本文選題：假體周圍感染 + 動物模型��； 參考：《山東大學》2017年博士論文

【摘要】：研究背景人工關節(jié)置換假體周圍感染(Periprosthetic joint infection,PJI)是關節(jié)置換術后災難性的并發(fā)癥,由于其復雜性,在臨床上是難以避免的并且常常導致不良的臨床預后。其治療往往需要2階段的關節(jié)翻修,這會給患者和醫(yī)療體系帶來更多的經(jīng)濟壓力和負擔,而且據(jù)報道表明翻修失敗率一直在增加,這最終將導致治療的失敗。如何及時準確的診斷假體周圍感染,對于避免診斷假陽性的病例術中進行不必要的2階段翻修和防止在假陰性病例中僅僅進行1階段修復而導致植入物再植失敗都是非常重要的。然而,在大部分患者中,關節(jié)假體周圍感染的診斷不是顯而易見的,其癥狀常常沒有特異性,部分發(fā)生假體周圍感染的病人沒有明顯的臨床表現(xiàn),因而其診斷一直是臨床上極具挑戰(zhàn)性的難題。預防是減少這種災難性的并發(fā)癥發(fā)生的首要的也是最好的方法。美國肌肉及骨骼感染學會(Musculoskeletal Infection Society,MSIS)已經(jīng)發(fā)現(xiàn)了這一臨床診斷上的缺陷所在,并且根據(jù)臨床癥狀表現(xiàn)、實驗室檢查及組織病理學提供了關節(jié)假體周圍感染的定義及診斷標準,但其需要1-2個主要指標或4-6個次要指標,雖然可以用于臨床,但其使用復雜且耗時長,不易于早期及時診斷及發(fā)現(xiàn)。關節(jié)置換術后假體周圍感染是影響手術成功的重要因素之一,在第一次進行關節(jié)置換患者人群中,發(fā)生于髖關節(jié)以及肩關節(jié)的感染的概率通常情況下不超過1%,發(fā)生于膝關節(jié)的感染概率一般不超過2%,而發(fā)生于肘關節(jié)感染率則通常較高,一般約為9%。對于進行關節(jié)翻修的患者,其感染率則遠遠高于一般患者。這種感染存在一定的致死率,據(jù)報道因感染而引起的患者的死亡率大概在1.0%～2.7%。診斷的理想方法是尋找一種可單獨并具有高度檢測敏感性和特異性而且容易掌握及理解的方法。目前,臨床醫(yī)生及科學家致力于通過檢測關節(jié)腔液中的生物標記物(biomarker)的方法來開發(fā)有效且簡單的診斷關節(jié)周圍感染的診斷工具。但是目前為止,仍舊沒有一種靈敏度及準確度均能被臨床所接受的生物標記物作為早期診斷的指標。防御素(defensins)是固有免疫系統(tǒng)的一部分,是一種具有抗病毒、細菌、真菌等微生物的的多肽,其功能主要為可以直接中和入侵的病原微生物。α-防御素(α-defensin)主要存在于分葉核中性粒細胞及包括單核中性粒細胞、淋巴細胞等細胞內(nèi),在自然殺傷細胞中也有一定表達。在分葉核中性粒細胞中,α-防御素在對被吞噬的病原體氧化應激依賴性殺傷作用中發(fā)揮重要作用。而且,α-防御素具有廣譜的抗微生物作用,對多種革蘭陰性及陽性菌均有殺傷作用。其在抗微生物中的重要作用表明,α-防御素在關節(jié)腔液中的表達也許可以作為假體周圍感染的診斷生物標記物。但是就這一點來說,α-防御素檢測在診斷假體周圍感染時仍沒有獨立機構確認,并且在不同報道中其敏感性及準確度均有一定爭議。目前,假體周圍感染已有大量的體外研究結果,但體外研究結果仍舊難以替代體內(nèi)情況,這就要求我們?nèi)ソ⒏咝矣杏玫膭游锬Ｐ?將各種體外研究成果向臨床轉化。為研究α-防御素檢測在診斷假體周圍感染的診斷價值,本實驗采用兔制備人工膝關節(jié)假體置換模型,通過向關節(jié)腔內(nèi)注射不同濃度的金黃色葡萄球菌,在不同時間點觀察關節(jié)感染情況和關節(jié)腔積液量,確定了建立假體周圍感染的合適菌量及處理時間。