早期诊断结核杆菌相关肾损伤的实验性研究
|
摘要
目的
结核病目前仍是危害人类健康的主要疾病之一,据世界卫生组织估计,目前全世界有近三分之一的人感染了结核,每年有870万新增病例,其中300万死亡,因此对该病的预防与治疗已成为研究的热点。肾结核病是常见的肺外结核病,肺结核死亡病人尸检显示,并发肾结核者占16-26%。而肾结核早期临床表现不典型,症状轻微,缺乏特异性,诊断比较困难。临床上漏诊误诊率高,使多数病人失去了早期病因治疗的机会,致使晚期进入终末期肾衰,威胁病人生命,故肾结核的早期诊断非常重要。目前尿结核杆菌培养阳性仍是肾结核诊断的金标准,但其时间长、阳性率低。结核菌产生分阶段性,所以早期肾结核的诊断率只占6%,不排菌的肾结核诊断率更低。因此在肾结核病感染的早期,快速的诊断是一项挑战性的课题。
结核杆菌对肾脏的损伤分为临床肾结核,病理肾结核和结核性变态反应综合征。所谓结核性变态反应综合征是指当机体对结核感染处于超敏状态时,可在远离原发灶的皮肤、关节、角膜、结膜、肾脏、浆膜腔等多处发生细胞免疫和体液免疫反应,引起类似系统性红斑狼疮,血管炎,角膜炎,关节炎,肾小球肾炎等多种临床表现。结核性变态反应综合征引起肾损伤,可以表现为蛋白尿,血尿,IgA肾病,肾病综合征等。其病理类型为：毛细血管内增生性肾小球肾炎、膜增生性肾小球肾炎、局灶节段性肾小球肾炎、系膜增生性肾小球肾炎、微小病变病等多种类型。结核杆菌感染做为这些疾病的病因。若不能及时诊断,及时行病因治疗,而单纯应用免疫抑制剂或激素治疗,结果会导致结核播散,疾病迁延不愈,给患者带来不能弥补的损伤或遗憾。
本试验将病例分为三组：肾结核组,非肾结核组,结核杆菌相关肾损伤疑诊组。从以下三个方面进行研究,以探讨结核性变态反应综合症导致的肾损伤的早期诊断方法：1应用实时定量PCR技术,对患者肾组织中结核分枝杆菌DNA表达进行定量分析。2研究上述三组患者肾组织中结核分枝杆菌早期分泌性蛋白抗原85(Ag85)及γ干扰素(IFN-γ)的表达,并分析二者的相关性。3研究结核分枝杆菌早期分泌性抗原靶(ESAT-6)在上述三组患者肾组织上的表达。
材料与方法
一、第一部分
1、研究对象与分组
病例选择：各组病例均来自2005年至2007年在中国医科大学附属第一医院住院患者,平均年龄为42.5±19.5岁,男58例,女32例。
肾结核组：因肾结核,行一侧肾切除、病理诊断为肾结核患者30例；
非肾结核组：临床诊断肾小球肾炎,无结核感染史,血结核抗体阴性,PPD
(—)患者30例。
结核杆菌相关肾损伤疑诊组30例：入选标准需具备以下6条：
①肾性血尿／蛋白尿；②结核中毒症状：不明原因低热、乏力>1月；③PPD(++—+++)；④肾外结核或结核史；⑤血γ球蛋白增高；⑥血结核抗体阳性。
以上病例均留取24小时尿沉渣在罗氏培养基行结核杆菌培养。
2、DNA提取
(1)将肾组织块剪碎,置于1.5ml离心管中
(2)加入消化液500ulTE PH8.0 445ul 10mmolTris/1mmol EDTA10%SDS 50ul终浓度为1%20mg／ml蛋白酶K 5ul终浓度为200ug／ml
(3)37C过夜
(4)加入等体积的饱和酚500ul,充分混合
(5)10000转离心5分钟
(6)将水相上一层移入一新管,再加入等体积饱和酚抽提一次
(7)将水相上一层移入一新管,加饱和酚和氯仿混合液1：1共500ul混合,10000转离心5分钟
(8)取水相加1／10体积的醋酸钠混合,再加2倍体积的-20C冻无水乙醇混合,可见固态结晶。
(9)10000转离心5分钟
(10)弃上清,加75%乙醇500ul洗一次,把DNA悬起即可
(11)10000转离心5分钟
(12)弃上清,吸干液体,37C,放置5分钟,干燥
(13)TE或去离子水50ul溶解,-20 C保存
DNA提取试剂盒(Sigma公司)
实时荧光定量试剂SYBR Green (Gene公司)
引物合成：以β-actin为内参,β-actin及结核杆菌正向、反向引物由TAKARA公司设计合成。
引物序列如下：使用特异性结核杆菌插入序列IS6110作为结核杆菌DNA扩增的目的基因,
β-actin正向引物5'-AGT TGC GTT ACA CCC TTT CTT G-3'反向引物5'-TCA CCT TCA CCG TTC CAG TTT-3'
结核杆菌正向引物5'-TTG GAA AGG ATG GGG TCA-3'反向引物5'-CGC AGC CAA CAC CAA GTA G-3'
预期扩增产物长度为156bp所有的Real-time PCR反应都是在ABI PRISM7700序列检测仪上进行。每一次试验都设立空白对照。应用随机带有的软件进行Real-time PCR数据分析。分别以35和40作为循环次数。
二、第二部分
1、研究对象与分组
本研究所选各组病例均来自2005年至2007年在中国医科大学附属第一医院住院患者,平均年龄为46.1±17.6岁,男40例,女33例。
肾结核组：因肾结核,行一侧肾切除、病理诊断为肾结核患者10例；
非肾结核组：临床诊断肾小球肾炎,无结核感染史,血结核抗体阴性,PPD(—)患者12例。
结核杆菌相关肾损伤疑诊组30例：入选标准同前。
以上病例均留取24小时尿沉渣在罗氏培养基行结核杆菌培养。
2、实验试剂
(1)Ag85
一抗,为鼠单克隆抗体,(Anti Mycobacterium tuberculosis Ag85, mouse monoclonal antibody HYT 27),购于丹麦AntibodyShop公司。
(2)IFN-γ
一抗,鼠抗人抗体,购于上海优宁维生物科技有限公司。
(3)免疫组化染色试剂盒
正常山羊血清封闭液,二抗山羊抗小鼠IgG,抗原修复液,0.1M PBS,0.01M枸橼酸盐缓冲液,酶-酶抗体复合物,甘氨酸-盐酸缓冲液,DAB显色试剂盒。购于武汉博士德公司。
3、实验步骤：免疫组织化学SABC法
(1)组织切片：用10%富尔马林固定组织,常规石蜡包埋。切片厚4-6gm,粘附于涂有1mg／ml多聚赖氨酸粘附剂的玻片上,入烤箱60-80℃,6-8小时。
(2)脱腊：100%乙醇Ⅰ5min,100%乙醇Ⅱ5min,95%乙醇5 min,90%乙醇5min,70%乙醇5 min,50%乙醇5 min,30%乙醇5 min。
(3)蒸馏水新鲜配置3%H2O2,室温5-10分钟以灭活内源性酶。蒸馏水洗3次。
(4)热修复抗原：将切片浸入0.01M枸橼酸盐缓冲液(PH6.0),微波炉加热至沸腾后断电,间隔5-10分钟后,反复1-2次。冷却后0.1M PBS洗涤2-3次。
(5)滴加正常山羊血清封闭液,室温30分钟。去除多余液体,不洗。
(6)滴加一抗：400倍稀释的一抗4℃过夜。0.1M PBS洗2分钟,3次。
(7)滴加生物素化山羊抗小鼠IgG,37℃30分钟。0.1M PBS洗2分钟,3次。
(8)滴加试剂SABC,37℃30分钟。0.1M PBS洗5分钟,4次。
(9)使用DAB显色试剂盒。取1ml蒸馏水,加试剂盒中A、B、C试剂各一滴,混均后加至切片。室温显色,镜下控制反应时间,一般在10分钟。蒸馏水洗涤。
(10)复染：苏木素轻度复染。脱水透明封片。显微镜观察。
4、免疫组织化学结果分析
应用中国医科大学医学图像分析系统检测,于肾皮质随机采集5个不含肾小球及动脉的视野(x40倍)测定阳性表达部分的平均光密度值,及其与整个视野面积的比值。
5、统计学处理
所有数据均用SPSS13.0统计软件进行分析,组间比较采用t检验,以P<0.05为差异具有显著性的标准。并计算Ag85和IFN-γ表达的相关性。
三、第三部分
1、研究对象与分组
同第二部分
2、实验试剂
ESAT-6,一抗,为鼠单克隆抗体,(Anti Mycobacterium tuberculosis, ESAT-6, mouse monoclonal antibody HYB 076-08),购于丹麦AntibodyShop公司。
二抗,山羊抗小鼠IgG、酶亲合素-过氧化物酶复合物SABC,复合消化液,抗原修复液,0.1M PBS,0.01M枸橼酸盐缓冲液,DAB显色试剂盒,购于武汉博士德公司。
3、实验步骤
免疫组织化学SABC法检测ESAT-6在肾组织上的表达,具体步骤同第二部分。
4、免疫组织化学结果分析
应用中国医科大学医学图像分析系统检测,于肾皮质随机采集5个不含肾小球及动脉的视野(×40倍)测定阳性表达部分的平均光密度值,及其与整个视野面积的比值。
5、统计学处理
所有数据均用SPSS13.0统计软件进行分析,组间比较采用t检验,以P<0.05为差异具有显著性的标准。
结果
一、第一部分
30例肾结核组病人中,7例尿培养为阳性,23例为阴性,非肾结核组病例尿培养结果全部为阴性,尿培养的敏感度为23.3%,特异度为100%。
实时定量PCR结果显示,以35作为循环次数,25例肾结核组病人阳性,非肾结核组病人4例阳性,敏感度为83.7%,特异度为86.7%；以40作为循环次数,28例肾结核组病人阳性,非肾结核组病人13例阳性,敏感度为93.3%,特异度为56.7%。
结核性肾损伤疑诊组：以35作为循环次数,9例肾结核组病人阳性；以40作为循环次数,14例肾结核组病人阳性。
肾结核组的CT值为19.64±1.15；结核性肾损伤疑诊组的CT值为23.32±5.32,二者比较P=0.09,无显著性差异。
我们跟踪随访了结核性肾损伤疑诊组9例阳性病例一年,其中3例在随后多次尿培养中找到结核杆菌；4例因常规治疗效果不佳,经患者同意后采用抗结核实验性治疗,病情好转；其余2例经常规治疗后病情好转。
二、第二部分
1、Ag85及IFN-γ免疫组织化学染色结果
肾结核组：肾组织中10例均可见Ag85及IFN-γ呈褐色颗粒状阳性物质沉积在肾间质、肾小管细胞的胞浆及部分肾小球系膜区、毛细血管基底膜。
非肾结核组：3例可见浅褐色物质片状分布于肾间质,其余病例均未见褐色物质沉积。
结核性肾损伤疑诊组：30例肾组织中14例可见褐色颗粒状阳性物质沉积在肾间质、肾小管细胞的胞浆及部分肾小球系膜区、毛细血管基底膜。肾脏病理损伤严重部位阳性物质沉积亦较多。
Ag85及IFN-γ表达部位一致。
肾结核组、结核性肾损伤疑诊组及非肾结核组Ag85的OD值分别为79.07±19.50,70.60±11.86,15.56±3.39；IFN-γ的OD值分别为50.88±11.61,46.19±14.43,5.93±2.01前两者比较差异无统计学意义(P>0.05),后两者比较差异有统计学意义(P<0.005)。
2、对Ag85及IFN-γ进行相关性分析
结果表明二者成直线正相关,r=0.9857,p<0.05。
3、尿培养及Ag85免疫组化的敏感性及特异性
10例肾结核组病人中,3例尿培养为阳性,7例为阴性,非肾结核组病例尿培养结果全部为阴性。尿培养的敏感度为30%,特异度为100%,Ag85免疫组化染色的敏感度为100%,特异度为75%。
三、第三部分
1、ESAT-6免疫组织化学染色结果
肾结核组：肾组织中10例均可见褐色颗粒状阳性物质沉积在肾间质、肾小管细胞的胞浆及部分肾小球系膜区、毛细血管基底膜。
非肾结核组：2例可见浅褐色物质沉积于肾间质,其余均未见褐色物质沉积。
结核性肾损伤疑诊组：30例肾组织中8例可见褐色颗粒状阳性物质沉积在肾间质、肾小管细胞的胞浆及部分肾小球系膜区、毛细血管基底膜。肾脏病理损伤严重部位阳性物质沉积亦较多。
肾结核组、结核性肾损伤疑诊组及非肾结核组OD值分别为53.67±14.21,48.5±9.26,6.47±1.61,前两者比较差异无统计学意义(P>0.05),后两者比较差异有统计学意义(P<0.005)。
2、尿培养及ESAT-6免疫组化的敏感性及特异性
尿培养的敏感度为30%,特异度为100%；ESAT-6免疫组化染色的敏感度为100%,特异度为83%。
结论
本研究结果表明：
1、荧光定量PCR技术检测肾组织中的结核分枝杆菌DNA,以35作为循环次数,敏感度为83.7%,特异度为86.7%。可以提高检测的特异性,灵敏度和可信性,为临床提供可靠的诊断依据。
2、结核分枝杆菌早期分泌性蛋白抗原85及γ干扰素(IFN-γ)在肾结核患者肾组织上的表达增强,且二者呈正相关,Ag85免疫组化染色的敏感度为100%,特异度为75%,Ag85表达阳性提示患者肾组织可能存在结核杆菌感染,临床上需密切观察此类病人,反复进行尿结核杆菌培养,以免出现漏诊及误诊。
3、结核分枝杆菌早期分泌性抗原靶ESAT-6在肾结核患者肾组织上的表达增强,ESAT-6免疫组化染色的敏感度为100%,特异度为83%,ESAT-6表达阳性提示患者可能感染致病性结核杆菌且特异度高于Ag85,可以为结核相关肾损伤早期诊断提供一定的价值。
Objective
Tuberculosis (TB) is a major global public health problem. The World Health Organization estimates that one-third of the world's population is infected with Mycobacterium tuberculosis (MTB), which is responsible for 8.7 million new TB cases and approximately 3 million deaths annually. Genitourinary TB is commonly a late manifestation of an earlier symptomatic or asymptomatic pulmonary TB infection. As one of the most common sites of involvement of extrapulmonary TB, genitourinary TB accounts for 15%-20% of infections outside of the lungs. Among genitourinary TB, diagnosis of renal tuberculosis (RTB) remains difficult, especially in the early stage due to the vagueness of chronic, intermittent, and non-specific urinary symptoms. As a result, many patients are not diagnosed or misdiagnosed and lose the chance of early treatment and progress to end-stage renal disease (ESRD), a life-threatening condition. Therefore, the early diagnosis of renal TB is very important to prevent progressive destruction of the kidney.
