【作者】 刘德鼎；

【导师】 康一凡；

【作者基本信息】 第二军医大学 ， 外科学（专业学位）， 2013， 博士

【摘要】 慢性骨髓炎一直是医学界公认的顽疾之一，被喻为“第二癌症”。尽管新一代抗菌药物抗菌活性不断提升，手术干预手段日趋完善，但慢性骨髓炎的复发率仍高达20-30%[1]，部分伤员最终不得不行截肢术，不仅给患者个人带来巨大的身心痛苦，而且给家庭及国家带来沉重的经济负担[1,2,3]。因此，缩短慢性骨髓炎的治疗时间，提高治愈率，降低致残率，具有重大的临床意义和社会意义。当前治疗慢性骨髓炎所形成的共识是：彻底清创和伤口引流的基础上给予抗菌药物治疗[3]。抗菌药物贯穿于慢性骨髓炎的治疗过程并决定着治疗的成败。近年来，虽然抗菌新药不断推出、给药方式不断改进，但治疗效果并不十分理想[4]。病灶内游离态抗菌药物浓度不足导致治愈率低和复发率高等问题始终未能彻底解决。臭氧是一种强氧化剂，具有广谱高效杀菌作用，近年来的实验研究表明，适当浓度的臭氧还具有杀灭细菌生物膜、调节机体氧化应激、改善组织微循环等作用。本课题首先建立靶点灌注臭氧治疗兔胫骨慢性骨髓炎模型，利用微透析取样技术对靶组织抗菌药物－万古霉素的药代动力学进行研究，探讨臭氧与抗菌药物的协同作用关系；其次，通过测定靶组织IL-6、TNF-α的含量，并通过影像学、病理学和微生物学手段评估疗效，摸索出臭氧局部治疗的有效方案。通过本实验研究，我们试图阐明局部应用臭氧治疗慢性骨髓炎的内在作用机制，从而为慢性骨髓炎的治疗开辟新思路与新途径。一、目的：建立兔慢性骨髓炎模型，静脉给予盐酸万古霉素，并在骨组织局部灌注臭氧，采用微透析技术联合反相高效液相色谱(HPLC)同步测定盐酸万古霉素在兔骨组织和血液中规定时间点的游离药物浓度，观察盐酸万古霉素在骨组织和血液的药代动力学变化特点，探讨臭氧与抗菌药物的协同作用关系；其次，定量分析骨髓炎病灶部位炎症因子（IL-6、TNF-α）的变化与臭氧的关系，初步揭示臭氧抗感染治疗的机制，为臭氧联合万古霉素治疗慢性骨髓炎提供依据。二、材料和方法：1、万古霉素的药动学研究部分：雄性新西兰大白兔18只，体重2-2.5Kg，随机分为3组，分别为正常组，慢性骨髓炎万古霉素治疗组，臭氧联合万古霉素治疗组。实验前18小时禁食不禁水。三个实验组均经兔耳缘静脉给予盐酸万古霉素30mg/Kg，臭氧干预组在静注万古霉素前给予臭氧2ml（40μ g/mL），采用微透析技术对骨组织和血液进行同步取样，联合反相高效液相色谱技术实时检测骨组织和血液中盐酸万古霉素的浓度变化，所得药物浓度数据用WinNonlin软件处理。2、臭氧抗感染治疗的机制研究：雄性新西兰大白兔18只，体重2-2.5Kg，随机分为3组，分别为正常组，慢性骨髓炎对照组，慢性骨髓炎臭氧干预组。臭氧干预组给予臭氧2ml（40μ g/mL），采用微透析技术对骨组织进行取样，联合ELISA法对样品中的IL-6、TNF-α进行定量分析。三、结果：1、药代动力学研究：正常兔血液中的游离万古霉素浓度在静注后10min达峰，为41.5±2.71μg/ml，骨组织也在10min时到达峰浓度40.57±2.50μg/ml，两者浓度在100min前始终保持接近；100min后，血液中的药物浓度开始高于骨组织，并持续至观察点结束。慢性骨髓炎组病灶部位骨组织中的万古霉素含量较血液明显降低，10min达峰浓度分别43.31±2.67μg/ml（血液），16.35±1.67μg/ml（骨）。臭氧干预治疗后，病灶骨组织的万古霉素浓度明显提高，达到44.37±2.31μg/ml（血液），20.58±4.85μg/ml（骨），但仍低于正常骨组织的药物浓度。血液、骨组织的药时曲线下面积AUC0-inf在三个实验组分别为：正常组：5280.31±205.73min*μg/ml （血液）和5065.03±209.56min*μg/ml （骨）；慢性骨髓炎万古霉素治疗组：5106.45±205.73min*μg/ml（血液）和1259.53±48.42min*μg/ml（骨）；臭氧联合万古霉素治疗组：5353.51±325.58min*μg/ml（血液）和1873.43±573.7min*μg/ml（骨）。2、炎症因子变化研究：IL-6、TNF-α在臭氧灌注后均明显下降，在慢性骨髓炎组，病灶部位IL-6和TNF-α的含量分别为：318.1±2.82pg/ml-361.42±8.52pg/ml，231.48±14.89pg/ml-267.13pg/ml；臭氧干预组病灶部位和正常组骨组织的含量分别为79.60±10.12pg/ml-277.16±10.02pg/ml，59.37±10.57pg/ml-112.06±14.85pg/ml（TNF-α）；127.36±13.83pg/ml-371.69±11.97pg/ml，91.91±10.57pg/ml-227.27±17.75pg/ml（IL-6）。四、结论：微透析采样技术具有良好的时间和空间分辨性，联合反相高效液相色谱检测方法可以准确客观地反映药物在慢性骨髓炎病灶组织和血液中的药代动力学特点，是进行药动学研究的良好工具。实验证明，静脉给药时盐酸万古霉素具有良好的骨组织穿透性。与血浆药动学参数相比，正常骨组织的达峰浓度及达峰时间无差别，但在慢性骨髓炎病灶部位，骨组织中的游离态药物浓度显著低于血浆药物浓度。臭氧干预后病灶组织的药物峰浓度和最低浓度较单纯应用万古霉素有所提高，显示臭氧与万古霉素间具有协同作用，该现象可能通过臭氧促进糖酵解酶类合成，加快糖代谢，增加NO、CO等物质生成，扩张微血管，改善局部循环完成。进一步研究显示臭氧可以调节炎症因子的表达，显著降低IL-6、TNF-α等炎症因子的表达水平，结合国内外研究，臭氧可通过引起α-synuclein蛋白在细胞内积聚，抑制核转录因子NF-KB的活性，使之不能进入细胞核内，抑制细胞表达IL-10、IL-6、TNF-α等炎症因子。虽然臭氧的协同作用无法将万古霉素的骨组织含量提高到正常水平，但可延长万古霉素在骨组织的驻留时间，这对于时间依赖性的抗菌药物具有重要意义。同时臭氧的应用可减轻局部慢性炎症的反复刺激，有利于炎症消除，并可抑制破骨细胞的活性，有利于骨组织重建。综上所述，病灶局部灌注臭氧治疗慢性骨髓炎具有积极意义，为慢性骨髓炎的治愈提供了一条新的途径。更多还原

【Abstract】 Chronic osteomyelitis has been acknowledged as an obstinate disease by medicalprofessionals, as such been called the “second cancer”. Although as the rising ofantimicrobial activity of the new generation antibiotics, and as the maturing of surgicalintervention, chronic osteomyelitis recurrence rate is still as high as20-30%[1], whichleads to amputation in some cases. It not only has broμght tremendous physical and mentalpain to the patients, but also heavy economic burden to the family and national healthcaresystem [1,2,3]. Therefore, shortening the treatment duration of chronic osteomyelitis,improving the cure rate and reducing morbidity have considerable clinical and socialsignificance.The current consensus on chronic osteomyelitis treatment is to apply antibiotictherapy [3] on the basis of thoroμgh