發(fā)布時(shí)間：2018-08-08 10:35

【摘要】：殼聚糖是甲殼素部分脫乙酰的產(chǎn)物,具有止血、抗菌、促進(jìn)傷口愈合和生物相容性好等優(yōu)點(diǎn),廣泛應(yīng)用于血管支架、創(chuàng)傷修復(fù)等組織工程領(lǐng)域。目前殼聚糖基止血材料取得巨大成功,其優(yōu)秀的促凝血效果在軍事和民用領(lǐng)域得到驗(yàn)證。然而,FDA批準(zhǔn)的現(xiàn)有殼聚糖基止血材料的應(yīng)用領(lǐng)域僅僅是外用,還不能作為可吸收止血材料長期體內(nèi)植入。研究表明,現(xiàn)有殼聚糖基止血材料存在以下兩個(gè)問題：(1)大都呈酸性,在動物體內(nèi)植入后會導(dǎo)致急性炎癥反應(yīng)和慢性炎癥,影響創(chuàng)傷的愈合；(2)降解較緩慢,由此產(chǎn)生組織粘連、纖維囊腫等副作用,并形成嚴(yán)重的疤痕組織。要使殼聚糖材料作為可吸收止血材料體內(nèi)植入,必須解決這兩個(gè)存在的缺陷。 (1)通過系統(tǒng)性研究殼聚糖分子量和脫乙酰度對殼聚糖降解速度的調(diào)控行為,采用均相氧化降解和非均相氧化降解法制備得到了重均分子量在36537-529652的系列小分子量的殼聚糖,采用甲殼素脫乙酰法和殼聚糖乙�；�法制備脫乙酰度在39.6%～59.6%的系列低脫乙酰度殼聚糖(酸堿滴定)。體外溶菌酶降解殼聚糖實(shí)驗(yàn)結(jié)果表明,脫乙酰度為39.6%的殼聚糖酶降解速度最快,比明膠海綿快。 (2)創(chuàng)新性的提出了仿手工抄紙工藝,從殼聚糖水凝膠懸浮液出發(fā),利用殼聚糖水凝膠顆粒之間的毛細(xì)作用和形成的氫鍵作用,使用高溫烘制或一次冷凍干燥成型,制備了殼聚糖多孔膜和多孔海綿材料。制得的殼聚糖多孔膜形狀規(guī)整、柔順性好并能夠與創(chuàng)面良好貼敷；殼聚糖多孔海綿具有高孔隙率和高表面粗糙度,這種高孔隙率和高表面粗糙度有利于促進(jìn)血液凝固。 (3)采用體外動態(tài)凝血法測定了不同脫乙酰度純殼聚糖促凝血性能,其中M0D3(D.D=48.07%,紅外)促凝效果最好。通過血小板吸附、紅細(xì)胞吸附、APTT和TT測試等方法初步研究可殼聚糖的促凝血機(jī)理。殼聚糖主要通過對血小板及紅細(xì)胞的吸附促進(jìn)血液凝固； (4)將脫乙酰度分別為39.6%,48.07%和93.6%的新型殼聚糖海綿進(jìn)行大鼠肝臟創(chuàng)傷止血實(shí)驗(yàn),結(jié)果顯示自制殼聚糖海綿在動物體內(nèi)也有止血效力,所有殼聚糖海綿止血效果都比明膠海綿好,總出血量都小于明膠海綿組,其中脫乙酰度為48.3%的殼聚糖止血效果最好。 (5)將脫乙酰度分別為39.6%,48.07%和93.6%(酸堿滴定)的新型殼聚糖海綿和明膠海綿植入大鼠肝臟創(chuàng)傷部位,在1周、4周、8周和24周時(shí)分別取各組大鼠的肝臟組織標(biāo)本,做HE.Masson's染色以及免疫組化染色(TGF-β1和IL-1p),檢查各種材料的吸收、炎癥、創(chuàng)傷愈合情況。結(jié)果表明,脫乙酰度39.6%的殼聚糖在第8周時(shí)完全被吸收；明膠海綿完全吸收的時(shí)間則大于8周,在24周時(shí)完全吸收；而脫乙酰度為48.07%和93.6%的殼聚糖在24周還未完全吸收。病理切片和IL-1β染色結(jié)果表明,脫乙酰度39.6%的殼聚糖體內(nèi)炎癥反應(yīng)最弱；,脫乙酰度39.6%的殼聚糖TGF-β1分泌水平低,膠原生成量適中,不形成疤痕性愈合；,脫乙酰度39.6%的殼聚糖,減少其他組織的粘連的幾率。
[Abstract]:Chitosan is a product of chitin partial deacetylation. It has many advantages, such as hemostasis, antibacterial, wound healing and good biocompatibility. It is widely used in the tissue engineering fields such as vascular scaffold and wound repair. At present, chitosan based hemostat has been a great success, and its excellent coagulation effect is verified in military and civil fields. However, FD The application of the existing chitosan based hemostat approved by A is only external and can not be implanted in the body for a long time as an absorbable hemostat. Research shows that the existing chitosan based hemostat has two problems: (1) most of them are acidic, and they can lead to acute inflammatory reaction and chronic inflammation after implantation in animals and affect the trauma. Healing; (2) slow degradation, resulting in tissue adhesions, fibrous cysts and other side effects, and forming severe scar tissue. To make the chitosan material as an absorbable hemostatic material to be implanted in the body, these two existing defects must be solved.