However, traditional laboratory techniques, such as direct microscopic observation and mycobacterial culture on semisolid or liquid medium, are far from being sufficiently sensitive and specific for rapid MTB identification. To test positive, an acid-fast bacilli (AFB) smear requires 5000-10000 AFB/ml. Although urine MTB culture is more sensitive than AFB smear, culture growth is slow; most samples do not show visible colonies of MTB before one month, thus delaying the diagnosis.
Genitourinary TB accounts for approximately15% of all extrapulmonary cases, and may involve any portion of the genitourinary tract. Local symptoms predominate. However, in the early stage of RTB, most patients may be asymptomatic and abnormal urinalyses are detected only as part of routine clinical evaluations or incidentally in the hospital while undergoing treatment for unrelated disorders. The urinalysis may reveal mild proteinuria, microscopic hematuria, and leucocyturia in acidic urine. Recently, due to some symptomatic treatment, the major clinical manifestation of some RTB patients gives priority to renal disease instead of cystitis. Several cases of glomerulonephritis associated with active TB have been described in the literature.
MTB can induce both cellular immune and humoral immune responses when bacilli invade the body. Studies have shown that MTB infection can lead to the disturbances of Thl/Th2 cells, which may give rise to nephritis. On the other hand, type III allergic reactions induced by immune complexes can cause tissue injury and may serve as the pathogenesis of TB. The role of disseminated tuberculosis in the pathogenesis of glomerulonephritis has been presumed to be dependent on humoral immunity and immune complexes have been reported detectable at high levels in the active phase of disseminated tuberculosis. As a result, some patients with early RTB show clinical features of chronic nephritis, such as hematuria, proteinuria, edema, and hypertension. Very often such patients are misdiagnosed and the disease is discovered only after severe destructive lesions of the kidneys have developed. In this study, urinalyses of all patients in the CS-RTB group showed proteinuria, hematuria, and leucocyturia without any symptoms of cystitis, such as urinary frequency, dysuria, and flank pain. We did not detect deposition of immune complexes in the renal biopsy. This may be because of the low level of immune complexes in the early stages of RTB.
Therefore, diagnostic tests devoted to the rapid, sensitive, and specific identification of MTB infections has been the key element for early diagnosis of RTB.
Our investigation included four aspects:①To detect MTB DNA in the renal biopsy specimens by real time PCR.②To study the expression of secretory antigen 85 (Ag85) of MTB and interferon-gamma (IFN-γ) in the renal tissues of patients with RTB; to discuss the relationship between the expression of Ag85 and IFN-γ.③To study the expression of early secretory antigen target-6 (ESAT-6) of MTB in the renal tissues of patients with RTB.④To discuss the sensitivity and specificity of Ag85, ESAT-6 and TB-DNA in the early diagnosis of renal tuberculosis.
Materials and Methods
The patients were divided into the following groups:renal tuberculosis (RTB); non-renal tuberculosis (N-RTB); and clinically suspected renal tuberculosis (CS-RTB).
Thirty patients were selected for the RTB group who underwent unilateral nephrectomy because of RTB, which was confirmed by pathologic diagnosis. Morning urine samples were collected from the patients for MTB culture before surgery.
The N-RTB group consisted of 30 patients, including seven patients with minimal change nephritic syndrome,13 patients with mesangioproliferative glomerulonephritis, five patients with membranous nephropathy, one patient with glomerulosclerosis, and four patients with focal segmental glomerulonephritis. No patients had pulmonary or extrapulmonary TB. The tuberculin skin tests and serum TB antibody titers were negative. The patients were diagnosed with primary glomerulonephritis and the condition improved after routine therapy.
The CS-RTB group was comprised of 30 patients who had extranephric TB, hematuria, and proteinuria. The tuberculin skin tests and serum TB antibody titers were positive, while the kidney ultrasonic examination and radiologic tests did not show any abnormalities.
Patients in the N-RTB and CS-RTB groups underwent renal biopsy and morning urine samples were collected for MTB culture.
All of the patients (52 males and 38 females with a mean age of 42.5±19.5 years) were selected from patients who were admitted to our hospital between 2004 and 2007.
Urine MTB culture
Urine samples were pre-treated by decontamination with 4%(w/v) NaOH and centrifugation at 1500 g for 10 min. The sediment was used for MTB culture. MTB culture was performed using in-house made Lowenstein-Jensen (LJ) solid medium with a maximum incubation period of 8 weeks. The results were read and reported weekly. Ziehl Neelsen (ZN) staining was performed to confirm the presence of mycobacteria when colonies were noted in the LJ medium.
1. Part 1
(1)DNA isolation
Two renal biopsies were obtained in all the cases and the length of each biopsy was approximately 12 mm. One specimen was divided into three parts for three different histopathologic diagnostic procedures. The other specimen was used for DNA extraction. DNA was extracted with the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The DNA concentration was measured and 100 ng of DNA was used for a real time PCR reaction, according to the instructions of the real time PCR kit.
(2) Real-time PCR
The primers were synthesized by TaKaRa Company (Dalian, China). The recommended concentration ranges were from 0.2-1.0μM. In this study, the final concentration of primers was 0.4μM, which has been shown to be a suitable concentration in preliminary experiments.
All PCRs were performed in the ABI PRISM 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with a 384-well plate. The thermal conditions were as follows:stage 1 (initial denaturation),95℃for 1 min,1 cycle; stage 2 (PCR),95℃for 5s and 60℃for 30s,40 cycles; and stage 3 (dissociation),95℃for 15s,60℃for 1 min, and 95℃for 15s,1 cycle. Every well was loaded with a PCR mixture containing SYBR Premix Ex TaqⅡ(2×; 10μl), PCR forward primer (10μM; 0.8μl), PCR reverse primer (10μM; 0.8μl), ROX reference dye (50×; 0.4μl), DNA template (2.0μl), and dH2O (6.0μl) in a total volume of 20μl. Each sample was tested in triplicate and data were collected in the last step for analysis with the SDS software version 2.0a23 (Applied Biosystems). Thirty-five and 40 were used as cycle threshold (CT) cut-off values. For the real-time experiments, post-PCR DNA melt curve analysis was performed to assess amplification specificity. In case of ambiguity, DNA gel electrophoresis was performed.
MTB DNA amplification was performed using insertion element IS6110 as a multi-copy target for molecular detection of the MTB complex.-actin was used as a housekeeper gene to control the DNA purification procedure and the existence of possible PCR inhibitors. The amplification of TB-DNA and-actin was performed in the same reaction. For each sample, we prepared two microtubes (one for TB-DNA amplification and one for-actin). The reaction system was the same, with the exception of the primers.
2. Part 2
To study the expression of Ag85 of MTB and IFN-yin renal tissues with immunohistochemistry method and discuss the relationship between the expression of Ag85 and IFN-y.
1. Deparaffinize and rehydrate sections as follows:3×3 min with xylene; 3×2 min with 100% ethanol; 2 min with 95% ethanol,2 min with 80% ethanol,2 min with 70% ethanol, and 5 min with PBS.
2. Retrieve antigen using one the antigen retrieval methods described below.
3. Block endogenous peroxidases by soaking slides in a solution of 90% methanol/3% H2O2 for 15 min at room temperature (RT). Then wash 3×5 min with PBS.
4. Shake and wipe off excess PBS. Circle all sections with a pap pen. Add 75 ul of Blocking buffer to each section immediately. Do not touch sections with tip.
5. Incubate 1 hr to overnight at RT in a humidified chamber. Do not let the slides touch each other.
6. Dilute primary antibody in Blocking buffer (dilutions will vary depending on tested antibody). Add 75 ml per section and incubate 1 hr to overnight at RT in a humidified chamber.
7. Drain primary antibody off section. Wash slides 3 x 10 min in PBS. You may have to wash slides in PBS+0.1%-0.5% Tween-20 for some primary antibodies.
8. Dilute secondary antibody 1:1000 in Blocking buffer without Tween-20. Add 75 ml per section and incubate for 1 hr at RT in a humidified chamber.
9. Drain secondary antibody and wash slides 5 x 10 min in PBS+0.1% Tween-20.(For secondary antibodies that are peroxidase conjugated, go to step 11.)
10. Make ABC according to Vector protocol 30 min before time of use (mix 5 ml of PBS with 2 drops of solution A and 2 drops of solution B). Incubate samples for 45 min at RT. Wash 5 min in PBS.
11. Make DAB according to Vector protocol in ddH2O. WEAR GLOVES:Mix 5 ml ddH2O with 2 drops of buffer,4 drops of DAB and 2 drops of H2O2. (If you want a gray-black stain, add 2 drops of the Nickel solution, and mix). Add immediately to slides and wait for color change (approximately 2-10 min). Drain slides and place into ddH2O for 5 min. Dispose of DAB waste with bleach.
12. Counterstain with methyl green (1 min) or hematoxylin (3 sec). Wash 3 times with ddH2O.
13. Immediately dehydrate in 70% ethanol,80% ethanol, and 100% ethanol (one dip each).
14. Mount with Permount and seal coverslip with nail polish.
3. Part 3
To study the expression of ESAT-6 of MTB in renal tissues with immunohistochemistry method.
The same protocol as above.
Results
1. Part 1
In the RTB group, seven patients were urine MTB culture-positive. For real-time PCR,25 samples tested positive by using 35 as the CT cut-off value and 28 samples tested positive by using 40 as the CT cut-off value.
In the N-RTB group, no patients were urine MTB culture-positive. Four patients were CT35 positive (two patients with focal segmental glomerulonephritis, one patient with glomerulosclerosis, and one patient with membranous nephropathy). Thirteen patients were CT40-positive (besides the four patients above, five patients with mesangioproliferative glomerulonephritis, two patients with minimal change nephritis syndrome, one patient with membranous nephropathy, and one patient with focal segmental glomerulonephritis). Neither granulomas nor evidence of acid-fast bacilli were noted in any of the positive biopsies. We followed those patients for 12 months and no patients developed RTB.
The sensitivity and specificity of the urine MTB culture were 23.3% and 100%, respectively. The sensitivity and specificity of real-time PCR (CT 40) were 93.3% and 56.7%, respectively. When using 35 as the CT cut off value, the sensitivity and specificity were 83.3% and 86.7%, respectively. Compared with CT 40, the specificity was significantly increased (Table 3).
In the CS-RTB group, when using 35 as the CT cut-off value of real-time PCR,9 samples tested positive. While using 40 as the CT cut-off value,14 samples tested positive.
2. Part 2
The expression of Ag85 and IFN-gamma were significantly increased in renal tuberculosis group,3 cases in non-renal tuberculosis group and 18 cases in experimental group. Compared with positive group, there was no significant difference in the optical density in experimental group. While compared with renal tuberculosis group, there was significant difference in experimental group. There was a positive linear correlation between ag85 and IFN-gamma. The sensitivity of ag85 was 100% and the specificity was 75%.
