發(fā)布時間：2018-02-11 15:00

  本文關鍵詞： 氧化再生纖維素 止血 膠原蛋白 靜電紡絲 聚己內(nèi)酯 細胞分化　出處：《哈爾濱工業(yè)大學》2016年博士論文　論文類型：學位論文

【摘要】：近年來,天然高分子止血材料的研制逐漸成為熱點,本文利用天然高分子,如纖維素、膠原蛋白等為原材料制備出一系列具有良好止血效果的復合型止血材料。其中,氧化再生纖維素膜材料具有新穎的物理形態(tài),對該材料的止血機制研究具有重要意義;膠原蛋白/氧化再生纖維素復合止血海綿具有優(yōu)異的止血效果,同時具有良好的抑菌性能;而聚己內(nèi)酯/膠原蛋白止血修復膜的制備則開啟了止血材料的新思路,使得快速止血、長效抑菌、組織修復等多重功能有機的結(jié)合在一起。氧化再生纖維是一種常見的止血材料,然而由于其物理形態(tài)的限制,關于其作用機制的研究較少,尚不完全明確。本文利用離子液體(1-烯丙基-3-甲基咪唑高氯酸鹽)作為溶劑,在NO2/CCl4體系下制備了不同氧化時間的氧化再生纖維素膜,此種形態(tài)非常利于細胞粘附研究。FT-IR和NMR譜圖顯示此體系在C6位上發(fā)生了高選擇性氧化反應,隨著氧化時間延長,羧基含量有一定程度增加。從接觸角分析中明顯看到,氧化后的再生纖維素膜表面潤濕性明顯提高,利于血液鋪展。在SEM照片中,可以得出氧化40 h的樣品粘附并激活的血小板數(shù)量最多,粘附的紅細胞數(shù)目也較多。本文推測了兩種可能的止血機制:一是氧化再生纖維素可吸收血液中液態(tài)物質(zhì),使血液粘稠,同時表面羧基可激活血小板,進而粘附紅細胞、纖維蛋白,形成血栓;二是表面負電荷與血液中的Fe2+發(fā)生了絡合反應,形成了棕色凝膠從而迅速封閉受傷血管,并保護其不受病毒和細菌的入侵。膠原蛋白是自然界中另一種生物相容性極好的材料,被廣泛用于傷口愈合和組織修復。然而,由于膠原蛋白抑菌性能和生物有效性有限,限制了其作為口腔止血和填塞材料的應用。本文所制備的超孔膠原蛋白/氧化再生纖維素復合物(Col/ORC)具有特殊的微觀結(jié)構(gòu)及優(yōu)異的止血、抑菌性能。SEM照片中發(fā)現(xiàn),束狀的氧化再生纖維素(ORC)纖維均勻地分布于膠原蛋白(Col)網(wǎng)狀結(jié)構(gòu)中。Col/ORC復合物的FT-IR圖完全不同于ORC,但是與Col極為相似。物理性能測試結(jié)果表明Col-0.25%ORC的潤濕性、孔隙率和吸水性最佳。MTT實驗表明Col和Col/ORC對活細胞無毒性,在兩種體內(nèi)損傷模型中對止血時間和出血量的評價顯示,Col-0.25%ORC生物有效性最佳,植入體內(nèi)后,3 w內(nèi)可降解完全,組織炎癥反應消失。另外,通過ELISA和理化性能結(jié)果分析首次推測了該材料的止血機制:1)具有超孔結(jié)構(gòu)的膠原蛋白海綿可以吸收大量血液,加速血液聚集;2)羧基可聚集并激活血小板;3)內(nèi)源性凝血途徑被激活。Col/ORC復合海綿的止血機制是膠原蛋白和氧化再生纖維素粉末物理吸附與生理止血共同的作用。在術后皮膚創(chuàng)傷恢復過程中,生長因子TGFβ1對于促進肌纖維細胞分化為具有可收縮活性的成肌纖維細胞,發(fā)揮了至關重要的作用。本文通過控制純化I型膠原蛋白(Col I)與聚己內(nèi)酯(PCL)紡絲原液配比及傷口敷料中的TGFβ1使用量,采用模擬細胞外基質(zhì)傳遞過程研究最為合適的參數(shù)配比。根據(jù)細胞CCK-8活性試驗、依托RT-PCR和熒光免疫染色的檢測方法,在宏觀層面、基因表達層面和蛋白質(zhì)表達層面加以研究,從而取得可以使得TGFβ1從靜電紡絲納米纖維止血修復膜中自主、持久地釋放的關鍵參數(shù),并優(yōu)化使得該止血修復膜具有最優(yōu)的細胞相容性和最適宜的細胞分化速率。同時,本文還研究了添加噬菌體后的止血修復膜的微觀形貌及抑菌性能,結(jié)果顯示,PCL-Col I B止血修復膜(40%/60%,w/w)具有最好的刺激細胞分化功能、較好的止血效果、優(yōu)異的抑菌性能及適宜的體內(nèi)降解速率。
[Abstract]:In recent years, the development of natural macromolecule hemostatic materials has become a hot spot, using natural polymers, such as cellulose, collagen and other raw materials for the preparation of a series of good hemostatic effect of compound hemostatic materials. The oxidized regenerated cellulose membrane materials with novel physical form, is of great significance to study the hemostatic mechanism of the material collagen; / oxidized regenerated cellulose composite has excellent hemostatic sponge hemostatic effect, and has good antibacterial properties; and polycaprolactone / collagen hemostatic membrane repair preparation is opening a new idea of hemostatic materials, making rapid hemostasis, long-term antibacterial function, multiple tissue repair combined with organic oxidation regeneration. Fiber is a kind of common hemostatic materials, but because of its physical form, research on the mechanism of the less, this is not entirely clear. Using ionic liquid paper (1- allyl -3- methyl imidazole perchlorate) as solvent in NO2/CCl4 system was prepared under different oxidation time of regenerated cellulose membrane, the shape is very conducive to the study of cell adhesion.FT-IR and NMR spectra showed that this system had high selective oxidation reaction in the C6 site, with the oxidation time, carboxyl the content has increased to a certain extent. From the contact angle analysis showed that the regenerated cellulose film surface wettability after oxidation was improved to blood spreading in SEM. In the photo, the number of platelet adhesion and oxidation of 40 h samples can be drawn and activate the most number of red blood cell adhesion is greater. This