fiber, PCL, PTMC There is no significant change in the characteristic peak, which has the characteristics of similar phase solubility. The electrostatic spinning tube prosthesis is prepared by dissolved organic solvent. The material is smooth, the tube wall and the cavity are uniform, the diameter is about 2mm, and the length about 5cm. is further clarified by the body cell and the electrostatic spinning. The frozen section of the embedded material is stained with HE. Two, after the implantation of PCL-PTMC tube type material, the implant material was stained with HE staining and stained with frozen section for HE staining. It was observed that there were a large number of inflammatory cells infiltrating in the early material surface and in the material. With the time of implantation, the number of cells around the material reached the peak at about 1 weeks, and then the number of cells decreased, and the number of cells decreased. The infiltrating cell layer formed between the fibrous connective tissue, the membrane and the electrospun material formed the infiltrating cell layer. In order to determine the surface cell type of the electrospun material and the influence of the polarization of the macrophage, the immunofluorescence staining was carried out in this study. Three, the results of immunofluorescence staining: the immunofluorescence of the implant materials. The results of cell chemofluorescence staining showed that CD68 labeled untyped macrophages, CD11b labeled inflammatory cells, CCR7 and CD206 labeled M1, M2 macrophages reached the peak at 1 weeks after the material implantation, and the number of surface adhesion cells with different mass ratio began to decrease as time went on, PCL-PTMC (3:1) and PCL-PTMC (3:2). The distribution of M1 macrophages and M2 macrophages on the surface of the materials was more uniform, and the biodegradable and tissue remodeling phenomena were relatively balanced in the scaffolds.

【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.08;R654

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