【作者】 王文；

【导师】 蔡锦方；

【作者基本信息】 第二军医大学 ， 外科学， 2008， 博士

【摘要】 研究背景:由于近年开放性外伤、高能量外伤急骤增加,伤情复杂,除骨折外多合并软组织损伤。处理不及时或内固定技术失误常引发骨感染。并且其的发生率呈逐年上升的趋势。对于骨感染的治疗,早期应用大剂量的敏感抗生素是一切治疗的基础。局部应用抗生素较全身应用抗生素,有其独特的优点:(1)可以准确地将抗生素投放到骨感染的部位;(2)可以在局部迅速达到峰值药物浓度并可维持较长时间,在局部形成几十甚至几百倍于全身应用抗生素时的药物浓度;(3)可以降低抗生素的毒副作用,虽然局部药物浓度极高,但用药总量远低于全身用药量,因而不会对全身重要脏器产生毒副作用;(4)可以避免局部缺血对抗生素作用的影响,并逐渐的被临床工作者接受。并成为现代医学的研究热点。现有局部应用抗生素的方法都存在着各种各样的缺点。靶向给药系统是指药物在载体作用下,通过局部给药或全身给药,主动或被动浓集、定位于靶器官、靶细胞或细胞内特定结构。最近,基于纳米技术发展起来的纳米载体介导的磁性载药系统,在外加磁场作用下,能实现位点特异的靶向给药目的,有利于提高病灶部位的局部药物浓度,同时使非治疗部位的药物浓度降低,从而进一步提高疗效,减少毒副作用。尤其是磁性靶向药物载体的研究,近年来受到了越来越多的关注,采用高分子材料将其与药物包裹起来形成磁性微球,通过静脉注射或口服进入体内,在外加磁场作用下,使其移向病变部位,达到定向治疗的目的。磁性给药载体的特点是在外加磁场作用下,能实现位点特异的靶向给药目的,有利于提高病灶部位的局部药物浓度,同时使非治疗部位的药物浓度降低,从而进一步提高疗效,减少毒副作用。该磁性纳米载药系统具有体外可控的特点,为骨感染的靶向治疗提供了一个新的方向。研究目的:一.制备具有磁场顺应性的万古霉素微球,并对其理化特性进行评价。二.观测具有磁场顺应性的万古霉素微球在体外对金黄色葡萄球菌的抗菌作用。三.制备出金黄色葡萄球菌感染的大鼠骨感染动物模型。四.研究具有磁场顺应性的万古霉素微球对大鼠骨感染的治疗效果及毒副作用。研究内容和方法:一.以壳聚糖作为万古霉素的载体,用乳化交联法制备具有磁场顺应性的万古霉素微球,通过扫描电镜及透射电镜观察微球形态,振动样品磁强计确证纳米微粒磁响应性的存在,并对纳米微球的平均粒径、载药量、包封率等进行评价。二.将具有磁场顺应性的万古霉素微球与金黄色葡萄球菌共育,观察体外抗菌效果。三.在大鼠胫骨内直接注射金黄色葡萄球菌,制备出大鼠急性骨感染动物模型。四.将具有磁场顺应性的万古霉素微球应用于大鼠急性骨感染动物模型中,观察外加磁场下具有磁场顺应性的万古霉素微球注射后对大鼠骨感染的影响、大鼠胫骨X线及病理的改变。研究结果:一.成功制备具有磁场顺应性的万古霉素微球,其外观呈规则球形,平均粒径298nm,平均载药量为32.36%,平均包封率为80.9%,体外具有较好的磁场顺应性。二.具有磁场顺应性的万古霉素微球与金黄色葡萄球菌共育48小时后表明,纳米微球在体外可抑制金黄色葡萄球菌生长;而不含万古霉素的磁性微球与金黄色葡萄球菌共育48小时后,未见其能抑制金黄色葡萄球菌的生长。结果表明具有磁场顺应性的万古霉素微球在体外有较好的抗菌活性。三.在大鼠胫骨平台下钻孔后,注射不同浓度的金黄色葡萄球菌一周后,发现当接种的金黄色葡萄球菌的浓度达到6.0×10~4CFU时,就出现了明显的骨质红肿、骨皮质增宽、炎性细胞浸润及出现死骨等骨感染典型改变。四.具有磁场顺应性的万古霉素微球应用于大鼠骨感染动物模型中。外部磁场介导下,具有磁场顺应性的万古霉素微球尾静脉注射后可聚集在病灶区,明显抑制骨感染。其万古霉素的实际使用剂量仅为全身应用万古霉素剂量的1/3。与全身应用万古霉素组相比较,发现无显著性差异,甚至个别参数还稍优于全身应用万古霉素的疗效。实验结论:一.成功制备可用于体内的具有磁场顺应性的万古霉素微球,其在体外具有较好的磁响应性。二.具有磁场顺应性的万古霉素微球有抗菌活性,在体外可抑制金黄色葡萄球菌的生长。三.当接种的金黄色葡萄球菌的浓度达到6.0×10~4CFU/5μl时,就可以成功的制备出大鼠骨感染动物模型。四.具有磁场顺应性的万古霉素微球,是一种安全可靠的新型药物靶向制剂。为局部应用抗生素治疗骨感染开拓了新思路。更多还原