3. Part 3
The expression of ESAT-6 were significantly increased in renal tuberculosis group, 4 cases in non-renal tuberculosis group and 15 cases in experimental group. Compared with renal tuberculosis group, there was no significant difference in the optical density in experimental group. While compared with non-renal tuberculosis group, there was significant difference in experimental group. The sensitivity of ESAT-6 was 100% and the specificity was 80%.
Conclusion
These data suggested that:
1. Detection of MTA DNA in renal biopsy tissues by real-time PCR is highly sensitive. Moreover, it can increase the diagnostic accuracy and provide valuable information which would complete other clinical data for the early diagnosis of RTB. Although urine MTB culture is still the gold standard for RTB diagnosis, we speculate that if real-time PCR is widely adopted within clinical practice, it will be a powerful tool for the rapid and accurate diagnosis of RTB.
2. The expression of Ag85 and IFN-gamma were significantly increased in renal tuberculosis group. There was a positive linear correlation between Ag85 and IFN-gamma. The sensitivity of Ag85 was 100% and the specificity was 75% which demonstrated that Ag85 played an important role in the early diagnosis of renal tuberculosis.
3. The expression of ESAT-6 were significantly increased in renal tuberculosis group, The sensitivity of ESAT-6 was 100% and the specificity was 80% which is higher than that of Ag85. It showed that ESAT-6 played an important role in the early diagnosis of renal tuberculosis.
结核病目前仍是危害人类健康的主要疾病之一,据世界卫生组织估计,目前全世界有近三分之一的人感染了结核,每年有870万新增病例,其中300万死亡,因此对该病的预防与治疗已成为研究的热点。肾结核病是常见的肺外结核病,肺结核死亡病人尸检显示,并发肾结核者占16-26%。而肾结核早期临床表现不典型,症状轻微,缺乏特异性,诊断比较困难。临床上漏诊误诊率高,使多数病人失去了早期病因治疗的机会,致使晚期进入终末期肾衰,威胁病人生命,故肾结核的早期诊断非常重要。目前尿结核杆菌培养阳性仍是肾结核诊断的金标准,但其时间长、阳性率低。结核菌产生分阶段性,所以早期肾结核的诊断率只占6%,不排菌的肾结核诊断率更低。因此在肾结核病感染的早期,快速的诊断是一项挑战性的课题。
结核杆菌对肾脏的损伤分为临床肾结核,病理肾结核和结核性变态反应综合征。所谓结核性变态反应综合征是指当机体对结核感染处于超敏状态时,可在远离原发灶的皮肤、关节、角膜、结膜、肾脏、浆膜腔等多处发生细胞免疫和体液免疫反应,引起类似系统性红斑狼疮,血管炎,角膜炎,关节炎,肾小球肾炎等多种临床表现。结核性变态反应综合征引起肾损伤,可以表现为蛋白尿,血尿,IgA肾病,肾病综合征等。其病理类型为：毛细血管内增生性肾小球肾炎、膜增生性肾小球肾炎、局灶节段性肾小球肾炎、系膜增生性肾小球肾炎、微小病变病等多种类型。结核杆菌感染做为这些疾病的病因。若不能及时诊断,及时行病因治疗,而单纯应用免疫抑制剂或激素治疗,结果会导致结核播散,疾病迁延不愈,给患者带来不能弥补的损伤或遗憾。
本试验将病例分为三组：肾结核组,非肾结核组,结核杆菌相关肾损伤疑诊组。从以下三个方面进行研究,以探讨结核性变态反应综合症导致的肾损伤的早期诊断方法：1应用实时定量PCR技术,对患者肾组织中结核分枝杆菌DNA表达进行定量分析。2研究上述三组患者肾组织中结核分枝杆菌早期分泌性蛋白抗原85(Ag85)及γ干扰素(IFN-γ)的表达,并分析二者的相关性。3研究结核分枝杆菌早期分泌性抗原靶(ESAT-6)在上述三组患者肾组织上的表达。
材料与方法
一、第一部分
1、研究对象与分组
病例选择：各组病例均来自2005年至2007年在中国医科大学附属第一医院住院患者,平均年龄为42.5±19.5岁,男58例,女32例。
肾结核组：因肾结核,行一侧肾切除、病理诊断为肾结核患者30例；
非肾结核组：临床诊断肾小球肾炎,无结核感染史,血结核抗体阴性,PPD
(—)患者30例。
结核杆菌相关肾损伤疑诊组30例：入选标准需具备以下6条：
①肾性血尿／蛋白尿；②结核中毒症状：不明原因低热、乏力>1月；③PPD(++—+++)；④肾外结核或结核史；⑤血γ球蛋白增高；⑥血结核抗体阳性。
以上病例均留取24小时尿沉渣在罗氏培养基行结核杆菌培养。
2、DNA提取
(1)将肾组织块剪碎,置于1.5ml离心管中
(2)加入消化液500ulTE PH8.0 445ul 10mmolTris/1mmol EDTA10%SDS 50ul终浓度为1%20mg／ml蛋白酶K 5ul终浓度为200ug／ml
(3)37C过夜
(4)加入等体积的饱和酚500ul,充分混合
(5)10000转离心5分钟
(6)将水相上一层移入一新管,再加入等体积饱和酚抽提一次
(7)将水相上一层移入一新管,加饱和酚和氯仿混合液1：1共500ul混合,10000转离心5分钟
(8)取水相加1／10体积的醋酸钠混合,再加2倍体积的-20C冻无水乙醇混合,可见固态结晶。
(9)10000转离心5分钟
(10)弃上清,加75%乙醇500ul洗一次,把DNA悬起即可
(11)10000转离心5分钟
(12)弃上清,吸干液体,37C,放置5分钟,干燥
(13)TE或去离子水50ul溶解,-20 C保存
DNA提取试剂盒(Sigma公司)
实时荧光定量试剂SYBR Green (Gene公司)
引物合成：以β-actin为内参,β-actin及结核杆菌正向、反向引物由TAKARA公司设计合成。
引物序列如下：使用特异性结核杆菌插入序列IS6110作为结核杆菌DNA扩增的目的基因,
β-actin正向引物5'-AGT TGC GTT ACA CCC TTT CTT G-3'反向引物5'-TCA CCT TCA CCG TTC CAG TTT-3'
结核杆菌正向引物5'-TTG GAA AGG ATG GGG TCA-3'反向引物5'-CGC AGC CAA CAC CAA GTA G-3'
预期扩增产物长度为156bp所有的Real-time PCR反应都是在ABI PRISM7700序列检测仪上进行。每一次试验都设立空白对照。应用随机带有的软件进行Real-time PCR数据分析。分别以35和40作为循环次数。
二、第二部分
1、研究对象与分组
本研究所选各组病例均来自2005年至2007年在中国医科大学附属第一医院住院患者,平均年龄为46.1±17.6岁,男40例,女33例。
肾结核组：因肾结核,行一侧肾切除、病理诊断为肾结核患者10例；
非肾结核组：临床诊断肾小球肾炎,无结核感染史,血结核抗体阴性,PPD(—)患者12例。
结核杆菌相关肾损伤疑诊组30例：入选标准同前。
以上病例均留取24小时尿沉渣在罗氏培养基行结核杆菌培养。
2、实验试剂
(1)Ag85
一抗,为鼠单克隆抗体,(Anti Mycobacterium tuberculosis Ag85, mouse monoclonal antibody HYT 27),购于丹麦AntibodyShop公司。
(2)IFN-γ
一抗,鼠抗人抗体,购于上海优宁维生物科技有限公司。
(3)免疫组化染色试剂盒
正常山羊血清封闭液,二抗山羊抗小鼠IgG,抗原修复液,0.1M PBS,0.01M枸橼酸盐缓冲液,酶-酶抗体复合物,甘氨酸-盐酸缓冲液,DAB显色试剂盒。购于武汉博士德公司。
3、实验步骤：免疫组织化学SABC法
(1)组织切片：用10%富尔马林固定组织,常规石蜡包埋。切片厚4-6gm,粘附于涂有1mg／ml多聚赖氨酸粘附剂的玻片上,入烤箱60-80℃,6-8小时。
(2)脱腊：100%乙醇Ⅰ5min,100%乙醇Ⅱ5min,95%乙醇5 min,90%乙醇5min,70%乙醇5 min,50%乙醇5 min,30%乙醇5 min。
(3)蒸馏水新鲜配置3%H2O2,室温5-10分钟以灭活内源性酶。蒸馏水洗3次。
(4)热修复抗原：将切片浸入0.01M枸橼酸盐缓冲液(PH6.0),微波炉加热至沸腾后断电,间隔5-10分钟后,反复1-2次。冷却后0.1M PBS洗涤2-3次。
(5)滴加正常山羊血清封闭液,室温30分钟。去除多余液体,不洗。
(6)滴加一抗：400倍稀释的一抗4℃过夜。0.1M PBS洗2分钟,3次。
(7)滴加生物素化山羊抗小鼠IgG,37℃30分钟。0.1M PBS洗2分钟,3次。
(8)滴加试剂SABC,37℃30分钟。0.1M PBS洗5分钟,4次。
(9)使用DAB显色试剂盒。取1ml蒸馏水,加试剂盒中A、B、C试剂各一滴,混均后加至切片。室温显色,镜下控制反应时间,一般在10分钟。蒸馏水洗涤。
(10)复染：苏木素轻度复染。脱水透明封片。显微镜观察。
4、免疫组织化学结果分析
应用中国医科大学医学图像分析系统检测,于肾皮质随机采集5个不含肾小球及动脉的视野(x40倍)测定阳性表达部分的平均光密度值,及其与整个视野面积的比值。
5、统计学处理
所有数据均用SPSS13.0统计软件进行分析,组间比较采用t检验,以P<0.05为差异具有显著性的标准。并计算Ag85和IFN-γ表达的相关性。
三、第三部分
1、研究对象与分组
同第二部分
2、实验试剂
ESAT-6,一抗,为鼠单克隆抗体,(Anti Mycobacterium tuberculosis, ESAT-6, mouse monoclonal antibody HYB 076-08),购于丹麦AntibodyShop公司。
二抗,山羊抗小鼠IgG、酶亲合素-过氧化物酶复合物SABC,复合消化液,抗原修复液,0.1M PBS,0.01M枸橼酸盐缓冲液,DAB显色试剂盒,购于武汉博士德公司。
3、实验步骤
免疫组织化学SABC法检测ESAT-6在肾组织上的表达,具体步骤同第二部分。
4、免疫组织化学结果分析
应用中国医科大学医学图像分析系统检测,于肾皮质随机采集5个不含肾小球及动脉的视野(×40倍)测定阳性表达部分的平均光密度值,及其与整个视野面积的比值。
5、统计学处理
所有数据均用SPSS13.0统计软件进行分析,组间比较采用t检验,以P<0.05为差异具有显著性的标准。
结果
一、第一部分
30例肾结核组病人中,7例尿培养为阳性,23例为阴性,非肾结核组病例尿培养结果全部为阴性,尿培养的敏感度为23.3%,特异度为100%。
实时定量PCR结果显示,以35作为循环次数,25例肾结核组病人阳性,非肾结核组病人4例阳性,敏感度为83.7%,特异度为86.7%；以40作为循环次数,28例肾结核组病人阳性,非肾结核组病人13例阳性,敏感度为93.3%,特异度为56.7%。
结核性肾损伤疑诊组：以35作为循环次数,9例肾结核组病人阳性；以40作为循环次数,14例肾结核组病人阳性。
肾结核组的CT值为19.64±1.15；结核性肾损伤疑诊组的CT值为23.32±5.32,二者比较P=0.09,无显著性差异。
我们跟踪随访了结核性肾损伤疑诊组9例阳性病例一年,其中3例在随后多次尿培养中找到结核杆菌；4例因常规治疗效果不佳,经患者同意后采用抗结核实验性治疗,病情好转；其余2例经常规治疗后病情好转。
二、第二部分
1、Ag85及IFN-γ免疫组织化学染色结果
肾结核组：肾组织中10例均可见Ag85及IFN-γ呈褐色颗粒状阳性物质沉积在肾间质、肾小管细胞的胞浆及部分肾小球系膜区、毛细血管基底膜。
非肾结核组：3例可见浅褐色物质片状分布于肾间质,其余病例均未见褐色物质沉积。
结核性肾损伤疑诊组：30例肾组织中14例可见褐色颗粒状阳性物质沉积在肾间质、肾小管细胞的胞浆及部分肾小球系膜区、毛细血管基底膜。肾脏病理损伤严重部位阳性物质沉积亦较多。
Ag85及IFN-γ表达部位一致。
肾结核组、结核性肾损伤疑诊组及非肾结核组Ag85的OD值分别为79.07±19.50,70.60±11.86,15.56±3.39；IFN-γ的OD值分别为50.88±11.61,46.19±14.43,5.93±2.01前两者比较差异无统计学意义(P>0.05),后两者比较差异有统计学意义(P<0.005)。
2、对Ag85及IFN-γ进行相关性分析
结果表明二者成直线正相关,r=0.9857,p<0.05。
3、尿培养及Ag85免疫组化的敏感性及特异性
10例肾结核组病人中,3例尿培养为阳性,7例为阴性,非肾结核组病例尿培养结果全部为阴性。尿培养的敏感度为30%,特异度为100%,Ag85免疫组化染色的敏感度为100%,特异度为75%。
三、第三部分
1、ESAT-6免疫组织化学染色结果
肾结核组：肾组织中10例均可见褐色颗粒状阳性物质沉积在肾间质、肾小管细胞的胞浆及部分肾小球系膜区、毛细血管基底膜。
非肾结核组：2例可见浅褐色物质沉积于肾间质,其余均未见褐色物质沉积。
结核性肾损伤疑诊组：30例肾组织中8例可见褐色颗粒状阳性物质沉积在肾间质、肾小管细胞的胞浆及部分肾小球系膜区、毛细血管基底膜。肾脏病理损伤严重部位阳性物质沉积亦较多。
肾结核组、结核性肾损伤疑诊组及非肾结核组OD值分别为53.67±14.21,48.5±9.26,6.47±1.61,前两者比较差异无统计学意义(P>0.05),后两者比较差异有统计学意义(P<0.005)。
2、尿培养及ESAT-6免疫组化的敏感性及特异性
尿培养的敏感度为30%,特异度为100%；ESAT-6免疫组化染色的敏感度为100%,特异度为83%。
结论
本研究结果表明：
1、荧光定量PCR技术检测肾组织中的结核分枝杆菌DNA,以35作为循环次数,敏感度为83.7%,特异度为86.7%。可以提高检测的特异性,灵敏度和可信性,为临床提供可靠的诊断依据。
2、结核分枝杆菌早期分泌性蛋白抗原85及γ干扰素(IFN-γ)在肾结核患者肾组织上的表达增强,且二者呈正相关,Ag85免疫组化染色的敏感度为100%,特异度为75%,Ag85表达阳性提示患者肾组织可能存在结核杆菌感染,临床上需密切观察此类病人,反复进行尿结核杆菌培养,以免出现漏诊及误诊。
3、结核分枝杆菌早期分泌性抗原靶ESAT-6在肾结核患者肾组织上的表达增强,ESAT-6免疫组化染色的敏感度为100%,特异度为83%,ESAT-6表达阳性提示患者可能感染致病性结核杆菌且特异度高于Ag85,可以为结核相关肾损伤早期诊断提供一定的价值。
Objective
Tuberculosis (TB) is a major global public health problem. The World Health Organization estimates that one-third of the world's population is infected with Mycobacterium tuberculosis (MTB), which is responsible for 8.7 million new TB cases and approximately 3 million deaths annually. Genitourinary TB is commonly a late manifestation of an earlier symptomatic or asymptomatic pulmonary TB infection. As one of the most common sites of involvement of extrapulmonary TB, genitourinary TB accounts for 15%-20% of infections outside of the lungs. Among genitourinary TB, diagnosis of renal tuberculosis (RTB) remains difficult, especially in the early stage due to the vagueness of chronic, intermittent, and non-specific urinary symptoms. As a result, many patients are not diagnosed or misdiagnosed and lose the chance of early treatment and progress to end-stage renal disease (ESRD), a life-threatening condition. Therefore, the early diagnosis of renal TB is very important to prevent progressive destruction of the kidney.