(1) by systematic study of the regulation of chitosan molecular weight and deacetylation degree on the degradation rate of chitosan, a series of small molecular weight chitosan with a heavy average molecular weight of 36537-529652 was prepared by means of homogeneous oxidation degradation and heterogeneous oxidation degradation. The deacetylation degree of chitosan was prepared by chitin deacetylation and chitosan acetylation. A series of low deacetylation degree chitosan (acid-base titration) from 39.6% to 59.6%. The results of in vitro lysozyme degradation of chitosan showed that the degradation rate of chitosan was 39.6% faster than that of gelatin sponge.
(2) an innovative handmade papermaking process was proposed. Starting from the chitosan hydrogel suspension, using the capillary action and the hydrogen bond between the chitosan hydrogel particles, the chitosan porous membrane and the porous sponge were prepared by high temperature drying or one freeze drying. The chitosan porous membrane was formed in shape and soft. The chitosan porous sponge has high porosity and high surface roughness. This high porosity and high surface roughness can help to promote blood coagulation.
(3) the procoagulant properties of pure chitosan with different deacetylation degree were measured by dynamic coagulation in vitro, in which the effect of M0D3 (D.D=48.07%, IR) was the best. The coagulation mechanism of chitosan was preliminarily studied by means of platelet adsorption, red cell adsorption, APTT and TT test. Chitosan was mainly used to promote the adsorption of platelets and red blood cells. Blood coagulation;
(4) a new chitosan sponge with a degree of deacetylation of 39.6%, 48.07% and 93.6% was carried out in the rat liver trauma test. The results showed that the self-made chitosan sponge had hemostatic effect in the animal body. All chitosan sponges had better hemostatic effect than gelatin sponge. The total amount of bleeding was less than that of Gelfoam group, and the degree of deacetylation of the chitosan sponge was 48.3%. The hemostatic effect of chitosan is the best.
(5) the deacetylation degree was 39.6%, 48.07% and 93.6% (acid-base titration), the new chitosan sponge and gelatin sponge were implanted in the rat liver trauma site. The liver tissue specimens of rats were collected at 1 weeks, 4 weeks, 8 weeks and 24 weeks respectively. HE.Masson's staining and immunohistochemical staining (TGF- beta 1 and IL-1p) were used to examine the absorption and inflammation of various materials. The results showed that the chitosan was completely absorbed at eighth weeks when the degree of deacetylation was 39.6%; the total absorption time of the gelatin sponge was more than 8 weeks and completely absorbed at 24 weeks. The chitosan with deacetylation of 48.07% and 93.6% was not completely absorbed at 24 weeks. Pathological section and IL-1 beta staining showed that the degree of deacetylation was 39.6% of the shell. The inflammatory reaction was the weakest in the body, and the degree of deacetylation of chitosan TGF- beta 1 was low, the amount of collagen production was moderate, and no scar healing was formed; and the degree of deacetylation of chitosan was 39.6%, reducing the risk of adhesion of other tissues.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：O636.1;R318.08

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