利用上述方法制備關節(jié)置換術后假體周圍感染的動物模型來檢測α-防御素在診斷中的準確性和特異性,并且和現(xiàn)有的常用實驗室檢查相比較,特別是白細胞計數(shù)(WBC)、中性粒細胞百分比(PMN%)、紅細胞沉降率(ESR)、C反應蛋白(CRP),從而用于探討α-防御素對假體周圍感染的診斷價值。由于人工關節(jié)置換術后假體周圍感染的診斷方法往往存在敏感性或特異性達不到要求或耗時較長等各種局限,同時由于近年來抗生素濫用嚴重,細菌在關節(jié)腔內(nèi)形成生物膜等,給診斷帶來了巨大的困難。α-防御素作為一種重要的抑菌肽,在病原體感染后由分葉核中性粒細胞及其他多種細胞分泌到關節(jié)液里面,然后整合到病原菌細胞膜上并快速殺死病原菌,從而給免疫系統(tǒng)提供抗菌支持,完成自發(fā)和適應性免疫反應這一過程。α-防御素的檢測可能成為一種新的現(xiàn)代分子生物學手段來攻克這道難關。第一部分關節(jié)置換術后適合關節(jié)腔抽液假體周圍感染動物模型的建立目的目前關于利用金黃色葡萄球菌構建關節(jié)置換術后假體周圍感染動物模型的研究已經(jīng)有部分報道,但細菌的具體用量、感染時間、產(chǎn)生的關節(jié)液量、構建模型的標準和實驗或臨床應用的方向等方面仍舊沒有明確、統(tǒng)一的說明。本研究的目的在于通過優(yōu)化細菌用量及感染時間等構建模型中的關鍵因素,建立關節(jié)置換術后假體周圍感染的模型,尋找在不同實驗條件下的關節(jié)感染狀態(tài)和滿足關節(jié)液抽取量等條件的最優(yōu)組合,探討該模型建立的可行性和應用價值。方法選取健康中國白兔64只,由同一研究員在兔左膝關節(jié)處做使用鈦合金螺釘行膝關節(jié)置換術,術后即刻行X線檢查,證實植入物位置良好。之后隨機分入對照組和實驗組(共4組,對照組A給予生理鹽水,實驗組B、C、D分別給予金葡菌液 1ml,濃度分別為 0.5×104cfu/ml,0.5×105cfu/ml,0.5×106cfu/ml)。之后通過進行大體評分、細菌培養(yǎng)鑒定、死亡率、感染率、關節(jié)腔穿刺抽液計量等檢測方法,按照MSIS的診斷標準和感染分級評分進行評分并判定感染,驗證模型建立的有效性、安全性和穩(wěn)定性。結果經(jīng)分組基線齊性檢驗,四組之間在實驗動物的體重、年齡、性別比例上沒有明顯的差異。在注射生理鹽水或金黃色葡萄球菌后,通過MRI檢測關節(jié)腔積液量,以橫徑×長徑的值反應關節(jié)腔積液的量:注射后第一周MRI測量關節(jié)腔積液量(A組無法檢測到,B組0.57±0.21,C組3.05±0.51,D組3.27±0.38),C組明顯高于B組(p0.001),D組也明顯高于B組(p0.001),但C、D兩組之間沒有明顯差異(p=0.38);注射后第二周MRI測量關節(jié)腔積液量(A組無法檢測到,B 組 1.58±0.32,C 組 6.18±0.66,D 組 6.25±0.58),C 組明顯高于 B組(p0.001),D組也明顯高于B組(p0.001),但C、D兩組之間沒有明顯差異(p=0.82)。實際抽取關節(jié)腔積液量,注射后第一周抽取關節(jié)腔積液量(A組難以抽取,B 組 0.12±0.12ml,C 組 0.25±0.32ml,D 組 0.37±0.15ml),C 組明顯高于B組(p0.001),D組也明顯高于B組(p0.001),但C、D兩組之間存在邊界性差異(p=0.042);注射后第二周抽取關節(jié)腔積液量(A組難以抽取,B組 0.14±0.22ml,C 組 0.76±0.11ml,D 組 0.67±0.11ml),C 組明顯高于 B 組(p0.001),D組也明顯高于B組(p0.001),但C、D兩組之間無統(tǒng)計學差異(p=0.15)。A組未出現(xiàn)實驗動物死亡也未出現(xiàn)假體周圍感染,B、C、D三組感染模型構建成功率分別為62.5%,100%,100%。