However, traditional laboratory techniques, such as direct microscopic observation and mycobacterial culture on semisolid or liquid medium, are far from being sufficiently sensitive and specific for rapid MTB identification. To test positive, an acid-fast bacilli (AFB) smear requires 5000-10000 AFB/ml. Although urine MTB culture is more sensitive than AFB smear, culture growth is slow; most samples do not show visible colonies of MTB before one month, thus delaying the diagnosis.
Genitourinary TB accounts for approximately15% of all extrapulmonary cases, and may involve any portion of the genitourinary tract. Local symptoms predominate. However, in the early stage of RTB, most patients may be asymptomatic and abnormal urinalyses are detected only as part of routine clinical evaluations or incidentally in the hospital while undergoing treatment for unrelated disorders. The urinalysis may reveal mild proteinuria, microscopic hematuria, and leucocyturia in acidic urine. Recently, due to some symptomatic treatment, the major clinical manifestation of some RTB patients gives priority to renal disease instead of cystitis. Several cases of glomerulonephritis associated with active TB have been described in the literature.
MTB can induce both cellular immune and humoral immune responses when bacilli invade the body. Studies have shown that MTB infection can lead to the disturbances of Thl/Th2 cells, which may give rise to nephritis. On the other hand, type III allergic reactions induced by immune complexes can cause tissue injury and may serve as the pathogenesis of TB. The role of disseminated tuberculosis in the pathogenesis of glomerulonephritis has been presumed to be dependent on humoral immunity and immune complexes have been reported detectable at high levels in the active phase of disseminated tuberculosis. As a result, some patients with early RTB show clinical features of chronic nephritis, such as hematuria, proteinuria, edema, and hypertension. Very often such patients are misdiagnosed and the disease is discovered only after severe destructive lesions of the kidneys have developed. In this study, urinalyses of all patients in the CS-RTB group showed proteinuria, hematuria, and leucocyturia without any symptoms of cystitis, such as urinary frequency, dysuria, and flank pain. We did not detect deposition of immune complexes in the renal biopsy. This may be because of the low level of immune complexes in the early stages of RTB.
Therefore, diagnostic tests devoted to the rapid, sensitive, and specific identification of MTB infections has been the key element for early diagnosis of RTB.
Our investigation included four aspects:①To detect MTB DNA in the renal biopsy specimens by real time PCR.②To study the expression of secretory antigen 85 (Ag85) of MTB and interferon-gamma (IFN-γ) in the renal tissues of patients with RTB; to discuss the relationship between the expression of Ag85 and IFN-γ.③To study the expression of early secretory antigen target-6 (ESAT-6) of MTB in the renal tissues of patients with RTB.④To discuss the sensitivity and specificity of Ag85, ESAT-6 and TB-DNA in the early diagnosis of renal tuberculosis.
Materials and Methods
The patients were divided into the following groups:renal tuberculosis (RTB); non-renal tuberculosis (N-RTB); and clinically suspected renal tuberculosis (CS-RTB).
Thirty patients were selected for the RTB group who underwent unilateral nephrectomy because of RTB, which was confirmed by pathologic diagnosis. Morning urine samples were collected from the patients for MTB culture before surgery.
The N-RTB group consisted of 30 patients, including seven patients with minimal change nephritic syndrome,13 patients with mesangioproliferative glomerulonephritis, five patients with membranous nephropathy, one patient with glomerulosclerosis, and four patients with focal segmental glomerulonephritis. No patients had pulmonary or extrapulmonary TB. The tuberculin skin tests and serum TB antibody titers were negative. The patients were diagnosed with primary glomerulonephritis and the condition improved after routine therapy.
The CS-RTB group was comprised of 30 patients who had extranephric TB, hematuria, and proteinuria. The tuberculin skin tests and serum TB antibody titers were positive, while the kidney ultrasonic examination and radiologic tests did not show any abnormalities.
Patients in the N-RTB and CS-RTB groups underwent renal biopsy and morning urine samples were collected for MTB culture.
All of the patients (52 males and 38 females with a mean age of 42.5±19.5 years) were selected from patients who were admitted to our hospital between 2004 and 2007.
Urine MTB culture
Urine samples were pre-treated by decontamination with 4%(w/v) NaOH and centrifugation at 1500 g for 10 min. The sediment was used for MTB culture. MTB culture was performed using in-house made Lowenstein-Jensen (LJ) solid medium with a maximum incubation period of 8 weeks. The results were read and reported weekly. Ziehl Neelsen (ZN) staining was performed to confirm the presence of mycobacteria when colonies were noted in the LJ medium.
1. Part 1
(1)DNA isolation
Two renal biopsies were obtained in all the cases and the length of each biopsy was approximately 12 mm. One specimen was divided into three parts for three different histopathologic diagnostic procedures. The other specimen was used for DNA extraction. DNA was extracted with the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The DNA concentration was measured and 100 ng of DNA was used for a real time PCR reaction, according to the instructions of the real time PCR kit.
(2) Real-time PCR
The primers were synthesized by TaKaRa Company (Dalian, China). The recommended concentration ranges were from 0.2-1.0μM. In this study, the final concentration of primers was 0.4μM, which has been shown to be a suitable concentration in preliminary experiments.
All PCRs were performed in the ABI PRISM 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with a 384-well plate. The thermal conditions were as follows:stage 1 (initial denaturation),95℃for 1 min,1 cycle; stage 2 (PCR),95℃for 5s and 60℃for 30s,40 cycles; and stage 3 (dissociation),95℃for 15s,60℃for 1 min, and 95℃for 15s,1 cycle. Every well was loaded with a PCR mixture containing SYBR Premix Ex TaqⅡ(2×; 10μl), PCR forward primer (10μM; 0.8μl), PCR reverse primer (10μM; 0.8μl), ROX reference dye (50×; 0.4μl), DNA template (2.0μl), and dH2O (6.0μl) in a total volume of 20μl. Each sample was tested in triplicate and data were collected in the last step for analysis with the SDS software version 2.0a23 (Applied Biosystems). Thirty-five and 40 were used as cycle threshold (CT) cut-off values. For the real-time experiments, post-PCR DNA melt curve analysis was performed to assess amplification specificity. In case of ambiguity, DNA gel electrophoresis was performed.
MTB DNA amplification was performed using insertion element IS6110 as a multi-copy target for molecular detection of the MTB complex.-actin was used as a housekeeper gene to control the DNA purification procedure and the existence of possible PCR inhibitors. The amplification of TB-DNA and-actin was performed in the same reaction. For each sample, we prepared two microtubes (one for TB-DNA amplification and one for-actin). The reaction system was the same, with the exception of the primers.
2. Part 2
To study the expression of Ag85 of MTB and IFN-yin renal tissues with immunohistochemistry method and discuss the relationship between the expression of Ag85 and IFN-y.
1. Deparaffinize and rehydrate sections as follows:3×3 min with xylene; 3×2 min with 100% ethanol; 2 min with 95% ethanol,2 min with 80% ethanol,2 min with 70% ethanol, and 5 min with PBS.
2. Retrieve antigen using one the antigen retrieval methods described below.
3. Block endogenous peroxidases by soaking slides in a solution of 90% methanol/3% H2O2 for 15 min at room temperature (RT). Then wash 3×5 min with PBS.
4. Shake and wipe off excess PBS. Circle all sections with a pap pen. Add 75 ul of Blocking buffer to each section immediately. Do not touch sections with tip.
5. Incubate 1 hr to overnight at RT in a humidified chamber. Do not let the slides touch each other.
6. Dilute primary antibody in Blocking buffer (dilutions will vary depending on tested antibody). Add 75 ml per section and incubate 1 hr to overnight at RT in a humidified chamber.
7. Drain primary antibody off section. Wash slides 3 x 10 min in PBS. You may have to wash slides in PBS+0.1%-0.5% Tween-20 for some primary antibodies.
8. Dilute secondary antibody 1:1000 in Blocking buffer without Tween-20. Add 75 ml per section and incubate for 1 hr at RT in a humidified chamber.
9. Drain secondary antibody and wash slides 5 x 10 min in PBS+0.1% Tween-20.(For secondary antibodies that are peroxidase conjugated, go to step 11.)
10. Make ABC according to Vector protocol 30 min before time of use (mix 5 ml of PBS with 2 drops of solution A and 2 drops of solution B). Incubate samples for 45 min at RT. Wash 5 min in PBS.
11. Make DAB according to Vector protocol in ddH2O. WEAR GLOVES:Mix 5 ml ddH2O with 2 drops of buffer,4 drops of DAB and 2 drops of H2O2. (If you want a gray-black stain, add 2 drops of the Nickel solution, and mix). Add immediately to slides and wait for color change (approximately 2-10 min). Drain slides and place into ddH2O for 5 min. Dispose of DAB waste with bleach.
12. Counterstain with methyl green (1 min) or hematoxylin (3 sec). Wash 3 times with ddH2O.
13. Immediately dehydrate in 70% ethanol,80% ethanol, and 100% ethanol (one dip each).
14. Mount with Permount and seal coverslip with nail polish.
3. Part 3
To study the expression of ESAT-6 of MTB in renal tissues with immunohistochemistry method.
The same protocol as above.
Results
1. Part 1
In the RTB group, seven patients were urine MTB culture-positive. For real-time PCR,25 samples tested positive by using 35 as the CT cut-off value and 28 samples tested positive by using 40 as the CT cut-off value.
