【摘要】：心血管疾病已經(jīng)成為世界上致死率和發(fā)病率最高的疾病之一,目前全球有超過30%的患者因心血管疾病而死亡。人工血管介入治療和血管搭橋手術(shù)是如今臨床上普遍使用的治療手段。由于血管狹窄嚴(yán)重或血管病變,在許多情況下不適合支架植入,血管搭橋手術(shù)常常受到自體血管來源不足的限制。隨著組織工程的發(fā)展,人工血管已經(jīng)成為研究的熱點(diǎn)。目前,已經(jīng)有商品化的大口徑人工血管應(yīng)用于臨床治療,且移植后長期通暢率較高；但是由于容易堵塞或內(nèi)膜增生,還沒有理想的小口徑人工血管(6mm)可應(yīng)用于臨床治療。本論文采用原位誘導(dǎo)人工血管在體內(nèi)快速內(nèi)皮化的思路,以期解決小口徑人工血管長期通暢性問題,并結(jié)合血管材料的結(jié)構(gòu)設(shè)計(jì),促進(jìn)血管體內(nèi)重構(gòu)過程,為構(gòu)建理想的小口徑人工血管奠定基礎(chǔ)。 研究證明,聚己內(nèi)酯(PCL)由于生物惰性和較慢的降解速度限制了其作為人工血管材料的應(yīng)用,而含有精氨酸-甘氨酸-天冬氨酸(RGD)的多肽的修飾可以促進(jìn)內(nèi)皮細(xì)胞(EC)的遷移、粘附和增殖。因此,本文發(fā)明了多種方法在PCL支架表面修飾RGD多肽。首先,通過引入2-氰基苯并噻唑(CBT)和D-半胱氨酸的縮合反應(yīng)成功地在PCL支架表面共價(jià)修飾了RGD多肽衍生物,這是一種新的化學(xué)選擇性連接作用,反應(yīng)條件溫和、收率高、無需催化劑,具有良好的生物相容性。研究結(jié)果證明這種方法是安全而便捷的,可有效改善PCL支架表面的親水性和細(xì)胞相容性。 再則,創(chuàng)新性地發(fā)展了一種可以在生理?xiàng)l件下進(jìn)行的溫和的表面涂覆技術(shù),利用“表面誘導(dǎo)自組裝”在PCL支架材料表面修飾Nap-FFGRGD多肽,該兩親性多肽分子可在疏水纖維表面自組裝,形成穩(wěn)定的生物活性涂層。修飾后的PCL支架在體外可抑制血小板聚集,促進(jìn)EC的粘附、鋪展和增殖。在植入兔頸動(dòng)脈后,該多肽的修飾改善了PCL人工血管的通暢性,抑制了血栓基質(zhì)的沉積,同時(shí)顯著促進(jìn)了內(nèi)皮化進(jìn)程和平滑肌再生。此外,通過對Nap-FFGRGD分子抑制血小板聚集機(jī)理的研究,作者證明了Nap-FFG可能是通過某種未知的配體-受體相互作用與血小板結(jié)合,并在血小板周圍自組裝形成納米纖維,通過靜電排斥作用抑制血小板聚集。因此,凝膠分子Nap-FFGRGD在PCL支架表面的修飾能夠同時(shí)實(shí)現(xiàn)原位誘導(dǎo)人工血管在體內(nèi)的抗凝血功能和快速內(nèi)皮化。 但是修飾后的支架材料由于電紡纖維間較小的孔徑限制了細(xì)胞浸潤和微血管新生,不利于人工血管的長期重構(gòu)。為此,本文結(jié)合電噴聚氧化乙烯(PEO)和電紡PCL技術(shù)制備了三層復(fù)合人工血管,中層為致孔層,孔徑大約40-50μm。兔頸動(dòng)脈移植結(jié)果表明三層PCL支架這種微米級孔和納米纖維結(jié)合的結(jié)構(gòu)顯著促進(jìn)了細(xì)胞浸潤和微血管新生,有效改善了支架材料的降解、內(nèi)皮化進(jìn)程和平滑肌再生。此外,單核/巨噬細(xì)胞在人工血管重構(gòu)過程中也發(fā)揮了重要的旁分泌作用。結(jié)果證明這是一種可應(yīng)用于構(gòu)建小口徑人工血管的良好結(jié)構(gòu)。 將構(gòu)建的RGD修飾的人工血管應(yīng)用于2型糖尿病大鼠體內(nèi)移植研究。雖然該血管材料在正常大鼠體內(nèi)再生效果良好,但是在2型糖尿病大鼠體內(nèi)的重構(gòu)明顯變差,表現(xiàn)在血小板粘附增多,內(nèi)皮化進(jìn)程變緩,并伴有早期鈣化和慢性炎癥反應(yīng)等方面。因此發(fā)展一種更好的小口徑人工血管以改善其在糖尿病患者體內(nèi)的再生是十分必要的,作者認(rèn)為動(dòng)物疾病模型是可以用于后續(xù)評價(jià)小口徑人工血管體內(nèi)表現(xiàn)的一種重要模型。 此外,本文創(chuàng)新性地合成了一氧化氮(NO)小分子水凝膠,具有酶控緩釋NO的性質(zhì)。NO的釋放速率是恒定的,并且可以通過改變p-半乳糖苷酶的濃度進(jìn)行調(diào)節(jié)。利用這種水凝膠對小鼠急性皮膚損傷治療7天后顯著促進(jìn)了血管新生和傷口愈合。這種具有酶控緩釋NO功能的新型小分子水凝膠在再生醫(yī)學(xué)和組織工程領(lǐng)域?qū)碛芯薮蟮膽?yīng)用前景。 綜上所述,本論文從多肽修飾和結(jié)構(gòu)設(shè)計(jì)兩方面對小口徑人工血管的構(gòu)建進(jìn)行了深入研究,構(gòu)建了原位誘導(dǎo)體內(nèi)重構(gòu)的小口徑人工血管,為制備理想的可臨床應(yīng)用的人工血管奠定了一定的基礎(chǔ)。
