【作者】 高金伟；

【导师】 刘万顺；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2009， 硕士

【摘要】 目的:肝脏至少承担着5000种以上的生理功能,是人体最重要的器官之一。由于肝脏是多血管的实质性脏器,外伤时常伴有大量的出血,常用的经典止血方法有直接压迫、结扎和电灼止血等,但死亡率仍达2%-8%,其中出血为最常见的原因。目前临床肝脏外科手术常用的止血材料包括可吸收明胶海绵、氧化纤维素、胶原蛋白海绵和纤维蛋白胶,但由于有些产品或与创面粘附性差,或其准备耗时,在肝脏外伤手术中使用受限。壳聚糖(Chitosan)是甲壳素脱乙酰基的产物,天然无毒、无抗原性、生物相容性好、可生物降解且具有一定的生物活性,壳聚糖经衍生化后具有良好水溶性、成膜性,抗炎、螯合重金属等作用,在医用材料领域应用前景广阔。本实验研究旨在讨论壳聚糖基纤维的止血和促进创面愈合的效果,并对其作用机理进行初步探讨,为其作为创伤止血愈创材料提供一定的理论依据。方法:1、实验所用壳聚糖基纤维性质评价,运用物理化学方法对纤维的理化性质进行评价,通过溶血实验、全身急性毒性实验、皮内刺激实验和细胞毒性实验评价了纤维的生物相容性;2、以新西兰白兔为实验动物,切除兔肝脏左叶部分组织,形成肝脏敞开型创面出血模型。设立实验组、对照组和空白组,实验组使用壳聚糖基纤维敷于创面处,空白组使用普通纱布,对照组使用速即纱。通过测定每组的止血成功率和出血量,评价纤维的止血效果;通过血液凝血酶原时间(PT)、部分凝血活酶时间(APTT)、凝血酶时间(TT)和纤维蛋白原(FIB)的测定、血液流变学、红细胞聚集和血小板粘附等实验研究了纤维对血液不同组分的作用,初步探讨了纤维的止血机理;3、以大白鼠为实验动物,在鼠肝脏左叶上制造5mm×3mm×2mm的创面,实验组将壳聚糖基纤维敷于创面上,对照组敷以速即纱,定期进行肉眼观察和行组织学检查,评价创面的粘连、炎症浸润和组织增生情况,以此反映纤维促进创面愈合的作用。以体外培养大鼠肝实质细胞BRL为研究对象,对纤维促肝细胞生长的作用进行评价。结果:1、实验所用壳聚糖基纤维符合医用材料要求,无菌无热原;溶血实验表明壳聚糖基纤维无溶血作用,相容性实验表明纤维无急性毒性和皮内刺激性,细胞毒性实验结果表明壳聚糖基纤维对L929成纤维细胞没有毒性作用,并且对L929细胞增殖有一定的促进作用,其中以100μg/mL浓度时对L929细胞的促增殖作用最为显著;2、以新西兰白兔肝脏左叶部分切除为出血模型的实验中,结果表明实验组的止血成功率为100%,出血量为0.443±0.030g,在止血率和出血量指标上均优于对照组和空白组(P<0.05);PT、FIB、APTT和TT的测定结果表明不同浓度的壳聚糖基纤维溶液对PT和APTT没有明显的作用,能使FIB浓度明显升高,且随着溶液浓度的增加FIB浓度逐渐增加,TT随溶液浓度的增加而缩短;血液流变学测定结果显示壳聚糖基纤维溶液在高、中、低切变率下均能使全血粘度升高;壳聚糖基纤维能显著促进红细胞的沉降,且随着溶液浓度的增加沉降量增加,同时能促进红细胞发生叠连;壳聚糖基纤维能够吸附血小板,促进血小板的聚集;3、在促进创面愈合的动物活体实验中,组织学切片观察结果表明实验组材料体内降解时间短、炎症细胞浸润少,创面愈合效果明显优于对照组;壳聚糖基纤维能促进肝细胞的的生长,在100μg/mL浓度时促进作用最为显著。结论:本研究中的壳聚糖基纤维符合生物医用材料的要求,具有良好的生物相容性和生物可降解性,在体内和体外止血作用显著,效果明显优于速即纱,粘附创面的能力强,用于肝创面止血后,局部刺激反应少,降解吸收快,能诱导肝细胞的再生,因而是一种良好、安全的止血和促进创面愈合的材料。更多还原

