本文選題：海藻酸鹽水凝膠粘附細(xì)胞法 + 組織工程血管移植物�。� 參考：《南京大學(xué)》2017年博士論文

【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年首次應(yīng)用于臨床后,血管旁路手術(shù)已是治療CVD最常用的方法之一。例如,冠狀動(dòng)脈旁路移植術(shù)(Coronary artery bypass grafting,CABG)現(xiàn)已成為冠心病(coronary artery disease,CAD)患者(特別是有多支血管病變的高�；颊�)血運(yùn)重建的金標(biāo)準(zhǔn)。在美國(guó),每年進(jìn)行140萬(wàn)例動(dòng)脈旁路手術(shù)治療缺血性心臟病及周圍血管疾病(包括約500,000例的CABG)。自體來(lái)源血管移植物與人造小直徑(內(nèi)徑6 mm)血管移植物(small-diameter vascular grafts,SDVGs)相比,有更好的近、遠(yuǎn)期通暢率。然而,由于某些原因,如:已存在的血管疾病、截肢、自體血管已被取用等,臨床上常沒(méi)有適合的自體動(dòng)靜脈可用。因此,構(gòu)建理想SDVGs替代自體血管是當(dāng)前的熱門(mén)課題。根據(jù)文獻(xiàn)報(bào)道,在植入機(jī)體前,于人工合成或天然移植物的支架表面預(yù)先種植細(xì)胞,是一種有效的構(gòu)建理想組織工程血管移植物(tissue engineered vascular graft,TEVG)的方法。但是,在種植細(xì)胞后,因等待細(xì)胞成熟而產(chǎn)生的時(shí)間間隔,仍然是預(yù)種植細(xì)胞移植物應(yīng)用于臨床的一個(gè)障礙。本研究中,我們應(yīng)用一種創(chuàng)新的人造血管支架細(xì)胞種植技術(shù),并通過(guò)此技術(shù)所構(gòu)建新型TEVG。從SD大鼠(Sprague-Dawley rats)身上提取大鼠脂肪干細(xì)胞(rat adipose-derived stem cells,rADSCs)并將其誘導(dǎo)培養(yǎng)成大鼠內(nèi)皮祖細(xì)胞(rat endothelial progenitor cells,rEPCs)作為構(gòu)建 TEVG 用的種子細(xì)胞。rEPCs通過(guò)免疫熒光技術(shù)及流式細(xì)胞技術(shù)對(duì)進(jìn)行鑒定。采用靜電紡絲技術(shù)制備聚己內(nèi)酯(polycaprolactone,PCL)支架,通過(guò)體內(nèi)外實(shí)驗(yàn),評(píng)估其纖維形貌、機(jī)械性能、生物適應(yīng)性方面的性能。使用海藻酸鹽水凝膠粘附細(xì)胞法(alginate hydrogel conglutinating cells,AHCC)將rEPCs種植于PCL支架上,成功通過(guò)此法構(gòu)建了TEVG。然后在體外通過(guò)AHCC法在PCL支架表面上種植rEPCs并進(jìn)行觀察,以驗(yàn)證AHCC法種植細(xì)胞的可行性;同時(shí)建立大鼠腎下腹主動(dòng)脈血管移植物移植的動(dòng)物模型,觀察短期內(nèi)該TEVG的通暢率、移植物表面內(nèi)皮化情況、組織形態(tài)學(xué)變化、免疫組化學(xué)變化、移植物表面組織的形貌等情況。實(shí)驗(yàn)結(jié)果如下所示。免疫熒光及流式細(xì)胞技術(shù)結(jié)果顯示,細(xì)胞明顯表面表達(dá)CD34、CD133、VWF抗原。且所誘導(dǎo)的rEPCs具有強(qiáng)大的增殖活性、保持rEPCs狀態(tài)傳代較長(zhǎng)、經(jīng)凍存后細(xì)胞生長(zhǎng)狀態(tài)仍可保持良好,是構(gòu)建TEVG的理想種子細(xì)胞。大鼠模型體內(nèi)外實(shí)驗(yàn)結(jié)果表明,靜電紡絲PCL支架具有亞微米級(jí)纖維直徑、良好的機(jī)械性能、無(wú)細(xì)胞毒性、良好的生物適應(yīng)性的特性,表明該支架是構(gòu)建TEVG的理想支架。體外實(shí)驗(yàn)結(jié)果表明,AHCC法組支架在相同時(shí)間點(diǎn)下具有更好的細(xì)胞粘附、增殖性,具有種植時(shí)間短、效率高、無(wú)毒性、預(yù)期通暢率高等特點(diǎn),是一種有效的細(xì)胞種植技術(shù)。大鼠模型體內(nèi)實(shí)驗(yàn)發(fā)現(xiàn),在大鼠腎下腹主動(dòng)脈植入的TEVG,其在1周、2周、4周時(shí)間點(diǎn)的觀察結(jié)果顯示,與對(duì)照組[空白PCL(blank PCL,BP)支架;空白海藻酸鹽水凝膠覆膜(blank alginate hydrogel coating,BAHC)支架;自然沉降種植細(xì)胞(natural sedimentation seeding cells,NSSC)支架]相比,AHCC組支架表面可觀察到較好的內(nèi)皮組織再生、排列情況,以及與天然血管內(nèi)膜形貌較相似的表面形貌。這些結(jié)果表明,與對(duì)照組相比,AHCC組支架具有較高的通暢率。通過(guò)AHCC法,種子細(xì)胞能夠直接粘附于血管支架表面,從而節(jié)省了等待細(xì)胞粘附的時(shí)間。根據(jù)本研究的實(shí)驗(yàn)結(jié)果,AHCC法可作為一種有效的種植細(xì)胞于血管支架的方法。我們能使用AHCC法在短時(shí)間內(nèi)快速構(gòu)建能夠迅速內(nèi)皮化的TEVG。AHCC法具有節(jié)省時(shí)間和較好的通暢率等優(yōu)點(diǎn)。
[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.

【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R318.08
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本文編號(hào)：1764762