In the N-RTB group, no patients were urine MTB culture-positive. Four patients were CT35 positive (two patients with focal segmental glomerulonephritis, one patient with glomerulosclerosis, and one patient with membranous nephropathy). Thirteen patients were CT40-positive (besides the four patients above, five patients with mesangioproliferative glomerulonephritis, two patients with minimal change nephritis syndrome, one patient with membranous nephropathy, and one patient with focal segmental glomerulonephritis). Neither granulomas nor evidence of acid-fast bacilli were noted in any of the positive biopsies. We followed those patients for 12 months and no patients developed RTB.
The sensitivity and specificity of the urine MTB culture were 23.3% and 100%, respectively. The sensitivity and specificity of real-time PCR (CT 40) were 93.3% and 56.7%, respectively. When using 35 as the CT cut off value, the sensitivity and specificity were 83.3% and 86.7%, respectively. Compared with CT 40, the specificity was significantly increased (Table 3).
In the CS-RTB group, when using 35 as the CT cut-off value of real-time PCR,9 samples tested positive. While using 40 as the CT cut-off value,14 samples tested positive.
2. Part 2
The expression of Ag85 and IFN-gamma were significantly increased in renal tuberculosis group,3 cases in non-renal tuberculosis group and 18 cases in experimental group. Compared with positive group, there was no significant difference in the optical density in experimental group. While compared with renal tuberculosis group, there was significant difference in experimental group. There was a positive linear correlation between ag85 and IFN-gamma. The sensitivity of ag85 was 100% and the specificity was 75%.
3. Part 3
The expression of ESAT-6 were significantly increased in renal tuberculosis group, 4 cases in non-renal tuberculosis group and 15 cases in experimental group. Compared with renal tuberculosis group, there was no significant difference in the optical density in experimental group. While compared with non-renal tuberculosis group, there was significant difference in experimental group. The sensitivity of ESAT-6 was 100% and the specificity was 80%.
Conclusion
These data suggested that:
1. Detection of MTA DNA in renal biopsy tissues by real-time PCR is highly sensitive. Moreover, it can increase the diagnostic accuracy and provide valuable information which would complete other clinical data for the early diagnosis of RTB. Although urine MTB culture is still the gold standard for RTB diagnosis, we speculate that if real-time PCR is widely adopted within clinical practice, it will be a powerful tool for the rapid and accurate diagnosis of RTB.
2. The expression of Ag85 and IFN-gamma were significantly increased in renal tuberculosis group. There was a positive linear correlation between Ag85 and IFN-gamma. The sensitivity of Ag85 was 100% and the specificity was 75% which demonstrated that Ag85 played an important role in the early diagnosis of renal tuberculosis.
3. The expression of ESAT-6 were significantly increased in renal tuberculosis group, The sensitivity of ESAT-6 was 100% and the specificity was 80% which is higher than that of Ag85. It showed that ESAT-6 played an important role in the early diagnosis of renal tuberculosis.
引文
1 Aziz MA, Wright A. The world health organization/international union against tuberculosis and lung disease global project on surveil-lance for anti-tuberculosis drug resistance:A model for other infectious diseases [J]. Clin Infect Dis,2005,41(suppl 4):S258-S262.
2 张慧洁,陈兰举,陈名武.T淋巴细胞亚群在结核感染免疫反应中的作用；实用儿科临床杂志，2006年10月第21卷第19期1342-1343.
3 王陇德.中国结核病控制现状及展望；中华结核和呼吸杂志,2006年8月第29卷第8期505-506.
4 张敦熔.结核病新概念,中国农业科技出版社,1998年8月第一版,310页.
5 崔洪伟肖青.呈慢性肾炎综合征表现的肾结核56例分析；中国综合临床,2002年5月第18卷第5期441.
6 马祖祥,旷寿金,鲍燕敏.结核杆菌相关性肾炎的实验研究；实用预防医学,2004年8月第11卷第4期749-751.
7 盛青唐林国 肖芃等.荧光定量PCR在结核分枝杆菌检测中的应用价值,现代医院,2007年7月第7卷第7期11-12.
8 Bennani S,Haliani M,Debbagh A,et al.Urogenital tuberculosis.diagnostic aspectS.J.Urol(Paris), 1995,101:187-190.
9 Baba K, Pathak S, Sviland L, Real-time quantitative PCR in the diagnosis of tuberculosis in formalin-fixed paraffin-embedded pleural tissue in patients from a high HIV endemic area. Diagn Mol Pathol.2008; 17(2):112-7.
10张跃堂.不典型肾结核的诊断；中原医刊2003年9月第30卷第18期42-43.
11方圻.现代内科学.北京：人民卫生出版社,1996.2945-296
12邹智聪.肾结核的诊断与治疗进展：海南医学,2006年第17卷第10期134-135.
13 Fujikawa H, Shimojima Y. Estimation of viable salmonella cell numbers in meat and meat product using real-time PCR. Shokuhin Eiseigaku Zasshi.2008; 49(4):261-5.
14 Koyuturk M, Ersoz M, Altiok N. Simvastatin induces proliferation inhibition and apoptosis in C6 glioma cells via c-jun N-terminal kinase[J]. Neurosci Lett,2004,370(2-3):212-217.
15辛茶香刘珍琼熊国亮.荧光定量PCR技术检测结核分支杆菌DNA的应用价值； 国际检验医学杂志,2007年3月第28卷第3期196-201
16 Remy-Jardin M, Remy J, Giraud F, et al. Pulmonary noduies:Detection with thick-section spiral CT versus conventional CT. Radiology 1993; 187:513-520.
17武忠弼.病理学[M].第3版.北京：北京人民卫生出版社,1990：526.
18 G. Kumar, P. K. Dagur, M. Singh, et al. Diagnostic potential of Ag85C in comparison to various secretory antigens for childhood tuberculosis. Scand J Immunol.2008; 68(2):177-83.
19 Rajesh G, Jim YK, Marcos AE. Responding to market failure in tuberculosis control [J]. Science,2001,293s 1049-1051.
20 Christopher D, Brian G, Williams MA. Erasing the world's slow stain, Strategies to beat multidrug-resistant tuberculosis [J]. Science,2003,295:2042-2046.
21 Glatman-FreedmanA. Advances in antibody-mediated immunity against Mycobacterium tuberculosis implications for a novel vaccine strategy. FEMS Immunol Med Microbiol.2003 Oct 24; 39(1):9-16.
22 Bhattacharya M, Dietrich S, Mosher L, et al. Cross-containation of specimens with Mycobacterium tuberculosis:clinical significance causes and prevention. Am J Clin Pathol. 1998 Mar:109(3):324-330.
23 Sanchez-Rodriguez C, Estrada-Chavez C, Garcia-Vigil J et al. An IgG antibody response to the antigen 85 complex is associated with good outcome in Mexican Totonaca Indians with pulmonary tuberculosis. Ind J Tub Lung Dis 2002; 6:706-12.
24 Abebe F, Holm-Hansen C, Wiker HG, Bjune G. Progress in serodiagnosis of Mycobacterium tuberculosis infection. Scand J Immunol 2007; 66:176-91.
25 Ma°len H, Softeland T, Wiker HG. Antigen analysis of Mycobacterium tuberculosis H37Rv culture filtrate proteins. Scand J Immunol 2008; 67:245-52.
26 Ulmer JB, Liu MA, Montgomery DL, et al. Expression and immunogenicity of Mycobacterium tuberculosis antigen 85 by DNA vaccination.Vaccine,1997,15:792-794.
27 Van-Lume DS, Souza JR, Melo WG, et al. Preliminary results in the immunodiagnosis of tuberculosis in children based on T cell responses to ESAT-6 and PPD antigens. Mem Inst Oswaldo Cruz.2008 Jun; 103(4):401-4.
28 Ge Y, El-Naggar M, Sze SK, et al. Top down characterization of secreted proteins from Mycobacterium tuberculosis by electron capture dissociation mass spectrometry. J Am Soc Mass Spectrom,2003; 14:253-261.
29 Pai M, Riley LW, Colford JM. Interferon-γ assays in the immunodiagnosis of tuberculosis:a systematic review. Lancet Infect Dis 2004,4:761-776.
30吴学琼.结核分枝杆菌保护性抗原的研究进展；中华结核和呼吸杂志,2006年5月第29卷第5期：340.
31 Pollock J M et al.J infect Dis,1997,175:1251-1254.
32李晖等,分支杆菌研究进展[J],《国外医学》预防诊断治疗用生物制品分册,2001,24(4)：145-147.
33 宫强 刘思国.结核杆菌特异性诊断抗原的研究；中国动物检疫,2003年第20卷第6期：45-46.
34 Weldingh K, Rosenkrands I, Okkels LM, et al. Assessing the serodiagnostic potential of 35 Mycobacterium tuberculosis proteins and identification or four novel serologic antigens. J Clin Microbiol 2005,43:57-65.
35 Lalvani A, Pathan AA, McShane H, et al. Rapid detection of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells. Am J Respir Crit Care Med.2001; 163(4): 824-8.
36 Teixeira HC, Abramo C, Munk ME. Diagnostico imunologico da tuberculose:problemas estrategias para o sucesso. JBras Pneumol 2007,33:323-334.
1 Aziz MA, Wright A. The world health organization/international union against tuberculosis and lung disease global project on surveil-lance for anti-tuberculosis drug resistance:A model for other infectious diseases [J]. Clin Infect Dis,2005,41(suppl 4):S258-S262.
2 张慧洁,陈兰举,陈名武.T淋巴细胞亚群在结核感染免疫反应中的作用；实用儿科临床杂志,2006年10月第21卷第19期1342-1343.
3 王陇德.中国结核病控制现状及展望；中华结核和呼吸杂志,2006年8月第29卷第8期505-506.
4 杨佩珍,闵伟琪,康淑娟等·结核感染与自身免疫疾病[J]·中华结核和呼吸杂志,1993,16(5)：261-263.
5 李志钢,康才,王景明,等·结核免疫反应性大动脉炎22例[J]·中华结核和呼吸杂志,1998,21(8)：511-511·
6 崔洪伟肖青.呈慢性肾炎综合征表现的肾结核56例分析；中国综合临床,2002年5月第18卷第5期441.
7 马祖祥,旷寿金,鲍燕敏.结核杆菌相关性肾炎的实验研究；实用预防医学,2004年8月第11卷第4期749-751.
8 王振运,吴金平,郑建龙等.非典型肾结核误诊分析；临床误诊误治,2007年3月第20卷第3期18-19.
9 张敦熔.结核病新概念,中国农业科技出版社,1998年8月第一版,310页.
10 Cole ST, Brosch R, Parkhill J, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998,393:537-544.
11 Ramakrishnan L, Federspiel NA, Falkow S. Granuloma-specific expression of mycobacterium virulence proteins from the glycine-rich PE-PGRS family. Science,2000,288:1430-1439.
12 Delogu G, Brennan MJ. Comparative immune response to PE and PE-PGRS antigens ofMycobacterium tuberculosis. Infect Immun,2001,59:5606-5611.
13王琳,史常旭.结核分枝杆菌的生物学特性与女性生殖器结核的发病机制；实用妇产科杂志2006年11月第22卷第11期641-642.
14 Flynn JL, Chan J. Tuberculosis:latency and reactivation. Infect Immun,2001,69:4195-4201.
15 Cooper AM, Dalton DK, StewartTA, et al. Disseminated tuberculosis in interferon-ygene-disrupted mice. J Exp Med,1993,178:2243-2247.
16 Roach DR, Briscoe H, Saunders B, et al. Secreted lymphetoxin-ais essential for the control of an intracellular bacterial infection. J Exp Med,2001,193:239-246.
17 Feldmann M, Maini RN. Anti-TNF-αtherapy of rheumatoid arthritis:what have we learned? Annu Rev Immunol,2001,19:163-196.
18 Kaufmann SHE. How can immunology contribute to the control of tuberculosis. Nat Rev Immun,2001,1:20-30.
19 Lewinsohn DM, Briden AL, Reed SG, et al. Mycobacterium tuberculosis reactive CD8+T lymphocyts:the relative contribution of classical versus nonclassical HLA restriction. J Immunol,2000,165:925-930.
20 Park SH, Bendelac A. CD1-restricted T-cell responses and microbial infection. Nature,2000, 406:788-792.
21 Shen Y, Zhou D, Qiu L, et al. Adaptive immune response of Vy2V82+T cells duringmycobacterial infections. Science,2002,295:2255-2258.
22 Andersen P, Andersen AB, Sorensen AL, et al. Recall of long-lived immunity to Mycobacterium tuberculosis infection in mice. J Immunol,1995,154:3359-3372.
23 Ge Y, El-Naggar M, Sze SK, et al. Top down characterization of secreted proteins from Mycobacterium tuberculosis by electron capture dissociation mass spectrometry. J Am Soc Mass Spectrom,2003,14:253-261.
