Alpha-防御素（α-defensin）作为诊断关节置换术后假体周围感染生物标记物的研究

发布时间：2018-05-17 20:59

本文选题：假体周围感染 + 动物模型　；参考：《山东大学》2017年博士论文

【摘要】：研究背景人工关节置换假体周围感染(Periprosthetic joint infection,PJI)是关节置换术后灾难性的并发症,由于其复杂性,在临床上是难以避免的并且常常导致不良的临床预后。其治疗往往需要2阶段的关节翻修,这会给患者和医疗体系带来更多的经济压力和负担,而且据报道表明翻修失败率一直在增加,这最终将导致治疗的失败。如何及时准确的诊断假体周围感染,对于避免诊断假阳性的病例术中进行不必要的2阶段翻修和防止在假阴性病例中仅仅进行1阶段修复而导致植入物再植失败都是非常重要的。然而,在大部分患者中,关节假体周围感染的诊断不是显而易见的,其症状常常没有特异性,部分发生假体周围感染的病人没有明显的临床表现,因而其诊断一直是临床上极具挑战性的难题。预防是减少这种灾难性的并发症发生的首要的也是最好的方法。美国肌肉及骨骼感染学会(Musculoskeletal Infection Society,MSIS)已经发现了这一临床诊断上的缺陷所在,并且根据临床症状表现、实验室检查及组织病理学提供了关节假体周围感染的定义及诊断标准,但其需要1-2个主要指标或4-6个次要指标,虽然可以用于临床,但其使用复杂且耗时长,不易于早期及时诊断及发现。关节置换术后假体周围感染是影响手术成功的重要因素之一,在第一次进行关节置换患者人群中,发生于髋关节以及肩关节的感染的概率通常情况下不超过1%,发生于膝关节的感染概率一般不超过2%,而发生于肘关节感染率则通常较高,一般约为9%。对于进行关节翻修的患者,其感染率则远远高于一般患者。这种感染存在一定的致死率,据报道因感染而引起的患者的死亡率大概在1.0%～2.7%。诊断的理想方法是寻找一种可单独并具有高度检测敏感性和特异性而且容易掌握及理解的方法。目前,临床医生及科学家致力于通过检测关节腔液中的生物标记物(biomarker)的方法来开发有效且简单的诊断关节周围感染的诊断工具。但是目前为止,仍旧没有一种灵敏度及准确度均能被临床所接受的生物标记物作为早期诊断的指标。防御素(defensins)是固有免疫系统的一部分,是一种具有抗病毒、细菌、真菌等微生物的的多肽,其功能主要为可以直接中和入侵的病原微生物。α-防御素(α-defensin)主要存在于分叶核中性粒细胞及包括单核中性粒细胞、淋巴细胞等细胞内,在自然杀伤细胞中也有一定表达。在分叶核中性粒细胞中,α-防御素在对被吞噬的病原体氧化应激依赖性杀伤作用中发挥重要作用。而且,α-防御素具有广谱的抗微生物作用,对多种革兰阴性及阳性菌均有杀伤作用。其在抗微生物中的重要作用表明,α-防御素在关节腔液中的表达也许可以作为假体周围感染的诊断生物标记物。但是就这一点来说,α-防御素检测在诊断假体周围感染时仍没有独立机构确认,并且在不同报道中其敏感性及准确度均有一定争议。目前,假体周围感染已有大量的体外研究结果,但体外研究结果仍旧难以替代体内情况,这就要求我们去建立高效且有用的动物模型,将各种体外研究成果向临床转化。为研究α-防御素检测在诊断假体周围感染的诊断价值,本实验采用兔制备人工膝关节假体置换模型,通过向关节腔内注射不同浓度的金黄色葡萄球菌,在不同时间点观察关节感染情况和关节腔积液量,确定了建立假体周围感染的合适菌量及处理时间。利用上述方法制备关节置换术后假体周围感染的动物模型来检测α-防御素在诊断中的准确性和特异性,并且和现有的常用实验室检查相比较,特别是白细胞计数(WBC)、中性粒细胞百分比(PMN%)、红细胞沉降率(ESR)、C反应蛋白(CRP),从而用于探讨α-防御素对假体周围感染的诊断价值。由于人工关节置换术后假体周围感染的诊断方法往往存在敏感性或特异性达不到要求或耗时较长等各种局限,同时由于近年来抗生素滥用严重,细菌在关节腔内形成生物膜等,给诊断带来了巨大的困难。α-防御素作为一种重要的抑菌肽,在病原体感染后由分叶核中性粒细胞及其他多种细胞分泌到关节液里面,然后整合到病原菌细胞膜上并快速杀死病原菌,从而给免疫系统提供抗菌支持,完成自发和适应性免疫反应这一过程。α-防御素的检测可能成为一种新的现代分子生物学手段来攻克这道难关。第一部分关节置换术后适合关节腔抽液假体周围感染动物模型的建立目的目前关于利用金黄色葡萄球菌构建关节置换术后假体周围感染动物模型的研究已经有部分报道,但细菌的具体用量、感染时间、产生的关节液量、构建模型的标准和实验或临床应用的方向等方面仍旧没有明确、统一的说明。本研究的目的在于通过优化细菌用量及感染时间等构建模型中的关键因素,建立关节置换术后假体周围感染的模型,寻找在不同实验条件下的关节感染状态和满足关节液抽取量等条件的最优组合,探讨该模型建立的可行性和应用价值。方法选取健康中国白兔64只,由同一研究员在兔左膝关节处做使用钛合金螺钉行膝关节置换术,术后即刻行X线检查,证实植入物位置良好。之后随机分入对照组和实验组(共4组,对照组A给予生理盐水,实验组B、C、D分别给予金葡菌液 1ml,浓度分别为 0.5×104cfu/ml,0.5×105cfu/ml,0.5×106cfu/ml)。之后通过进行大体评分、细菌培养鉴定、死亡率、感染率、关节腔穿刺抽液计量等检测方法,按照MSIS的诊断标准和感染分级评分进行评分并判定感染,验证模型建立的有效性、安全性和稳定性。结果经分组基线齐性检验,四组之间在实验动物的体重、年龄、性别比例上没有明显的差异。在注射生理盐水或金黄色葡萄球菌后,通过MRI检测关节腔积液量,以横径×长径的值反应关节腔积液的量:注射后第一周MRI测量关节腔积液量(A组无法检测到,B组0.57±0.21,C组3.05±0.51,D组3.27±0.38),C组明显高于B组(p0.001),D组也明显高于B组(p0.001),但C、D两组之间没有明显差异(p=0.38);注射后第二周MRI测量关节腔积液量(A组无法检测到,B 组 1.58±0.32,C 组 6.18±0.66,D 组 6.25±0.58),C 组明显高于 B组(p0.001),D组也明显高于B组(p0.001),但C、D两组之间没有明显差异(p=0.82)。实际抽取关节腔积液量,注射后第一周抽取关节腔积液量(A组难以抽取,B 组 0.12±0.12ml,C 组 0.25±0.32ml,D 组 0.37±0.15ml),C 组明显高于B组(p0.001),D组也明显高于B组(p0.001),但C、D两组之间存在边界性差异(p=0.042);注射后第二周抽取关节腔积液量(A组难以抽取,B组 0.14±0.22ml,C 组 0.76±0.11ml,D 组 0.67±0.11ml),C 组明显高于 B 组(p0.001),D组也明显高于B组(p0.001),但C、D两组之间无统计学差异(p=0.15)。A组未出现实验动物死亡也未出现假体周围感染,B、C、D三组感染模型构建成功率分别为62.5%,100%,100%。尽管C、D两组的感染率均为100%,但C组死亡率为0%,而D组死亡率则为18.75%。同时,实验结果显示,在感染后第二周关节液量足够进行检测,且能保证大部分实验动物存活状态良好。结论本研究发现,家兔膝关节腔内注射0.5×105cfu/ml的金黄色葡萄球菌1ml能够成功构建稳定的关节置换术后假体周围感染动物模型,并且在感染后的两周可以获得合适的关节腔积液量用于假体周围感染的体内实验研究。第二部分Alpha-防御素在关节置换术后假体周围感染诊断中的价值目的本研究通过兔关节置换术后假体周围感染模型探讨α-防御素在关节置换术后假体周围感染诊断中的价值,明确诊断阈值,并与其他诊断指标进行比较,为α-防御素在临床上的应用提供实验基础。方法将90只白兔随机分为三组:A组为空白对照组,B组为实验对照组,C组为术后感染组。模型制备成功后两周,耳缘静脉取血用于白细胞、中性粒细胞百分比、C反应蛋白、血沉等常规实验室指标检测。空气栓塞处死实验动物后,C组关节腔穿刺抽取关节液样本,A、B组穿刺入关节腔后注射1ml无菌生理盐水,灌洗后再抽出以获取样本。采用双抗体夹心酶联免疫吸附试验法检测α-防御素在关节腔液中的表达,并分别取关节腔积液、关节内滑膜组织、假体周围炎性肉芽组织和假体螺钉行细菌培养及检测,利用ROC曲线和Pearson correlation分析来评估α-防御素作为生物标记物对关节置换后假体周围感染的诊断价值。