海藻酸盐水凝胶粘附细胞于静电纺丝聚己内酯血管支架的机体研究

发布时间：2018-04-17 18:29

  本文选题：海藻酸盐水凝胶粘附细胞法 + 组织工程血管移植物　； 参考：《南京大学》2017年博士论文

【摘要】：心血管疾病(Cardiovascular disease,CVD)是全世界主要的死亡原因之一。自1949年首次应用于临床后,血管旁路手术已是治疗CVD最常用的方法之一。例如,冠状动脉旁路移植术(Coronary artery bypass grafting,CABG)现已成为冠心病(coronary artery disease,CAD)患者(特别是有多支血管病变的高危患者)血运重建的金标准。在美国,每年进行140万例动脉旁路手术治疗缺血性心脏病及周围血管疾病(包括约500,000例的CABG)。自体来源血管移植物与人造小直径(内径6 mm)血管移植物(small-diameter vascular grafts,SDVGs)相比,有更好的近、远期通畅率。然而,由于某些原因,如:已存在的血管疾病、截肢、自体血管已被取用等,临床上常没有适合的自体动静脉可用。因此,构建理想SDVGs替代自体血管是当前的热门课题。根据文献报道,在植入机体前,于人工合成或天然移植物的支架表面预先种植细胞,是一种有效的构建理想组织工程血管移植物(tissue engineered vascular graft,TEVG)的方法。但是,在种植细胞后,因等待细胞成熟而产生的时间间隔,仍然是预种植细胞移植物应用于临床的一个障碍。本研究中,我们应用一种创新的人造血管支架细胞种植技术,并通过此技术所构建新型TEVG。从SD大鼠(Sprague-Dawley rats)身上提取大鼠脂肪干细胞(rat adipose-derived stem cells,rADSCs)并将其诱导培养成大鼠内皮祖细胞(rat endothelial progenitor cells,rEPCs)作为构建 TEVG 用的种子细胞。rEPCs通过免疫荧光技术及流式细胞技术对进行鉴定。采用静电纺丝技术制备聚己内酯(polycaprolactone,PCL)支架,通过体内外实验,评估其纤维形貌、机械性能、生物适应性方面的性能。使用海藻酸盐水凝胶粘附细胞法(alginate hydrogel conglutinating cells,AHCC)将rEPCs种植于PCL支架上,成功通过此法构建了TEVG。然后在体外通过AHCC法在PCL支架表面上种植rEPCs并进行观察,以验证AHCC法种植细胞的可行性;同时建立大鼠肾下腹主动脉血管移植物移植的动物模型,观察短期内该TEVG的通畅率、移植物表面内皮化情况、组织形态学变化、免疫组化学变化、移植物表面组织的形貌等情况。实验结果如下所示。免疫荧光及流式细胞技术结果显示,细胞明显表面表达CD34、CD133、VWF抗原。且所诱导的rEPCs具有强大的增殖活性、保持rEPCs状态传代较长、经冻存后细胞生长状态仍可保持良好,是构建TEVG的理想种子细胞。大鼠模型体内外实验结果表明,静电纺丝PCL支架具有亚微米级纤维直径、良好的机械性能、无细胞毒性、良好的生物适应性的特性,表明该支架是构建TEVG的理想支架。体外实验结果表明,AHCC法组支架在相同时间点下具有更好的细胞粘附、增殖性,具有种植时间短、效率高、无毒性、预期通畅率高等特点,是一种有效的细胞种植技术。大鼠模型体内实验发现,在大鼠肾下腹主动脉植入的TEVG,其在1周、2周、4周时间点的观察结果显示,与对照组[空白PCL(blank PCL,BP)支架;空白海藻酸盐水凝胶覆膜(blank alginate hydrogel coating,BAHC)支架;自然沉降种植细胞(natural sedimentation seeding cells,NSSC)支架]相比,AHCC组支架表面可观察到较好的内皮组织再生、排列情况,以及与天然血管内膜形貌较相似的表面形貌。这些结果表明,与对照组相比,AHCC组支架具有较高的通畅率。通过AHCC法,种子细胞能够直接粘附于血管支架表面,从而节省了等待细胞粘附的时间。根据本研究的实验结果,AHCC法可作为一种有效的种植细胞于血管支架的方法。我们能使用AHCC法在短时间内快速构建能够迅速内皮化的TEVG。AHCC法具有节省时间和较好的通畅率等优点。
[Abstract]:Cardiovascular disease (Cardiovascular disease CVD) is one of the main causes of death in the world. For the first time since 1949 for clinical application after vascular bypass surgery is one of the most commonly used method for the treatment of CVD. For example, coronary artery bypass grafting (Coronary artery bypass grafting CABG (coronary artery) has become the coronary heart disease disease, CAD) patients (especially with multivessel disease in high-risk patients) gold standard revascularization. In the United States, 1 million 400 thousand cases of arterial bypass surgery for the treatment of ischemic heart disease and peripheral vascular disease each year (including about 500000 cases of CABG). Autologous vascular grafts and artificial small diameter (diameter 6 mm) vascular graft (small-diameter vascular grafts, SDVGs) compared with better near and long-term patency rate. However, due to some reasons, such as: amputation of existing vascular disease, has been taken by autologous blood vessels, clinical Often there is no suitable arteriovenous available. Therefore, to construct an ideal alternative to autologous vascular SDVGs is a hot topic at present. According to the literature, implanted in the body before, onto the surface of synthetic or natural graft pre plant cells, is an effective construction of ideal tissue engineering vascular graft (tissue engineered vascular graft, TEVG) method. However, in plant cells, and waiting for the maturation time interval, is still an obstacle to pre implant cell