24陆宇,朱莉贞,段连山结核分支杆菌抗原85复合物的研究及应用前景；中华结核和呼吸杂志,2003年8月第26卷第8期481.
25 Belisle JT, Vissa VD, Sievert T, et al. Role of the major antigen of my cubaeterium tuberculosis in cell wall biogenesis. Science,1997,276(5317):1420
26 lazes E, Huygen K, Content J, et al. Immunogenicity and efficacy of a tuberculosis DNA vaccine encoding the components of the secreted antigen 85 complex. Vaccine,1997,15(8): 830
27 Bentley Hibbert SI, Quan X, Newrnan T, et al, pathophysiology of antigen 85 in patients with active tuberculosis:antigen 85 circulates, complexes withfibronectin and immunoglobulin G Infect Immun,1999,67(2):581
28. Harth G, Lee BY, Wang J, et al. Novel insights into the genetics, biochemistry, and immunocytochemistry of the 30-kilodalton majorextracellular protein of Mycobacterium tuberculosis. Infect Immun,1996,64:3038-3047.
29 MustafaAS, Shaban FA, AbalAT, et al. Identification and HLA restriction of naturally derived Thl-cell epitopes from the secreted mycobacterium tuberculosis antigen 85B recognized by antigen-specific human CD+4T-cell lines. Infect Immun,2000,68:3933-3940.
30 Ulmer JB, Liu MA, Montgomery DL, et al. Expression and immunogenicity of Mycobacterium tuberculosis antigen 85 by DNA vaccination.Vaccine,1997,15:792-794.
31 Buddle B M. Use of esat-6 in the IFN-γ test for diagnosis of bovine tuberculosis following skin testing.Vet Microbiology,2001,80:37-46.
32 Pollock J M et al.J infect Dis,1997,175:1251-1254.
33 李晖等,分支杆菌研究进展[J],《国外医学》预防诊断治疗用生物制品分册,2001,24(4)：
145-147.
34 Dillon DC, AldersonMR, Day CH, et al. Molecular and immunological characterization of Mycobacterium tuberculosisCFP10, an immunodiagnostic antigen missing in Mycobacterium bovis BCG[J]. J Clin Microbio,l 2000,38(9):3285-3290.
35 VordermeierHM, WhelanA, Cockle PJ, et al. Use of synthetic petides derived from the antigen sESAT-6 and CFP10 for different diagnosis of bovine tuberculosis in cattle [J].Clin Diagn Lab Immnol 2001,8(3):571-578.
36 Sorensen AL, Nagai S, Houen G, et al. Purification and characterization of low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis [J]. Infect Immun,1995,63(5): 1710-1717.
37 Lefevre P, Braibant M, de Wit L, et al. Three different putative phosphate transport receptors are encoded by the Mycobacterium tuberculosis genome and are present at the surface of Mycobacterium bovis BCG.J Bacteriol,1997,179:2900-2906.
38 Tanghe A, Lefevre P, Denis O, et al. Immunogenicity and protective efficacy of tuberculosis DNA vaccines encoding putative phosphate transport receptors. J Immunol,1999,162: 1113-1119.
39 Skeiky YA, Lodes MJ, Guderian JA, et al. Cloning, expression, and immunological evaluation of two putative secreted serine protease antigens of Mycobacterium tuberculosis. Infect Immun, 1999,67:3998-4007.
40 XU DZ, HUANG K L, ZHAO K, et al. Vaccination with recombinant HBsAg-HBIG complex in healthy adults [J].Vaccine,2005,23(20):2658-2664.
41 PATRICK MARCELLIN,GEORGE K K LAU, FERRUCCIO BONINO, et al. Peginterferon alfa-2a alone, lamivudine alone, and the two in combination in patients with HBeAg-Negative chronic hepatitis B [J]. N Engl J Med,2004,35(12):12061.
42杨汉春.动物免疫学[M].2版：北京：中国农业出版社,2004：98.
43 Kwakami K, Qureshi M H, Zhang T T, et al. Interferon-gamma (IFN-γ)-dependent protection and synthesis of chemoattractants for mononuclear leucocytes caused by IL-12 in the lungs of mice infected withCryptococcus neoformans [J]. Clin Exl Immunol,1999,117(1):113-122.
44 Murray H W. Interferon-gamma and host antimicrobial defense:current and future clinical applications [J]. Am J Med,1994,97(5):459-467.
45 Rajesh G, Jim YK, Marcos AE. Responding to market failure in tuberculosis control [J]. Science,2001,293s 1049-1051.
46 Christopher D, Brian G, Williams MA. Erasing the world's slow stain, Strategies to beat multidrug-resistant tuberculosis [J]. Science,2003,295:2042-2046.
47陈敬,董德琼,杨渝洁,等.白细胞介素12对结核病患者TH-1／TH-2平衡的影响[J].中华结核和呼吸杂志,2006,29(5)：292-295.
48 Hwang S A, KruzelM L, Actor JK. Lactoferrin augments BCG vaccine efficacy to generate T helper response and subsequent protection against challenge with vivulentmycobacterium tuberculosis[J]. Int lmmunopharmaco,2005,5(3):591-599.
49 Feng C G, Jankovic D, Kul lbergM, et al. Maintenance of Pulmonary Thl EffectorFunction in Chronic Tuberculosis Requires Persistent IL-12 Production [J]. J Immuno,2005,174 (7): 4185-4192.
50 LascoTM, YamamotoT, YoshimuraT. EffectofMycobacterium bovis BCG vaccination on Mycobacterium specific cellular prol-iferation and tumor necrosis factor alpha production from distinct guinea pig leukocyte populations [J]. Infect Immun,2003,71(12):7035-7042.
51 Saunders B M, Briscoe H, Britton W J, et al. T cell-derived tumour necrosis factor is essentia, butnot sufficient, forprotection againstMycobacterium tuberculosis infection[J]. Clin Exp Immuno,2004,137(2):279-287.
52 Ehlers S J, Benini S, Kutsch R, et al. Fatal granuloma necrosis without exacerbated-mycobacterial growth in tumor necrosis factor receptor p55gene-deficientmice intravenously infected with Mycobacterium avium [J]. Infect Immun,2000,68(7):3571-3579.
53 EngeleM E, StobetaelK, Castiglione N, et al. Induction of TNF in human alveolar-macrophages as a potentialevasionmechanism of virulentMycobacterium tuberculosis[J]. J Immun,2006,172:1328-1337.
54 PriceNM, GilmanRH, Uddin J, et al. Unopposedmatrixmetal-loproteinase-9 expression in human tuberculosis granuloma and the role of TNF-alpha dependentmonocyte networks[J]. J Im-muno,2003,171(10):5579-5586.
55 Demissie A, AbebeM, Aseffa A, et al. Healthy individuals that control a latent infection with mycobacterium tuberculosis express high levels of Thl cytokines and the IL-4 antagonist IL-4 delta2 [J]. J Immuno,2006,174(11):6938-6943.
56 SeahG T, RookG A. IL-4 influence apoptosis of mycobacterium reactive lymphocytes in the presence of TNF-alpha [J]. J Immuno,2001,137(3); 1230-1237.
57 Swanination S, Gong J, ZhangM, et al. Cytokine production in children with tuberculosis infection and disease [J]. Clin Infect Dis,2000,29(6):1290-1293.
58 North R J.Mice in capable of making IL-4 or IL-10 display normal resistance to infectionwithmycobacterium tuberculosis [J]. Clin Exp Immuno,1998,113(1):55-58.
59 Leal IS, Smedegard B, Andersen P, et al. interleukin-6 and interleukin-12 participate in induction of a type 1 protective T-cell response during vaccination with a tuberculosis subunit vaccine[J]. Infect Immun,1999,67(11):5747-5754.
60 LadelCH, Blum C, DreherA, et al. Lethal tuberculosis in interleukin-6 deficient mutantmice [J]. Infect Immun,1997,65; (11):4843-4849.
61 BernadetteM, Saddners, Anthony A. Interleukin-6 induces early gamma interferon production in the infected lung but is not required generation ofspecific immunity tomycobacterium
tuberculosis infection [J]. Infect Immun,2006,74(6):3322-3326.
62 De la Barrera S, AlemanM, Musella R, et al. IL-10 down-regulate costimulatory molecule on mycobacterium tuberculosis pulsedmacrophages and impair the lytic activity ofCD4and CD8 CTL in tuberculosis patlents [J]. Clin Exp Immuno,12004,138 (1):128-138.
63 Tuner J, Ginzalez-JuarreroM, EllisD L. In vivo IL-10 production reactivates chronic pulmonary tuberculosis in C57BL/6 mice [J]. J Immuno,2006,173(11):6343-6351.
64 Fortsch D, RollinghoffM, StengerS, et al. IL-10 converts human dendritic cells intomacrophage-like cellswith increased antibacterial activity against virulentMycobacterium tuberculosis [J]. J Immuno,2000,165(2):978-987.
65陈灏珠.实用内科学.第10版.北京：人民卫生出版社,1997.472.
66 Chijioke A. Current concepts on pathogenesis of renal tuberculosis. West Afr J Med.2001 Apr-Jun; 20(2):107-110
67 Glatman-FreedmanA. Advances in antibody-mediated immunity against Mycobacterium tuberculosis implications for a novel vaccine strategy. FEMS Immunol Med Microbiol.2003 Oct 24; 39(1):9-16
68 Bhattacharya M, Dietrich S, Mosher L, et al. Cross-containation of specimens with Mycobacterium tuberculosis:clinical significance causes and prevention. Am J Clin Pathol. 1998 Mar:109(3):324-330.
69 Matssuzawa N, Nakabayashi K. Nagasawa T, et al. Nephrotic IgA nephropathy associated with disseminated tuberculosis. Clin Nephrol.2002 Jan; 57(1):63-68.
70 Chijioke A. Current concepts on pathogenesis of renal tuberculosis. West Afr J Med.2001 Apr-Jun; 20(2):107-110.
71 Mercadal L, Martinez F, Barrou B et al. Retroperitoneal fibrosis and membranous nephropathy. Clin Nephrol.2000 Jan; 53(1):71-74.
72 Review Pecchini F, Bufano G, Ghiringhelli P. Membranoproliferative glomerulonephritis secondary to tuberculosis. Clin Nephrol.1997 Jan; 47(1):63-64.
73 Thomashow DF, Huang Z, Sanchez MA, et al. Granulomatous panniculitis associated with crescentic glomerulonephritis. Nephron.1994; 67(3):374-376.
74 Kala U, Milner LS, Jacobs D, et al. Impacdt of tuberculosis in children with idiopathic nephritic syndrome. Pediatr Nephrol.1993 Aug; 7(4):392-395.
75 Sopena B, Sobrado J, Javier Perez A, et al. Rapidly progressive glomerulonephritis and pulmonary tuberculosis. Nephron.1991; 57(2):232-235.
76 Pbrien AA, Kelly P, Gaffney EF, et al. Immune complex glomerulonephritis secondary to tuberculosis. Ir J Med Sci.1990 Jun; 159 (6):187.
77 Rodriguez-Garcia JL, Fraile G, Mampaso F, et al. Pulmonary tuberculosis associated with membranous nephropathy. Nephron.1990; 55(2):218-219.
78 Peene I, Meheus L, Veys EM, et al. Diagnostic associations in a large and consecutively identified population positive for anti-SSA and/or anti-SSB:the range of associated diseases differs according to the detailed serotype. Ann Rheum Dis.2002 Dec; 61(12):1090-1094.
79 Elkayam O, Gat A, Lidgi M, et al. Atyoical cutaneous findings in a patient with systemic lupus erythematosus. Lupus.2003; 12(5):413-417.
80 Cooper GS, Dookey MA, Treadwell EL, et al. Risk factors for development of systemic lupus erythematosus:allergies, infections, and family history. J Clin Epidemiol.2002 Oct:55(10): 982-989.
81 Dong X, Wang J, Kabir FN, et al. Autoantibodies to DEK oncoprotein in human inflammatory disease. Arthritis Rheum.2000 Jan; 43(1):85-93.
82 Rovensky J, Kovalancik M, Kristufek P, et al. Contribution to the problem of occurrence of tuberculosis in patients with systemic lupus erythematosus. Z Rheumatol.1996 May-Jun; 55(3):180-187.
83 Tasneem S, Islam N, Ali R. Crossreactivity of SLE autoantibodies with 70 kDa heat shock proteins of Mycobacterium tuberculosis. Microbiol Ummunol.2001; 45(12):841-846.
84张跃堂.不典型肾结核的诊断；中原医刊2003年9月第30卷第18期42-43.
85阿孜古丽,梁兰青.警惕肾结核的误诊误治；临床误诊误治,2006年5月第19卷第6期69.
86邹智聪.肾结核的诊断与治疗进展；海南医学,2006年第17卷第10期134-135.
87方圻.现代内科学.北京：人民卫生出版社,1996.2945-296.