结果A、B、C三组每组各30只动物。根据培养结果,C组动物关节腔组织细菌培养可见大量金黄色葡萄球菌生长,A、B两组培养均为阴性,证明引起感染的为金黄色葡萄球菌,利用MSIS标准判定C组动物假体周围感染模型建立成功。术后感染组的白细胞数明显高于实验对照和空白对照组(A组6.59±1.2×109/L,B 组 10.15±1.07×109/L,C 组 11.76±1.55×109/L,P0.001),敏感性及特异性分别为96%及67%。中性粒细胞百分比在三组之间也存在明显差异(A组41.78±10.45%,B 组 56.00±11.09%,C 组 69.17±10.26%,p0.001),诊断的敏感性和特异性分别为100%及64%。血沉术后感染组也明显高于其他两组(A组2.94±0.50mm/h,B 组 13.53±0.91mm/h,C 组 17.48±2.71mm/h,P0.001),诊断的敏感性及特异性分别为82%及67%。CRP在三组之间也存在明显差异(A组1.67±0.15mg/L,B 组 1.82±0.15mg/L,C 组 2.20±0.27mg/L,p0.001),诊断的特异性及敏感性分别为82%及87%。α-防御素的检测结果在C组中明显高于另外两组(A 组 36.56±0.87ng/μL,B 组 37.91±1.02ng/μL,C 组 59.15±5.64ng/μL,p0.001)。α-防御素在诊断的特异性及敏感性上分别达到100%及95%,远远高于其他指标。结论 α-防御素作为关节置换术后假体周围感染的诊断指标具有极高的敏感性(100%)及特异性(95%),是一种理想的假体周围感染的诊断标准,但其是否受其他因素影响,仍需其他疾病动物模型及临床试验验证。
[Abstract]:Background artificial joint replacement prosthesis (Periprosthetic joint infection, PJI) is a catastrophic complication after arthroplasty. Because of its complexity, it is clinically difficult to avoid and often leads to adverse clinical outcomes. The treatment often requires 2 stages of joint revision, which will bring more patients and medical systems. Many economic pressures and burdens, and it is reported that the failure rate of refurbishment has been increasing, which will eventually lead to the failure of the treatment. How to diagnose the infection around the prosthesis in a timely and accurate way, to avoid the 2 stage refurbishment in the case of false positive cases, and to prevent only 1 stages of repair in false negative cases. The failure of implant replantation is very important. However, in most patients, the diagnosis of the periprosthetic infection is not obvious, the symptoms are often unspecific, and the patients with periprosthetic infection are not obviously clinical, so the diagnosis is a very challenging problem in the clinic. Prevention is a reduction. The first and foremost method of this catastrophic complication is also the best method. The Musculoskeletal Infection Society (MSIS) has found this clinical diagnostic defect, and based on clinical symptoms, laboratory tests and histopathology provide a definition of periprosthetic infection. And diagnostic criteria, but they need 1-2 main or 4-6 secondary indicators, although it can be used in clinical, but its use is complex and time-consuming, it is not easy to diagnose and discover early and timely. Infection of the periprosthesis after arthroplasty is one of the important factors that affect the success of the operation, and occurs in the hip replacement patients in the first time. The probability of infection of the joint and shoulder joint is usually not more than 1%, the incidence of infection in the knee joint is generally not more than 2%, and the incidence of infection in the elbow joint is usually high. It is generally about 9%. for patients undergoing joint revision, and the infection rate is much higher than that of the general patients. The ideal method for the 1% to 2.7%. diagnosis of infection caused by infection is to find a method that can be isolated and highly sensitive and specific and is easy to understand and understand. Currently, clinicians and scientists are committed to developing a method of detection of biomarkers (biomarker) in the articular fluid. An effective and simple diagnostic tool for the diagnosis of infection around the joint. But so far, there is still no sensitivity and accuracy that can be clinically acceptable as an indicator of early diagnosis. Defensins is part of the