paper suggested two possible the hemostatic mechanism: one is the oxidized regenerated cellulose can absorb liquid substances in the blood, the blood viscosity, and the surface carboxyl groups can activate platelets, red blood cells and adhesion, fibrin, The formation of thrombus; two is the negatively charged surface complexation reaction with Fe2+ in blood, forming a brown gel which quickly closed the injured vessel, and to protect against viruses and bacteria invasion. Collagen is another excellent biocompatible materials in nature, is widely used in wound healing and tissue repair. However, the antibacterial properties and bioavailability of collagen Co., limited its application as oral hemostatic and packing materials. The preparation of compound collagen / oxidized regenerated cellulose super hole preparation (Col/ORC) with micro structure and excellent hemostatic special antibacterial properties, found.SEM photo, oxidized regenerated cellulose bundle (ORC) fiber evenly distributed in collagen (Col).Col/ORC complexes in the network structure of FT-IR is different from ORC and Col, but are very similar. Test results show that the physical properties of Col-0 The wettability of.25%ORC, porosity and water absorption of the best.MTT experiments showed that Col and Col/ORC in living cells is non-toxic, display on the bleeding time and bleeding volume in the evaluation of two kinds of internal damage model, the bioavailability of Col-0.25%ORC best, after implantation can be completely degraded within 3 W, disappearance of inflammation. In addition, through ELISA and physicochemical properties analysis results for the first time that the hemostatic mechanism of the material: 1) with collagen sponge pore structure can absorb large amounts of blood, accelerate blood aggregation; 2) can activate platelet aggregation and carboxyl group; 3) endogenous coagulation pathway is activated the hemostatic mechanism of.Col/ORC composite sponge is the role of collagen and oxidized regenerated cellulose powder physical adsorption and hemostasis. Common in skin trauma and postoperative recovery process, growth factor beta 1 TGF to promote the differentiation of muscle cells with contractile activity into The muscle cells, play a crucial role in this paper. By controlling the purification of type I collagen (Col I) and polycaprolactone (PCL) spinning dope ratio and wound dressing in beta 1 TGF usage, the simulation of extracellular matrix transfer process is the most suitable parameter ratio. According to the CCK-8 cell activity test, detection methods based on the RT-PCR and immunofluorescence staining, at the macro level, gene expression level and protein expression level to study, so as to achieve to make TGF beta 1 from electrospun fibrous membrane in hemostasis and repair are key parameters, sustained release, and the optimization of hemostasis and repair membrane has the optimal cell compatibility and the most the suitable cell differentiation rate. At the same time, this paper also studies the microstructure and antibacterial properties, add hemostasis and repair after the phage membrane showed that PCL-Col I B (40%/60%, w/w, hemostasis and repair membrane) out It has the best function of stimulating cell differentiation, better hemostatic effect, excellent bacteriostasis and suitable degradation rate in the body.

【學位授予單位】：哈爾濱工業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R318.08;TQ317

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