debridement and wound drainage. Antibiotics, being adetermining factor, is used throμghout the treatment. In recent years, althoμgh antibioticsand the ways of its application have been continuously improving, the result hasn’t beenvery satisfactory [4]. Low concentration of free-state antibiotics in the nidus has resulted inlow cure rate and high recurrence of this disease. Ozone, being a strong oxidizing agent,has broad and strong antibacterial effect. Recent experiments have also shown that certainconcentration of ozone can kill bacteria biofilm, regulate oxidative stress, and improvemicrocirculation. This subject, firstly, establishes the rabbit tibia model of chronicosteomyelitis by infusing ozone to the target tissue, in order to study the pharmacokineticsof vancomycin, the antibiotics applied to the target tissue, throμgh microdialysis samplingtechnique. Secondly, the subject intends to explore effective methods of local treatmentwith ozone via measuring the content of IL-6and TNF-α in the target tissue, and viaassessing the treatment effect throμgh imaging, pathology and microbiological methods.Throμgh this study, we intend to clarify the intrinsic mechanism of topical application ofozone treatment on chronic osteomyelitis, so as to discover new ideas and methods to treatthis disease..Objective:To establish rabbit models of chronic osteomyelitis, and apply intravenous injection ofvancomycin hydrochlorid and ozone perfusion of local bone tissue. To use microdialysis technique combined with reversed-phase high performance liquid chromatography (HPLC)to perform synchronous detection of free-state vancomycin hydrochloride concentration intarget tissue and blood at specified time spots, so as to observe the pharmacokineticsdynamics of vancomycin hydrochloride in the bone tissue and blood, and to investigate thesynergy of the ozone and antibiotics. In addition, to conduct quantitative analysis of theevolution related with ozone on inflammatory factors (IL-6and TNF-α) in the targetosteomyelitis tissue, in order to reveal the mechanism of the ozone anti-infection treatment,and to provide evidence for the treatment of chronic osteomyelitis by ozone combined withvancomycin..Methods:1. For the sutdy of vancomycin pharmacokinetics:18male New Zealand white rabbits,weight between2to2.5Kg, to be randomly divided into3groups as normal group,chronic osteomyelitis vancomycin treatment group, and ozone vancomycin treatmentgroup respectively. To ensure18hours of fasting except for water before the experiment.To apply, via rabbit ear vein,30mg/Kg of vancomycin hydrochloride on all three groups.To apply2ml of ozone (40μg/mL) before injection of vancomycin hydrochloride on theozone vancomycin treatment group. Using microdialysis technique to conduct synchronoussampling of bone tissue and blood. Combined with joint reversed-phase high performanceliquid chromatography, to record real time change of concentration of hydrochloridevancomycin in target tissue and blood. And to process the recorded data with WinNonlinsoftware.2. Study of ozone anti-infective therapy mechanism:18male New Zealand whiterabbits, weight between2to2.5Kg, to be randomly divided into3groups as normal group,chronic osteomyelitis group and ozone treatment group. To apply2ml of ozone (40μg/mL)in ozone treatment group. To perform sampling using microdialysis technique in allanimals. And to conduct quantitative analysis on IL-6and TNF-α in the samples togetherwith ELISA method.Results:1, pharmacokinetic