88 Bennani S, Haliani M, Debbagh A, et al. Urogenital tuberculosis, diagnostic aspectS. J. Urol(Paris),1995,101:187-190.
89廖利民,石炳毅,梁春泉等.聚合酶链反应技术诊断泌尿系结核[J].中华泌尿外科杂志,1995,16：41-43.
90 MunkME, Orme IM, SaxenaRK-Use of ESAT-6 and CFP-10 antigens for diagnosis of extra pulmonary tuberculosis [J]. J Infect Dis,2001,183(1):175-176.
91 Remy-Jardin M, Remy J, Giraud F, et al. Pulmonary noduies:Detection with thick-section spiral CT versus conventional CT. Radiology 1993; 187:513-520.
92武忠弼.病理学[M].第3版.北京：北京人民卫生出版社,1990：526.
93盛青,唐林国,肖,芃等.荧光定量PCR在结核分枝杆菌检测中的应用价值；现代医院,2007年7月第7卷第7期,11-12.
94 Koyuturk M, Ersoz M, Altiok N. Simvastatin induces proliferation inhibition and apoptosis in C6 glioma cells via c-jun N-terminal kinase[J]. Neurosci Lett,2004,370(2-3):212-217.
95 Shellman YG, Ribble D, Miller L, et al. Lovastatin-induced apoptosis in human melanoma cell lines [J]. Melanoma Res,2005,15(2):83-89.
96辛茶香,刘珍琼,熊国亮.荧光定量PCR技术检测结核分支杆菌DNA的应用价值；国际 检验医学杂志,2007年3月第28卷第3期,196-201.
2 张慧洁,陈兰举,陈名武.T淋巴细胞亚群在结核感染免疫反应中的作用；实用儿科临床杂志，2006年10月第21卷第19期1342-1343.
3 王陇德.中国结核病控制现状及展望；中华结核和呼吸杂志,2006年8月第29卷第8期505-506.
4 张敦熔.结核病新概念,中国农业科技出版社,1998年8月第一版,310页.
5 崔洪伟肖青.呈慢性肾炎综合征表现的肾结核56例分析；中国综合临床,2002年5月第18卷第5期441.
6 马祖祥,旷寿金,鲍燕敏.结核杆菌相关性肾炎的实验研究；实用预防医学,2004年8月第11卷第4期749-751.
7 盛青唐林国 肖芃等.荧光定量PCR在结核分枝杆菌检测中的应用价值,现代医院,2007年7月第7卷第7期11-12.
8 Bennani S,Haliani M,Debbagh A,et al.Urogenital tuberculosis.diagnostic aspectS.J.Urol(Paris), 1995,101:187-190.
9 Baba K, Pathak S, Sviland L, Real-time quantitative PCR in the diagnosis of tuberculosis in formalin-fixed paraffin-embedded pleural tissue in patients from a high HIV endemic area. Diagn Mol Pathol.2008; 17(2):112-7.
10张跃堂.不典型肾结核的诊断；中原医刊2003年9月第30卷第18期42-43.
11方圻.现代内科学.北京：人民卫生出版社,1996.2945-296
12邹智聪.肾结核的诊断与治疗进展：海南医学,2006年第17卷第10期134-135.
13 Fujikawa H, Shimojima Y. Estimation of viable salmonella cell numbers in meat and meat product using real-time PCR. Shokuhin Eiseigaku Zasshi.2008; 49(4):261-5.
14 Koyuturk M, Ersoz M, Altiok N. Simvastatin induces proliferation inhibition and apoptosis in C6 glioma cells via c-jun N-terminal kinase[J]. Neurosci Lett,2004,370(2-3):212-217.
15辛茶香刘珍琼熊国亮.荧光定量PCR技术检测结核分支杆菌DNA的应用价值； 国际检验医学杂志,2007年3月第28卷第3期196-201
16 Remy-Jardin M, Remy J, Giraud F, et al. Pulmonary noduies:Detection with thick-section spiral CT versus conventional CT. Radiology 1993; 187:513-520.
17武忠弼.病理学[M].第3版.北京：北京人民卫生出版社,1990：526.
18 G. Kumar, P. K. Dagur, M. Singh, et al. Diagnostic potential of Ag85C in comparison to various secretory antigens for childhood tuberculosis. Scand J Immunol.2008; 68(2):177-83.
19 Rajesh G, Jim YK, Marcos AE. Responding to market failure in tuberculosis control [J]. Science,2001,293s 1049-1051.
20 Christopher D, Brian G, Williams MA. Erasing the world's slow stain, Strategies to beat multidrug-resistant tuberculosis [J]. Science,2003,295:2042-2046.
21 Glatman-FreedmanA. Advances in antibody-mediated immunity against Mycobacterium tuberculosis implications for a novel vaccine strategy. FEMS Immunol Med Microbiol.2003 Oct 24; 39(1):9-16.
22 Bhattacharya M, Dietrich S, Mosher L, et al. Cross-containation of specimens with Mycobacterium tuberculosis:clinical significance causes and prevention. Am J Clin Pathol. 1998 Mar:109(3):324-330.
23 Sanchez-Rodriguez C, Estrada-Chavez C, Garcia-Vigil J et al. An IgG antibody response to the antigen 85 complex is associated with good outcome in Mexican Totonaca Indians with pulmonary tuberculosis. Ind J Tub Lung Dis 2002; 6:706-12.
24 Abebe F, Holm-Hansen C, Wiker HG, Bjune G. Progress in serodiagnosis of Mycobacterium tuberculosis infection. Scand J Immunol 2007; 66:176-91.
25 Ma°len H, Softeland T, Wiker HG. Antigen analysis of Mycobacterium tuberculosis H37Rv culture filtrate proteins. Scand J Immunol 2008; 67:245-52.
26 Ulmer JB, Liu MA, Montgomery DL, et al. Expression and immunogenicity of Mycobacterium tuberculosis antigen 85 by DNA vaccination.Vaccine,1997,15:792-794.
27 Van-Lume DS, Souza JR, Melo WG, et al. Preliminary results in the immunodiagnosis of tuberculosis in children based on T cell responses to ESAT-6 and PPD antigens. Mem Inst Oswaldo Cruz.2008 Jun; 103(4):401-4.
28 Ge Y, El-Naggar M, Sze SK, et al. Top down characterization of secreted proteins from Mycobacterium tuberculosis by electron capture dissociation mass spectrometry. J Am Soc Mass Spectrom,2003; 14:253-261.
29 Pai M, Riley LW, Colford JM. Interferon-γ assays in the immunodiagnosis of tuberculosis:a systematic review. Lancet Infect Dis 2004,4:761-776.
30吴学琼.结核分枝杆菌保护性抗原的研究进展；中华结核和呼吸杂志,2006年5月第29卷第5期：340.
31 Pollock J M et al.J infect Dis,1997,175:1251-1254.
32李晖等,分支杆菌研究进展[J],《国外医学》预防诊断治疗用生物制品分册,2001,24(4)：145-147.
33 宫强 刘思国.结核杆菌特异性诊断抗原的研究；中国动物检疫,2003年第20卷第6期：45-46.
34 Weldingh K, Rosenkrands I, Okkels LM, et al. Assessing the serodiagnostic potential of 35 Mycobacterium tuberculosis proteins and identification or four novel serologic antigens. J Clin Microbiol 2005,43:57-65.
35 Lalvani A, Pathan AA, McShane H, et al. Rapid detection of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells. Am J Respir Crit Care Med.2001; 163(4): 824-8.
36 Teixeira HC, Abramo C, Munk ME. Diagnostico imunologico da tuberculose:problemas estrategias para o sucesso. JBras Pneumol 2007,33:323-334.
1 Aziz MA, Wright A. The world health organization/international union against tuberculosis and lung disease global project on surveil-lance for anti-tuberculosis drug resistance:A model for other infectious diseases [J]. Clin Infect Dis,2005,41(suppl 4):S258-S262.
2 张慧洁,陈兰举,陈名武.T淋巴细胞亚群在结核感染免疫反应中的作用；实用儿科临床杂志,2006年10月第21卷第19期1342-1343.
3 王陇德.中国结核病控制现状及展望；中华结核和呼吸杂志,2006年8月第29卷第8期505-506.
4 杨佩珍,闵伟琪,康淑娟等·结核感染与自身免疫疾病[J]·中华结核和呼吸杂志,1993,16(5)：261-263.
5 李志钢,康才,王景明,等·结核免疫反应性大动脉炎22例[J]·中华结核和呼吸杂志,1998,21(8)：511-511·
6 崔洪伟肖青.呈慢性肾炎综合征表现的肾结核56例分析；中国综合临床,2002年5月第18卷第5期441.
7 马祖祥,旷寿金,鲍燕敏.结核杆菌相关性肾炎的实验研究；实用预防医学,2004年8月第11卷第4期749-751.
8 王振运,吴金平,郑建龙等.非典型肾结核误诊分析；临床误诊误治,2007年3月第20卷第3期18-19.
9 张敦熔.结核病新概念,中国农业科技出版社,1998年8月第一版,310页.
10 Cole ST, Brosch R, Parkhill J, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998,393:537-544.
11 Ramakrishnan L, Federspiel NA, Falkow S. Granuloma-specific expression of mycobacterium virulence proteins from the glycine-rich PE-PGRS family. Science,2000,288:1430-1439.
12 Delogu G, Brennan MJ. Comparative immune response to PE and PE-PGRS antigens ofMycobacterium tuberculosis. Infect Immun,2001,59:5606-5611.
13王琳,史常旭.结核分枝杆菌的生物学特性与女性生殖器结核的发病机制；实用妇产科杂志2006年11月第22卷第11期641-642.
14 Flynn JL, Chan J. Tuberculosis:latency and reactivation. Infect Immun,2001,69:4195-4201.
15 Cooper AM, Dalton DK, StewartTA, et al. Disseminated tuberculosis in interferon-ygene-disrupted mice. J Exp Med,1993,178:2243-2247.
16 Roach DR, Briscoe H, Saunders B, et al. Secreted lymphetoxin-ais essential for the control of an intracellular bacterial infection. J Exp Med,2001,193:239-246.
17 Feldmann M, Maini RN. Anti-TNF-αtherapy of rheumatoid arthritis:what have we learned? Annu Rev Immunol,2001,19:163-196.
18 Kaufmann SHE. How can immunology contribute to the control of tuberculosis. Nat Rev Immun,2001,1:20-30.
19 Lewinsohn DM, Briden AL, Reed SG, et al. Mycobacterium tuberculosis reactive CD8+T lymphocyts:the relative contribution of classical versus nonclassical HLA restriction. J Immunol,2000,165:925-930.
20 Park SH, Bendelac A. CD1-restricted T-cell responses and microbial infection. Nature,2000, 406:788-792.
21 Shen Y, Zhou D, Qiu L, et al. Adaptive immune response of Vy2V82+T cells duringmycobacterial infections. Science,2002,295:2255-2258.
22 Andersen P, Andersen AB, Sorensen AL, et al. Recall of long-lived immunity to Mycobacterium tuberculosis infection in mice. J Immunol,1995,154:3359-3372.
23 Ge Y, El-Naggar M, Sze SK, et al. Top down characterization of secreted proteins from Mycobacterium tuberculosis by electron capture dissociation mass spectrometry. J Am Soc Mass Spectrom,2003,14:253-261.
24陆宇,朱莉贞,段连山结核分支杆菌抗原85复合物的研究及应用前景；中华结核和呼吸杂志,2003年8月第26卷第8期481.
25 Belisle JT, Vissa VD, Sievert T, et al. Role of the major antigen of my cubaeterium tuberculosis in cell wall biogenesis. Science,1997,276(5317):1420
26 lazes E, Huygen K, Content J, et al. Immunogenicity and efficacy of a tuberculosis DNA vaccine encoding the components of the secreted antigen 85 complex. Vaccine,1997,15(8): 830
27 Bentley Hibbert SI, Quan X, Newrnan T, et al, pathophysiology of antigen 85 in patients with active tuberculosis:antigen 85 circulates, complexes withfibronectin and immunoglobulin G Infect Immun,1999,67(2):581
28. Harth G, Lee BY, Wang J, et al. Novel insights into the genetics, biochemistry, and immunocytochemistry of the 30-kilodalton majorextracellular protein of Mycobacterium tuberculosis. Infect Immun,1996,64:3038-3047.
29 MustafaAS, Shaban FA, AbalAT, et al. Identification and HLA restriction of naturally derived Thl-cell epitopes from the secreted mycobacterium tuberculosis antigen 85B recognized by antigen-specific human CD+4T-cell lines. Infect Immun,2000,68:3933-3940.
30 Ulmer JB, Liu MA, Montgomery DL, et al. Expression and immunogenicity of Mycobacterium tuberculosis antigen 85 by DNA vaccination.Vaccine,1997,15:792-794.