【Abstract】 Objective: Liver is one of the most important organs,which undertakes more than 5000 physiological functions.Because of vascularity,liver trauma is often accompanied by massive hemorrhage.The classical methods of hemostasis include direct compression, ligation, electric cauterization and so on ,but these methods are ineffective.The mortality rate is still as high as 2%-8%.Bleeding is the most common cause. Presently, hemostatic material including absorbable gelatin sponge, oxidized cellulose, collagen sponge and fibrin glue are used limited in clinical hepatic operation, not only because it consumes time, but also it’s easily washed away by active bleeding.Chitosan is the product of chitin after deacetylation, which is natural non-toxic, non-antigetic, biocompatible and has unique biological activities. Chitosan derivatives also have good water-solude, film-forming , anti-inflammatory,so they have a wide application prospect in the field of medical materials.This experiment aims at discussing the hemostatic and healing effects of chitosan-based fibre, including acting mechanism, providing the theoretical basis as hemostasia repair material.Methods: 1.Evalute the physicochemical properties of chitosan-based fibre by using methods such as potentiometric titration and infrared spectrum. Examine the biocompatibility by hemolysis test, acute toxicity test, intradermal irritation and cytotoxicity test. 2. Establish the model of hepatic hemorrhage of New Zealand White rabbits with part of liver left lobe resection and an open-type wound.All rabbits are randomly divided into three groups: experimental group, control group and blank group. Hemostasis is performed with chitosan-based fibre in experimental group, Surgicel in control group and common gauze in blank group. The hemostatic effects and mechanism are evaluated by hemostatic rate, blood loss, prothrombin time, partial thromboplastin time, thrombin time, fibrinogen, hemorheology, erythrocyte aggregation and platelet adhesion tests. 3.Two types(chitosan-based fibre and Surgicel )are used in a model of hepatic trauma of rats. Regular macroscopic and histological observation are carried out to examine operative incision adhesion, inflammatory infiltration and tissue proliferation.The effect of the chitosan-based fibre on BRL is evaluated by using MTT.Results: 1.Chitosan-based fibre is aseptic and consistent with the standard of medical use. Experiment results show that the fibre has no hemolytic reaction, acute toxicity, intradermal irritation or cytotoxicity.The Chitosan-based fibre solutions with different densities can promote L929 proliferation,significantly for 100ug/mL. 2. Animal experiment shows that the effects of Chitosan-based fibre are better than Surgicel in hemostatic rate and blood loss(P<0.05).The hemostatic rate of Chitosan-based fibre is 100% and the blood loss is only 0.443±0.030g.Different densities of Chitosan-based fibre have no significant effect on APTT and PT,but can increase the FIB and decrease the TT. The density is directly proportional to FIB and inversely proportional to TT. The Hemorrheology test shows that Chitosan-based fibre can increase the whole blood viscosity in low stree, middle stree and high stree. The promotions of erythrocyte sedimentation and platelet aggregation are remarkable. 3. By comparing materials degradation time, inflammatory cell infiltration and wound healing through histological examination, the experimental group is much better than the control group. Chitosan-based fibre can promote BRL proliferation. The optimal concentration is 100ug/mL.Conclusions:Chitosan-based fibre is proved in those experiments to have met the biomedical materials requirements .It has outstanding biocompatibility and biodegradability. Animal experiment shows that chitosan-based fibre has effective hemostatic effect and stimulating cell proliferation. Chitosan-based fibre is a biosafetive and bioactive material with effective hemostatic and healing functions.更多还原