[Abstract]:Cardiovascular disease has become one of the world's highest morbidity and morbidity, and more than 30% of the world's patients now die due to cardiovascular disease. Artificial blood vessel interventional therapy and vascular bypass surgery are currently widely used in clinical practice. Because of the severe or vascular stenosis of the vessel, in many cases it is not suitable for stent implantation, and the vascular bypass surgery is often limited by the insufficient source of the autologous blood vessel. With the development of the tissue engineering, the artificial blood vessel has become the hot spot of the research. At present, the commercial large-caliber artificial blood vessel has been applied to the clinical treatment, and the long-term patency rate after the transplantation is high; however, the small-caliber artificial blood vessel (6 mm), which is not ideal because of being easy to block or hyperplasia, can be used for clinical treatment. In order to solve the problem of long-term patency of the small-caliber artificial blood vessel, and to combine the structural design of the vascular material and promote the in-vivo reconstruction of the artificial blood vessel, the paper lays the foundation for the construction of the ideal small-caliber artificial blood vessel. Studies have shown that polycaprolactone (PCL) has limited its application as an artificial blood vessel material due to the biological inertia and the slower degradation, and the modification of the polypeptide containing arginine-glycine-aspartic acid (RGD) can promote the migration, adhesion and adhesion of the endothelial cells (EC), Thus, a variety of methods have been invented to modify the RGD on the surface of the PCL scaffold, Firstly, the RGD polypeptide derivative is successfully modified on the surface of the PCL scaffold by the condensation reaction of 2-cyanogen-based benzo-cyanophenyl (CBT) and D-cysteine, which is a new chemical selective connection effect, the reaction conditions are mild, the yield is high, The agent has a good biological phase. The results of the study show that the method is safe and convenient, and can effectively improve the hydrophilic and cellular phase of the surface of the PCL scaffold. In addition, a mild surface coating technique which can be carried out under physiological conditions is creatively developed, the Nap-FFGRGD polypeptide can be modified on the surface of the PCL support material by using the 鈥淪urface-induced self-assembly鈥，

本文編號：2329265