本文選題：巨噬細胞 + 生物可降解材料　； 參考：《蘭州大學》2017年碩士論文

【摘要】：周圍血管性疾病是炎癥性、階段性和反復發(fā)作的慢性閉塞性疾病。該病常侵襲四肢中小動靜脈,以下肢多見,多次發(fā)作后癥狀逐漸明顯和加重,其治療方法主要包括非手術療法和手術療法。非手術治療即選用抗血小板聚集與擴血管藥物、高壓氧倉治療和中醫(yī)療法;手術治療的目的是重建動脈血流通道,增加肢體血供,改善缺血。局部血管發(fā)生嚴重病變,不能保證血液的正常供應且不適于保守治療時,則需進行外科血管移植治療。在實際生活中,血管損傷也是臨床工作中經(jīng)常遇到的疾病,受傷原因主要包括炸傷、槍傷、機器致傷、車禍傷及刀傷等,受傷類型主要有血管完全或部分斷裂、創(chuàng)傷性動脈瘤、創(chuàng)傷性動靜脈瘺等,因此也常常需要外科血管移植治療。目前,臨床上應用的血管移植物主要是自體血管,雖然自體血管手術效果較好,但常常因來源有限而面臨無血管可用的問題。因此,人們不得不把目光集中到人工血管替代物上。目前臨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)或聚對苯二甲酸乙二醇酯(polyethylene terephthalate,PET),即滌綸(Dacron)制成的人工合成血管。這些血管替代物用于中等直徑(6-10 mm,ID)和大直徑的(10 mm,ID)血管是比較成功的,但當用于小口徑血管(6 mm ID),易造成血栓形成和內膜增生,其有效性受到嚴重限制,遠不能滿足臨床需要。聚己內酯(polycaprolactone,PCL)被美國FDA批準作為植入人體的可降解材料。它具有出眾的力學性能,易于組織工程支架的加工制作;具有良好的生物相容性,支持多種細胞的粘附生長;可以在體內緩慢降解,且降解產(chǎn)物能夠被組織很快吸收。聚三亞甲基碳酸酯(poly(1,3-trimethylene carbonate),PTMC)與PCL相似,同屬線性脂肪族聚酯。體內的生物相容性和毒性分析顯示,PTMC對心、肝、腎等重要器官無影響,采用PTMC制備的多孔支架支持人臍靜脈內皮細胞,平滑肌細胞和間充質干細胞等多種細胞的黏附與生長。PTMC較之PCL的主要區(qū)別在于PTMC具有更好的彈性,且能在體內快速降解,因此聚三亞甲基碳酸酯作為原位組織工程血管支架材料具有潛在優(yōu)勢。本實驗以PCL-PTMC材料為研究對象,自行制備不同質量比PCL-PTMC(3:1、3:2、1:1)和PCL四組管型靜電紡絲材料,通過小鼠皮下組織埋植實驗,在預先設計的時間點(3天,1周,3周,5周,7周)取材,采用冰凍組織切片技術,以HE染色及巨噬細胞分類標記的結果為基礎,研究不同質量比PCL-PTMC靜電紡絲材料在動物體內降解速率變化情況,并觀察不同質量比材料對巨噬細胞極化分型和組織再生的影響,初步揭示該材料降解速率、巨噬細胞行為與組織重塑三者之間的內在聯(lián)系,評價其作為小口徑血管替代物的可能性,為獲得理想的血管移植物材料提供實驗基礎和理論依據(jù)。主要實驗結果如下:一、PCL-PTMC靜電紡絲物理特性靜電紡絲拉伸實驗表明,該納米材料具有一定彈性和收縮特性,與自體中小型動脈血管物理特性相似。PTMC與PCL具有類似的化學結構,在PCL-PTMC纖維材料中,PCL、PTMC的特征峰不發(fā)生顯著變化,兩者具有相似相溶性的特點,經(jīng)有機溶劑溶解后制備靜電紡絲管型假體,材料外表光滑,管壁及管腔均勻,直徑約2mm,長約5cm。為進一步明確機體細胞與靜電紡絲相融特性,本研究對埋植材料進行冰凍切片行HE染色,觀察細胞浸潤情況。二、PCL-PTMC管型材料埋植后HE染色結果埋植材料經(jīng)冰凍切片行HE染色,經(jīng)觀察發(fā)現(xiàn)早期材料表面及材料內部有大量的炎癥細胞浸潤,隨埋植時間延長,材料周圍細胞數(shù)量在1周左右時達到高峰,隨后細胞數(shù)量減少,周圍形成纖維結締組織包膜,包膜與靜電紡絲材料之間形成浸潤細胞層。為明確靜電紡絲材料表面細胞類型以及對巨噬細胞分型極化的影響,本研究進行了免疫組織細胞化學熒光染色。三、免疫組織化學熒光染色結果:埋植材料行免疫組織細胞化學熒光染色結果顯示,分別由CD68標記未分型巨噬細胞、CD11b標記的炎癥細胞、CCR7和CD206標記的M1、M2型巨噬細胞在材料植入后1周達到高峰,隨時間推移,不同質量比的生物材料表面粘附細胞數(shù)量均開始減少,PCL-PTMC(3:1)和PCL-PTMC(3:2)組材料表面M1型巨噬細胞和M2型巨噬細胞分布較均勻,支架材料內形成比較平衡的生物降解和組織重塑現(xiàn)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.

【學位授予單位】：蘭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R318.08;R654

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