inherent immune system and is a kind of antiviral, bacterial, fungal and other microorganisms. The main function of the peptide is to directly neutralize the invasive pathogenic microorganism. Alpha defensin (alpha -defensin) is mainly found in the lobular nucleus neutrophils, including mononuclear neutrophils, lymphocytes and other cells, and is also expressed in natural killer cells. In the lobular nucleus, the alpha defensin is in the phagocytic pathogen. Oxidative stress plays an important role in the dependent killing effect. Moreover, the alpha defensin has a broad-spectrum antimicrobial effect and has a killing effect on a variety of gram-negative and positive bacteria. Its important role in the anti microorganism indicates that the expression of alpha defensin in the articular cavity may be a diagnostic biomarker for the infection around the prosthesis. But in this case, there is no independent confirmation of alpha defensin detection in the diagnosis of infection around the prosthesis, and there are some disputes in the sensitivity and accuracy of different reports. There are a lot of in vitro research results around the infection of the prosthesis, but the results in vitro are still difficult to replace the body, which requires me. In order to study the diagnostic value of alpha defensin detection in the diagnosis of periprosthetic infection, we used rabbits to prepare artificial knee prosthesis replacement model by injecting different concentrations of Staphylococcus aureus to the articular cavity at different time points. Observe the condition of joint infection and the amount of effusion of the articular cavity, determine the appropriate amount of bacteria and the time to deal with the infection around the prosthesis. The animal model of the periprosthetic infection after arthroplasty is used to detect the accuracy and specificity of the alpha defensin in the diagnosis, and is compared with the common laboratory examination, especially Leukocyte count (WBC), percentage of neutrophils (PMN%), erythrocyte sedimentation rate (ESR) and C reactive protein (CRP) are used to explore the diagnostic value of alpha defensin on periprosthetic infection. At the same time, because of the serious abuse of antibiotics in recent years, bacteria have formed a biomembrane in the joint cavity. The alpha defensin, as an important antimicrobial peptide, is secreted into the joint fluid from the lobular nucleus neutrophils and a variety of other cells after the infection of the pathogen and then integrated into the membrane of the pathogen. And quickly kill the pathogenic bacteria, thus providing antibacterial support to the immune system and completing the process of spontaneous and adaptive immune response. The detection of alpha defensin may become a new modern molecular biological means to overcome this difficulty. Part 1 after arthroplasty is suitable for the establishment of animal models of the infection around the joint cavity suction prosthesis. Objective the study of animal models of periprosthetic infection after arthroplasty with Staphylococcus aureus has been reported in part, but the specific dosage of the bacteria, the time of the infection, the amount of joint fluid produced, the standard of building the model and the direction of the experiment or clinical application are still not clear. This study is a unified explanation. The aim is to establish the key factors in the model by optimizing the dosage of bacteria and the time of infection, to establish the model of the infection around the prosthesis after arthroplasty, to find the optimal combination of the condition of the joint infection and the extraction of the joint fluid under different experimental conditions, and to discuss the feasibility and application value of the model. 