盡管C、D兩組的感染率均為100%,但C組死亡率為0%,而D組死亡率則為18.75%。同時,實驗結果顯示,在感染后第二周關節(jié)液量足夠進行檢測,且能保證大部分實驗動物存活狀態(tài)良好。結論本研究發(fā)現(xiàn),家兔膝關節(jié)腔內(nèi)注射0.5×105cfu/ml的金黃色葡萄球菌1ml能夠成功構建穩(wěn)定的關節(jié)置換術后假體周圍感染動物模型,并且在感染后的兩周可以獲得合適的關節(jié)腔積液量用于假體周圍感染的體內(nèi)實驗研究。第二部分Alpha-防御素在關節(jié)置換術后假體周圍感染診斷中的價值目的本研究通過兔關節(jié)置換術后假體周圍感染模型探討α-防御素在關節(jié)置換術后假體周圍感染診斷中的價值,明確診斷閾值,并與其他診斷指標進行比較,為α-防御素在臨床上的應用提供實驗基礎。方法將90只白兔隨機分為三組:A組為空白對照組,B組為實驗對照組,C組為術后感染組。模型制備成功后兩周,耳緣靜脈取血用于白細胞、中性粒細胞百分比、C反應蛋白、血沉等常規(guī)實驗室指標檢測�？諝馑ㄈ幩缹嶒瀯游锖�,C組關節(jié)腔穿刺抽取關節(jié)液樣本,A、B組穿刺入關節(jié)腔后注射1ml無菌生理鹽水,灌洗后再抽出以獲取樣本。采用雙抗體夾心酶聯(lián)免疫吸附試驗法檢測α-防御素在關節(jié)腔液中的表達,并分別取關節(jié)腔積液、關節(jié)內(nèi)滑膜組織、假體周圍炎性肉芽組織和假體螺釘行細菌培養(yǎng)及檢測,利用ROC曲線和Pearson correlation分析來評估α-防御素作為生物標記物對關節(jié)置換后假體周圍感染的診斷價值。結果A、B、C三組每組各30只動物。根據(jù)培養(yǎng)結果,C組動物關節(jié)腔組織細菌培養(yǎng)可見大量金黃色葡萄球菌生長,A、B兩組培養(yǎng)均為陰性,證明引起感染的為金黃色葡萄球菌,利用MSIS標準判定C組動物假體周圍感染模型建立成功。術后感染組的白細胞數(shù)明顯高于實驗對照和空白對照組(A組6.59±1.2×109/L,B 組 10.15±1.07×109/L,C 組 11.76±1.55×109/L,P0.001),敏感性及特異性分別為96%及67%。中性粒細胞百分比在三組之間也存在明顯差異(A組41.78±10.45%,B 組 56.00±11.09%,C 組 69.17±10.26%,p0.001),診斷的敏感性和特異性分別為100%及64%。血沉術后感染組也明顯高于其他兩組(A組2.94±0.50mm/h,B 組 13.53±0.91mm/h,C 組 17.48±2.71mm/h,P0.001),診斷的敏感性及特異性分別為82%及67%。CRP在三組之間也存在明顯差異(A組1.67±0.15mg/L,B 組 1.82±0.15mg/L,C 組 2.20±0.27mg/L,p0.001),診斷的特異性及敏感性分別為82%及87%。α-防御素的檢測結果在C組中明顯高于另外兩組(A 組 36.56±0.87ng/μL,B 組 37.91±1.02ng/μL,C 組 59.15±5.64ng/μL,p0.001)。α-防御素在診斷的特異性及敏感性上分別達到100%及95%,遠遠高于其他指標。結論 α-防御素作為關節(jié)置換術后假體周圍感染的診斷指標具有極高的敏感性(100%)及特異性(95%),是一種理想的假體周圍感染的診斷標準,但其是否受其他因素影響,仍需其他疾病動物模型及臨床試驗驗證。
[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.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R687.4

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