grafts for clinical application. In this study, we used cell planting artificial blood vessel support an innovative technology, and through the construction of new technology from TEVG. SD rats (Sprague-Dawley rats) from the rat adipose derived stem cells on the body (rat adipose-derived stem cells, rADSCs) and induced cultured rat endothelial progenitor cells (rat endothelial progenitor cel Ls rEPCs,.REPCs TEVG) as seed cells for construction by immunofluorescence and flow cytometry were used to identify. Electrospun polycaprolactone (polycaprolactone, PCL) scaffolds in vivo. To assess the fiber morphology, mechanical properties and biological properties of the adaptability. The use of alginate hydrogels cell adhesion method (alginate hydrogel conglutinating cells, AHCC) rEPCs seeded on the PCL scaffolds, successfully by this method to construct the TEVG. and then in the PCL on the surface of the bracket and rEPCs were observed in vitro grown by AHCC method and AHCC method to verify the feasibility of planting cells; animal model and establish the infrarenal abdominal aorta of rats vascular graft the observation of the short-term TEVG graft patency rate, surface endothelialization, morphological changes, immunohistochemical changes, plant tissue surface shape shift The appearance and so on. The experimental results are shown. Immunofluorescence and flow cytometry showed that cell surface expression was CD34, CD133, VWF and induced by antigen. REPCs has strong proliferation activity, maintain the state of the rEPCs were longer, after frozen to maintain good growth state of cells can survive, is the ideal seed cells for construction the TEVG rat model in vivo. The experimental results show that the electrospun PCL scaffold with submicron fiber diameter, good mechanical properties, cell toxicity, good biocompatibility characteristics, showed that the scaffold is an ideal scaffold in vitro TEVG. Experimental results show that AHCC method has better support group at the same time points the cell adhesion, proliferation, with planting time is short, high efficiency, non-toxic, high expected patency rate is an effective cell seeding technology. Rats model experiment showed that in the kidney of rats The abdominal aorta of TEVG implantation in 1 weeks, 2 weeks, 4 weeks of observation time points, and the control group (blank PCL, BP PCL blank) stent; blank alginate gel film (blank alginate hydrogel coating, BAHC) stent; natural sedimentation cell (natural sedimentation seeding cells plantation, NSSC) compared with group AHCC] stents, stent were observed on the surface of endothelial tissue regeneration, better arrangement, surface morphology and morphology were similar with natural vascular intima. These results show that, compared with the control group, AHCC group support has high patency rate. By AHCC method, the seed cells can direct adhesion on the stent surface thus, saving the waiting time for cell adhesion. According to the results of this study, AHCC method can be planted as an effective cell in vascular stents. We can use the AHCC method to quickly build in a short period of time The TEVG.AHCC method, which is fast enough to endothelialization, has the advantages of saving time and better patency.

【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R318.08
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本文编号：1764762