64 healthy Chinese white rabbits were selected by the same researcher at the left knee joint of the rabbit. The X-ray examination was performed on the left knee joint of the rabbit. The position of the implant was confirmed immediately after the operation. Then it was randomly divided into the control group and the experimental group (4 groups, the control group A gave the physiological saline, the experimental group B, C, D respectively gave 1ml of Staphylococcus aureus solution, the concentration points respectively. " Don't be 0.5 x 104cfu/ml, 0.5 x 105cfu/ml, 0.5 x 106cfu/ml). Then by gross score, bacterial culture identification, mortality, infection rate, joint cavity puncture measurement and other detection methods, according to the diagnostic criteria of MSIS and infection grading score and determine the infection, verify the validity, safety and stability of the model establishment. After group baseline homogeneity test, there was no significant difference in weight, age and sex ratio between the four groups. After injection of saline or Staphylococcus aureus, the amount of joint effusion was measured by MRI, and the amount of joint effusion was measured at the value of transverse diameter by MRI. The volume of joint effusion was measured at the first week after the injection (group A was not available. Group B was 0.57 + 0.21, group C was 3.05 + 0.51, group D was 3.27 + 0.38), group C was significantly higher than group B (p0.001), D group was also significantly higher than B group (p0.001), but there was no significant difference between C, D two groups (p=0.38), and second weeks after injection (1.58 + 0.32, 6.18 + 0.66, 6.25 + 0.58). Group (p0.001) and group D were also significantly higher than group B (p0.001), but there was no significant difference between groups of C and D (p=0.82). The volume of joint effusion was extracted from the joint cavity, and the volume of joint effusion was extracted in the first week after injection (group A was difficult to be extracted, B group 0.12 + 0.12ml, C group 0.25 + 0.32ml, 0.37 +), and the group was obviously higher than that of the group. But there was a boundary difference (p=0.042) between the two groups of C, D, and second weeks after the injection (group A was difficult to extract, B group 0.14 + 0.22ml, C group 0.76 + 0.11ml, D group 0.67 + 0.11ml), C group was significantly higher than that of the B group, but there was no statistical difference between the two groups. There was no infection around the prosthesis. The success rate of B, C, D three group infection models was 62.5%, 100% respectively. Although C, D two, the infection rate was 100%, but the death rate in the C group was 0%, while the D group mortality was 18.75%. simultaneously. The experimental results showed that the joint volume was enough to be tested at second weeks after the infection and could guarantee most of the experiments. The survival state of the animals is good. Conclusion this study found that the injection of 0.5 * 105cfu/ml Staphylococcus aureus 1ml in the rabbit's knee joint can successfully construct a stable animal model of periprosthetic infection after arthroplasty, and the appropriate joint cavity volume can be obtained for the experimental research of the infection around the