【Abstract】 Background:Fractures are frequently accompanied by soft tissue injury in recent years because open trauma and high-energy trauma increase.Treatment of fracture is not timely and wrong fixation could cause bone infection.The incidence of bone infection is gradually rising in recent years.Using large doses of sensitive antibiotics early is basis of treating bone infection.Topical application of antibiotics has its own unique advantages,and is accepted gradually by clinical workers.Although topical application of antibiotics for the treatment of bone infection is more attended,it has not been widely applied in clinical, because the existing methods have strong side effects,low effect and bad bone targeting. With the development of nanotechnology,there has been a new magnetism drug-carried system mediated by nanovector.In the magnetic field,the system can implement site-specific targeting drug administertion,which can raise drug concentration in the focus of infection,elevate therapeutic effect,and decrease side effect all over the body.The new magnetism drug-loading system has the controllable characteristic in vitro.We wish the idea can provide a new direction of bone infection therapy.Objective:1.Research the technique for magnetic field compliance vancomycin microspheres and to evaluate the characteristics of the prepared nanoparticles.2.Investigate anti-staphylococcus aureus effect of magnetic field compliance vancomycin microspheres in vivo.3.Establish the rat model of acute staphylococcal osteomyelitis.4.Investigate antibacterial effect of magnetic field compliance vancomycin microspheres in the rat model of acute staphylococcal osteomyelitis.Method:1.Take chitosan(CTS)as the vector of vancomycin.The magnetic field compliance vancomycin microspheres were prepared by emulsion-chemical crosslink.We observe the shape of nanoparticles by scanning electron microscope(SEM) and transmission electronmicroscope(TEM),determine the magnetism by vibrating samplemagnetometer (VSM) and evaluate the mean diameter,encapsulation ratio,drug loading,and so on.2.To verify antibacterial effect of magnetic field compliance vancomycin microspheres in vivo,the staphylococcus aureus(SA) were cultured in Petri dish,which contained magnetic field compliance vancomycin microspheres. 3.The staphylococcus aureus was inoculated directly into tibial bone of rat,and We assessed the relationship between inoculation dose and histological,radiological,and microbiological changes in the acute phase(1 week after inoculation) using this rat osteomyelitis model.4.To research therapeutic efficacy of the magnetic field compliance vancomycin microspheres to bone infection by observating the histological,radiological,and microbiological changes in the acute phase of rat model.Result:1.The magnetic field compliance vancomycin microspheres have been prepared succeed.The microspheres had regular spherical surfaces,the eighty percent of microsphereswere found to have the mean diameter within the range of 100-350nm,the drug loading was 32.36%,the mean encapsulation ratio was 80.9%,and had a good magnetic response in vitro.2.The staphylococcus aureus(SA) were cultured in Petri dish for 48 hours,which contained the magnetic field compliance vancomycin microspheres.The microspheres could effectively inhibit the growth of SA in vivo.the staphylococcus aureus(SA) were cultured in Petri dish for 48 hours,which contained the magnetic field compliance microspheres without vancomycin.The microspheres without vancomycin could not inhibit the growth of SA in vivo.The results showed that the magnetic field compliance vancomycin microspheres,could effectively inhibit the growth of SA in vivo.3.Development of significant histological and radiological signs of osteomyelitis required an inoculum of at least 6.0×10~2CFU/5μl staphylococcus aureus.The rat model demonstrated clear infective destruction in the tibia at a dose of 6.0×10~4CFU/5μl,with special reference to the acute stage only(1 week after infection).The rat model of acute staphylococcal osteomyelitis was established successfully.4.The application of the magnetic field compliance vancomycin microspheres in the treatment of bone infection was better,The actual vancomycin amount dosage of the magnetic field compliance vancomycin microspheres group was 1/3 of general usage group.There was no significant difference of the magnetic field compliance vancomycin microspheres group vs general usage group.Conclusions:1.The magnetic field compliance vancomycin microspheres have been prepared succeed.The method for preparing the magnetic field compliance vancomycin microspheres is simple and can meet the requirement of pharmaceutics,The microspheres had a good magnetic response.2.The magnetic field compliance vancomycin microspheres could effectively inhibit the growth of staphylococcus aureus in vivo.3.To established successfully the rat model of acute staphylococcal osteomyelitis,the staphylococcus aureus of at least 6.0×10~4CFU/5μl was inoculated directly into tibial bone of rat.4.In the field of magnetic,the magnetic field compliance vancomycin microspheres have satisfactory magnetic responsibility in vivo and in vitro,could obviously elevate drug concentration in targeting intraorganic,reinforce vancomycin effect,and relieve or avoid side effect.It is a dependable targeting preparation.更多还原