studies: free vancomycin concentration in the blood of normalrabbit reached its peak after10min of intravenous injection to41.5±2.71μg/ml, same as in bone tissue to40.57±2.50μg/ml; both concentration data stay close within100min afterthe injection. While once pass the100min threshold, free vancomycin concentration inblood stays above that in bone tissue until the end of the observation. The vancomycincontent in bone tissue of chronic osteomyelitis lesion was significantly lower than that inblood;10min peak concentration was43.31±2.67μg/ml (in blood) and16.35±1.67μg/ml(in bone tissue). After ozone intervention therapy, vancomycin concentration in theinfected bone tissue significantly increased to44.37±2.31μg/ml (in blood) and20.58±4.85μg/ml (in bone tissue), which were still lower than that in normal bone tissue. Theunder curve area of blood and bone tissue after medication in all three groupswere: normal group:5280.31±205.73min*μg/ml (in blood) and5065.03±209.56min*μg/ml (in bone tissue); vancomycin treatment group:5106.45±205.73min*μg/ml (inblood), and1259.53±48.42min*μg/ml (in bone tissue); ozone joint interventiontreatment group:5353.51±325.58min*μg/ml (in blood) and1873.43+573.7min*μg/ml (in bone tissue).2. Change of inflammatory cytokines: IL-6and TNF-α level showed a significantdownward trend after ozone perfusion, where they were318.1±2.82pg/ml-361.42±8.52pg/ml (IL-6) and231.48±14.89pg/ml-267.13pg/ml (TNF-α)in chronic osteomyelitis group,79.60±10.12pg/ml-277.16±10.02pg/ml (TNF-α) and127.36±13.83pg/ml-371.69±11.97pg/ml (IL-6) in ozone treatment group and59.37±10.57pg/ml-112.06±14.85pg/ml (TNF-α),91.91±10.57pg/ml-227.27±17.75pg/ml（IL-6）in normal group.Conclusion:Microdialysis sampling technique has high time and spatial resolution. Combinedwith reverse phase HPLC method, they can accurately and objectively reflect thepharmacokinetic characteristics of drμgs in chronic osteomyelitis lesions tissues and inblood as effective tools of study. The experiments show that vancomycin hydrochloride,under intravenous injection, can well penetrate bone tissues. Compared with the plasmapharmacokinetic parameters, the peak concentration level and the duration to peak level ofthe drμg concentration show no difference in normal bone tissues; while in chronicosteomyelitis lesion, drμg concentration in bone tissues was significantly lower than that inplasma. Althoμgh after the ozoen intervention, peak and troμgh drμg concentration level in the leison both increased compare to that of pure vancomycin therapy, which showed asynergy between the ozone and vancomycin yet no strong enoμgh to achieve normal bonetissue drμg concentration. Comparing the normal group and the experimented groups,in the bone tissue was smaller than that in plasma, while there was no significantstatistical difference in the drμg mean residence time (MRT) and drμg half-life.To make a conclusion according to the dynamics of IL-6, ozone inhibits the activity ofnuclear transcription factor NF-KB by causing the accumulation of α-synuclein protein incells. Thus prevents NF-KB from entering the nucleus, represses cell expression ofinflammatory cytokines such as IL-10, IL-6and TNF-α, enhances the synthesis ofglycolytic enzymes, and accelerates glucose metabolism; thereby increases generation ofsubstances such as NO and CO, expands capillaries, and improves local circulation, whichattribute to the increasing of vancomycin concentration in the lesion bone tissues.更多还原