prosthesis for two weeks after the infection. The value of the second part of the Alpha- defensin in the diagnosis of periprosthetic periprosthetic infection after arthroplasty: a study of the value of alpha defensin in the diagnosis of periprosthetic infection after arthroplasty after arthroplasty and the value of the diagnostic threshold for the diagnosis of periprosthetic infection after arthroplasty. Methods 90 white rabbits were randomly divided into three groups: the A group was a blank control group, the B group was the experimental control group, the C group was the postoperative infection group. The model preparation was two weeks after the success of the model preparation. The ear vein blood was used for the white blood cells, the percentage of neutrophils, the C anti stress protein, the erythrocyte sedimentation rate and so on. After the experimental animals were killed, the articular fluid samples were extracted from the joint cavity of group C. The group of A, group B were injected into the articular cavity and injected 1ml aseptic saline, and then extracted to obtain the samples. The double antibody sandwich enzyme linked immunosorbent assay was used to detect the expression of alpha defensin in the articular cavity fluid, and the joint cavity effusion and synovial tissue in the joint were taken respectively. Bacterial culture and detection of periprosthetic periprosthetic granulation tissue and prosthesis screw were used to assess the value of alpha defensin as a biomarker in the diagnosis of periprosthetic infection after arthroplasty by ROC curve and Pearson correlation analysis. Results 30 animals in each group of three groups of A, B, C were cultured. The root culture results, the joint cavity tissue culture of group C animals The growth of Staphylococcus aureus was seen in a large number of Staphylococcus aureus, A and B two groups were negative. It proved that the infection was Staphylococcus aureus. The model of peripheral infection in group C was successfully established by MSIS standard. The number of leukocytes in the postoperative infection group was significantly higher than that of the experimental and empty white control group (group A 6.59 + 1.2 x 109/L, B group 10.15 + 1.07 X. 109/L, group C, 11.76 + 1.55 x 109/L, P0.001), the sensitivity and specificity of 96% and 67%. neutrophils were also significantly different among the three groups (A group 41.78 + 10.45%, B group 56 + 11.09%, C group 69.17 + 10.26%, p0.001), and the sensitivity and specificity of diagnosis were also significantly higher than those of the group 100% and 64%. ESR. In the two groups (group A 2.94 + 0.50mm/h, group B 13.53 + 0.91mm/h, C group 17.48 + 2.71mm/h, P0.001), the sensitivity and specificity of diagnosis were 82% and 67%.CRP in three groups were also significantly different (A group 1.67 + 0.15mg/L, B group 1.82 + 0.15mg/L, 2.20 + 13.53), and 82% and sensitivity alpha defensin, respectively. The detection results were significantly higher in the C group than in the other two groups (group A, 36.56 + 0.87ng/, L, B group 37.91 + 1.02ng/ mu L, C group 59.15 + 5.64ng/ L, p0.001). The specificity and sensitivity of alpha defensin reached 100% and 95%, respectively. Conclusion alpha defensin was the diagnosis of periprosthetic infection after arthroplasty. It has high sensitivity (100%) and specificity (95%). It is an ideal diagnostic criteria for periprosthetic infection.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R687.4

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