31 Buddle B M. Use of esat-6 in the IFN-γ test for diagnosis of bovine tuberculosis following skin testing.Vet Microbiology,2001,80:37-46.
32 Pollock J M et al.J infect Dis,1997,175:1251-1254.
33 李晖等,分支杆菌研究进展[J],《国外医学》预防诊断治疗用生物制品分册,2001,24(4)：
145-147.
34 Dillon DC, AldersonMR, Day CH, et al. Molecular and immunological characterization of Mycobacterium tuberculosisCFP10, an immunodiagnostic antigen missing in Mycobacterium bovis BCG[J]. J Clin Microbio,l 2000,38(9):3285-3290.
35 VordermeierHM, WhelanA, Cockle PJ, et al. Use of synthetic petides derived from the antigen sESAT-6 and CFP10 for different diagnosis of bovine tuberculosis in cattle [J].Clin Diagn Lab Immnol 2001,8(3):571-578.
36 Sorensen AL, Nagai S, Houen G, et al. Purification and characterization of low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis [J]. Infect Immun,1995,63(5): 1710-1717.
37 Lefevre P, Braibant M, de Wit L, et al. Three different putative phosphate transport receptors are encoded by the Mycobacterium tuberculosis genome and are present at the surface of Mycobacterium bovis BCG.J Bacteriol,1997,179:2900-2906.
38 Tanghe A, Lefevre P, Denis O, et al. Immunogenicity and protective efficacy of tuberculosis DNA vaccines encoding putative phosphate transport receptors. J Immunol,1999,162: 1113-1119.
39 Skeiky YA, Lodes MJ, Guderian JA, et al. Cloning, expression, and immunological evaluation of two putative secreted serine protease antigens of Mycobacterium tuberculosis. Infect Immun, 1999,67:3998-4007.
40 XU DZ, HUANG K L, ZHAO K, et al. Vaccination with recombinant HBsAg-HBIG complex in healthy adults [J].Vaccine,2005,23(20):2658-2664.
41 PATRICK MARCELLIN,GEORGE K K LAU, FERRUCCIO BONINO, et al. Peginterferon alfa-2a alone, lamivudine alone, and the two in combination in patients with HBeAg-Negative chronic hepatitis B [J]. N Engl J Med,2004,35(12):12061.
42杨汉春.动物免疫学[M].2版：北京：中国农业出版社,2004：98.
43 Kwakami K, Qureshi M H, Zhang T T, et al. Interferon-gamma (IFN-γ)-dependent protection and synthesis of chemoattractants for mononuclear leucocytes caused by IL-12 in the lungs of mice infected withCryptococcus neoformans [J]. Clin Exl Immunol,1999,117(1):113-122.
44 Murray H W. Interferon-gamma and host antimicrobial defense:current and future clinical applications [J]. Am J Med,1994,97(5):459-467.
45 Rajesh G, Jim YK, Marcos AE. Responding to market failure in tuberculosis control [J]. Science,2001,293s 1049-1051.
46 Christopher D, Brian G, Williams MA. Erasing the world's slow stain, Strategies to beat multidrug-resistant tuberculosis [J]. Science,2003,295:2042-2046.
47陈敬,董德琼,杨渝洁,等.白细胞介素12对结核病患者TH-1／TH-2平衡的影响[J].中华结核和呼吸杂志,2006,29(5)：292-295.
48 Hwang S A, KruzelM L, Actor JK. Lactoferrin augments BCG vaccine efficacy to generate T helper response and subsequent protection against challenge with vivulentmycobacterium tuberculosis[J]. Int lmmunopharmaco,2005,5(3):591-599.
49 Feng C G, Jankovic D, Kul lbergM, et al. Maintenance of Pulmonary Thl EffectorFunction in Chronic Tuberculosis Requires Persistent IL-12 Production [J]. J Immuno,2005,174 (7): 4185-4192.
50 LascoTM, YamamotoT, YoshimuraT. EffectofMycobacterium bovis BCG vaccination on Mycobacterium specific cellular prol-iferation and tumor necrosis factor alpha production from distinct guinea pig leukocyte populations [J]. Infect Immun,2003,71(12):7035-7042.
51 Saunders B M, Briscoe H, Britton W J, et al. T cell-derived tumour necrosis factor is essentia, butnot sufficient, forprotection againstMycobacterium tuberculosis infection[J]. Clin Exp Immuno,2004,137(2):279-287.
52 Ehlers S J, Benini S, Kutsch R, et al. Fatal granuloma necrosis without exacerbated-mycobacterial growth in tumor necrosis factor receptor p55gene-deficientmice intravenously infected with Mycobacterium avium [J]. Infect Immun,2000,68(7):3571-3579.
53 EngeleM E, StobetaelK, Castiglione N, et al. Induction of TNF in human alveolar-macrophages as a potentialevasionmechanism of virulentMycobacterium tuberculosis[J]. J Immun,2006,172:1328-1337.
54 PriceNM, GilmanRH, Uddin J, et al. Unopposedmatrixmetal-loproteinase-9 expression in human tuberculosis granuloma and the role of TNF-alpha dependentmonocyte networks[J]. J Im-muno,2003,171(10):5579-5586.
55 Demissie A, AbebeM, Aseffa A, et al. Healthy individuals that control a latent infection with mycobacterium tuberculosis express high levels of Thl cytokines and the IL-4 antagonist IL-4 delta2 [J]. J Immuno,2006,174(11):6938-6943.
56 SeahG T, RookG A. IL-4 influence apoptosis of mycobacterium reactive lymphocytes in the presence of TNF-alpha [J]. J Immuno,2001,137(3); 1230-1237.
57 Swanination S, Gong J, ZhangM, et al. Cytokine production in children with tuberculosis infection and disease [J]. Clin Infect Dis,2000,29(6):1290-1293.
58 North R J.Mice in capable of making IL-4 or IL-10 display normal resistance to infectionwithmycobacterium tuberculosis [J]. Clin Exp Immuno,1998,113(1):55-58.
59 Leal IS, Smedegard B, Andersen P, et al. interleukin-6 and interleukin-12 participate in induction of a type 1 protective T-cell response during vaccination with a tuberculosis subunit vaccine[J]. Infect Immun,1999,67(11):5747-5754.
60 LadelCH, Blum C, DreherA, et al. Lethal tuberculosis in interleukin-6 deficient mutantmice [J]. Infect Immun,1997,65; (11):4843-4849.
61 BernadetteM, Saddners, Anthony A. Interleukin-6 induces early gamma interferon production in the infected lung but is not required generation ofspecific immunity tomycobacterium
tuberculosis infection [J]. Infect Immun,2006,74(6):3322-3326.
62 De la Barrera S, AlemanM, Musella R, et al. IL-10 down-regulate costimulatory molecule on mycobacterium tuberculosis pulsedmacrophages and impair the lytic activity ofCD4and CD8 CTL in tuberculosis patlents [J]. Clin Exp Immuno,12004,138 (1):128-138.
63 Tuner J, Ginzalez-JuarreroM, EllisD L. In vivo IL-10 production reactivates chronic pulmonary tuberculosis in C57BL/6 mice [J]. J Immuno,2006,173(11):6343-6351.
64 Fortsch D, RollinghoffM, StengerS, et al. IL-10 converts human dendritic cells intomacrophage-like cellswith increased antibacterial activity against virulentMycobacterium tuberculosis [J]. J Immuno,2000,165(2):978-987.
65陈灏珠.实用内科学.第10版.北京：人民卫生出版社,1997.472.
66 Chijioke A. Current concepts on pathogenesis of renal tuberculosis. West Afr J Med.2001 Apr-Jun; 20(2):107-110
67 Glatman-FreedmanA. Advances in antibody-mediated immunity against Mycobacterium tuberculosis implications for a novel vaccine strategy. FEMS Immunol Med Microbiol.2003 Oct 24; 39(1):9-16
68 Bhattacharya M, Dietrich S, Mosher L, et al. Cross-containation of specimens with Mycobacterium tuberculosis:clinical significance causes and prevention. Am J Clin Pathol. 1998 Mar:109(3):324-330.
69 Matssuzawa N, Nakabayashi K. Nagasawa T, et al. Nephrotic IgA nephropathy associated with disseminated tuberculosis. Clin Nephrol.2002 Jan; 57(1):63-68.
70 Chijioke A. Current concepts on pathogenesis of renal tuberculosis. West Afr J Med.2001 Apr-Jun; 20(2):107-110.
71 Mercadal L, Martinez F, Barrou B et al. Retroperitoneal fibrosis and membranous nephropathy. Clin Nephrol.2000 Jan; 53(1):71-74.
72 Review Pecchini F, Bufano G, Ghiringhelli P. Membranoproliferative glomerulonephritis secondary to tuberculosis. Clin Nephrol.1997 Jan; 47(1):63-64.
73 Thomashow DF, Huang Z, Sanchez MA, et al. Granulomatous panniculitis associated with crescentic glomerulonephritis. Nephron.1994; 67(3):374-376.
74 Kala U, Milner LS, Jacobs D, et al. Impacdt of tuberculosis in children with idiopathic nephritic syndrome. Pediatr Nephrol.1993 Aug; 7(4):392-395.
75 Sopena B, Sobrado J, Javier Perez A, et al. Rapidly progressive glomerulonephritis and pulmonary tuberculosis. Nephron.1991; 57(2):232-235.
76 Pbrien AA, Kelly P, Gaffney EF, et al. Immune complex glomerulonephritis secondary to tuberculosis. Ir J Med Sci.1990 Jun; 159 (6):187.
77 Rodriguez-Garcia JL, Fraile G, Mampaso F, et al. Pulmonary tuberculosis associated with membranous nephropathy. Nephron.1990; 55(2):218-219.
78 Peene I, Meheus L, Veys EM, et al. Diagnostic associations in a large and consecutively identified population positive for anti-SSA and/or anti-SSB:the range of associated diseases differs according to the detailed serotype. Ann Rheum Dis.2002 Dec; 61(12):1090-1094.
79 Elkayam O, Gat A, Lidgi M, et al. Atyoical cutaneous findings in a patient with systemic lupus erythematosus. Lupus.2003; 12(5):413-417.
80 Cooper GS, Dookey MA, Treadwell EL, et al. Risk factors for development of systemic lupus erythematosus:allergies, infections, and family history. J Clin Epidemiol.2002 Oct:55(10): 982-989.
81 Dong X, Wang J, Kabir FN, et al. Autoantibodies to DEK oncoprotein in human inflammatory disease. Arthritis Rheum.2000 Jan; 43(1):85-93.
82 Rovensky J, Kovalancik M, Kristufek P, et al. Contribution to the problem of occurrence of tuberculosis in patients with systemic lupus erythematosus. Z Rheumatol.1996 May-Jun; 55(3):180-187.
83 Tasneem S, Islam N, Ali R. Crossreactivity of SLE autoantibodies with 70 kDa heat shock proteins of Mycobacterium tuberculosis. Microbiol Ummunol.2001; 45(12):841-846.
84张跃堂.不典型肾结核的诊断；中原医刊2003年9月第30卷第18期42-43.
85阿孜古丽,梁兰青.警惕肾结核的误诊误治；临床误诊误治,2006年5月第19卷第6期69.
86邹智聪.肾结核的诊断与治疗进展；海南医学,2006年第17卷第10期134-135.
87方圻.现代内科学.北京：人民卫生出版社,1996.2945-296.
88 Bennani S, Haliani M, Debbagh A, et al. Urogenital tuberculosis, diagnostic aspectS. J. Urol(Paris),1995,101:187-190.
89廖利民,石炳毅,梁春泉等.聚合酶链反应技术诊断泌尿系结核[J].中华泌尿外科杂志,1995,16：41-43.
90 MunkME, Orme IM, SaxenaRK-Use of ESAT-6 and CFP-10 antigens for diagnosis of extra pulmonary tuberculosis [J]. J Infect Dis,2001,183(1):175-176.
91 Remy-Jardin M, Remy J, Giraud F, et al. Pulmonary noduies:Detection with thick-section spiral CT versus conventional CT. Radiology 1993; 187:513-520.
92武忠弼.病理学[M].第3版.北京：北京人民卫生出版社,1990：526.
93盛青,唐林国,肖,芃等.荧光定量PCR在结核分枝杆菌检测中的应用价值；现代医院,2007年7月第7卷第7期,11-12.
94 Koyuturk M, Ersoz M, Altiok N. Simvastatin induces proliferation inhibition and apoptosis in C6 glioma cells via c-jun N-terminal kinase[J]. Neurosci Lett,2004,370(2-3):212-217.
95 Shellman YG, Ribble D, Miller L, et al. Lovastatin-induced apoptosis in human melanoma cell lines [J]. Melanoma Res,2005,15(2):83-89.
96辛茶香,刘珍琼,熊国亮.荧光定量PCR技术检测结核分支杆菌DNA的应用价值；国际 检验医学杂志,2007年3月第28卷第3期,196-201.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |