脊柱结核课件_16

1、Monocyte Chemotactic Protein-1 -2518 Gene Polymorphism and Susceptibility to Spinal Tuberculosis 单核细胞趋化蛋白-1-2518的基因多态性与脊柱结核的敏感性。 2、Enzyme-linked immunospot assay response to recombinant CFP-10/ESAT-6 fusion protein among patients with spinal tuberculosis: implications for diagnosis and monitoring of surgical therapy 酶联免疫斑点技术对脊柱结核患者中重组CFP-10/ESAT-6 融合蛋白的反应性：诊断与外科手术治疗监测的意义。,1、Monocyte Chemotactic Protein-1 -2518 Gene Polymorphism and Susceptibility to Spinal Tuberculosis 单核细胞趋化蛋白-1-2518的基因多态性与脊柱结核的敏感性。 （Department of Spine Surgery, Xiangya Hospital, Central South University, Hunan, China Clinical Laboratory, Secondary Affiliated Hospital, Fujian Medical University, Fujian, China） Archives of Medical Research 45 (2014),Background and Aims. Monocyte chemotactic protein-1 (MCP-1) gene polymorphisms play important roles in regulating immunological reactions and may be associated with pulmonary tuberculosis. However, the relationship between the MCP-1 -2518 gene polymorphism and susceptibility to spinal tuberculosis remains unknown. 背景与目的 单核细胞趋化蛋白-1（MCP-1）基因多态性在调节免疫反应中发挥重要作用，可能与肺结核的发生密切相关。然而，在MCP-1-2518基因多态性与其对脊柱结核敏感性之间的关系尚不清楚。,We undertook this study to investigate the relationships between MCP-1 promoter 2518 genotype frequency and allele polymorphisms and susceptibility to spinal tuberculosis in a Chinese Han population. 本研究旨在探讨中国汉族人群中MCP-1基因启动子2518基因型频率和等位基因多态性与脊柱结核易感性的关系。,Methods. Patients with spinal tuberculosis and healthy volunteers were enrolled between December 2004 and December 2010. MCP-1 -2518 polymorphisms in both groups were detected using polymerase chain reaction and DNA sequencing. MCP-1 genotype was analyzed in all patients. Differences in genotype frequencies between groups were compared using tests. 方法： 选取2004年12月至2010年12之间的脊柱结核病人和 健康志愿者纳入到本研究中。两组均采用聚合酶链反应和DNA序列测定来检测MCP-1 -2518 基因多态性，所有患者的MCP-1基因型均进行了分析。采用检验来比较组间基因型频率差异。,Results. A total of 208 patients with spinal tuberculosis and 210 healthy volunteers were included. The distribution frequencies of MCP-1 -2518 GG, GA and AA genotypes were 36.1, 50.9 and 13.0%, respectively, in the case group and 25.2, 53.8 and 21.0%, respectively, in the control group (p0.05). MCP-1 -2518 GG genotype was significantly associated with the onset of spinal tuberculosis (OR=52.306, 95% CI=51.273e4.178). 结果： 共208例脊柱结核患者和210例健康志愿者纳入到本研究中。单核细胞趋化蛋白-1（MCP-1）- 2518 GG，GA和AA基因型的分布频率：在病例组分别为36.1%、50.9 %和13.0%，在对照组分别为25.2 % 、53.8 %和21.0%（P0.05）。单核细胞趋化蛋白-1 - 2518 GG基因型与脊柱结核发病率显著相关（OR=52.306，95% CI =51.273e4.178）。,The G and A allele frequencies were 61.5% and 38.5%, respectively, in the case group, and 52.1% and 47.9% in the control group (p0.05), the allele “G” of MCP-1 -2518 showed an association with an increased risk for spinal tuberculosis: OR=1.777, 95% CI=1.053-2.999,p=0.03 in the dominant model; OR=1.67, 95% CI=5 1.097-2.544,p=0.016 in the recessive model. G和A等位基因频率在病例组分别为61.5%和38.5%，在对照组分别为52.1%和47.9% (p0.05)；研究表明，单核细胞趋化蛋白-1（MCP-1）- 2518的等位基因“G”与脊柱结核发生的危险因素增加有关：在显性模式中OR = 177.7，95 % CI = 1.053-2.999，P = 0.03；在隐性模式中OR= 1.67，95 % CI = 5 1.097-2.544，P = 0.016。,Conclusions. The MCP-1 -2518 GG genotype and presence of the G allele may be associated with susceptibility to spinal tuberculosis in the Chinese Han population. 结论： 单核细胞趋化蛋白-1 - 2518 GG基因型和G等位基因的存在可能与中国汉族人群脊柱结核易感性相关。,Introduction Spinal tuberculosis is associated with high morbidity and a high disability rate. An increase in the incidence of multidrug-resistant strains of Mycobacterium tuberculosis indicates that the treatment needs of patients with spinal tuberculosis have changed. A thorough understanding of the individual factors influencing the host antituberculosis immunological reaction is therefore needed to help prevent and treat spinal tuberculosis. 引言 ： 脊柱结核具有高发病率和高致残率，在耐多药结核分枝杆菌菌株出现的增加表明脊柱结核患者的治疗需求已经改变。因此需要深入了解影响宿主的抗结核免疫反应的个体因素，从而有助于预防和治疗脊柱结核。,Monocyte chemotactic protein-1 (MCP-1) is a monocyte and macrophage chemotactic factor that induces and activates monocytes. MCP-1 can increase the free oxygen concentration in monocyte fluid to enhance the killing effects of monocytes against bacteria (1). Moreover, MCP-1 can mediate cell migration and phagocytosis and other adhesion-dependent functions by regulating cell surface adhesion molecule expression and cytokine production(2). 单核细胞趋化蛋白-1（MCP-1）是单核细胞和巨噬细胞趋化因子，能诱导和激活单核细胞。MCP-1可以增加单核细胞液体中的游离氧浓度，增强单核细胞对细菌的杀伤作用。此外，单核细胞趋化蛋白-1可通过调节细胞表面粘附分子的表达和细胞因子的产生来介导细胞迁移、吞噬作用和粘附依赖等功能。,MCP-1 can restrict the in vivo spreading of M. tuberculosis and regulate secondary immunological reactions and is thus closely related to the occurrence and progression of tuberculosis (3).A previous study demonstrated that the MCP-1 -2518A/G polymorphism was associated with increased susceptibility to pulmonary tuberculosis in the Chinese Han population(4); MCP-1可以抑制在体内结核杆菌的传播并调节二次免疫反应，从而与结核病的发生和发展密切相关。先前的研究表明，在中国汉族人群中单核细胞趋化蛋白-1 - 2518 A/G多态性与肺结核易感性的增加相关,however, the correlation between the gene polymorphism and spinal tuberculosis remains poorly understood. The present study therefore aimed to clarify the relationship between MCP-1 -2518 polymorphisms and the incidence of spinal tuberculosis in the Chinese Han population. 然而，基因多态性与脊柱结核之间的相关性仍知之甚少，因此本研究旨在阐明MCP-1 - 2518基因多态性与中国汉族人群脊柱结核的发病率之间的关系。,Subjects and Methods Participants The case group comprised new patients with spinal tuberculosis admitted to our hospital between December 2004 and December 2010. 对象和方法 试验对象：病例组包括2004年12月至2010年12月我院新收治的脊柱结核患者。,The inclusion criteria were spinal tuberculosis infection confirmed by preoperative imaging and serological examinations, preoperative lung imaging showing no obvious signs of pulmonary tuberculosis infection, exclusion of other extrapulmonary tuberculosis, pathological examination of surgical sections showing tuberculosis infection; and etiological examination positive for acid fast staining or M. tuberculosis in exudate. 纳入标准：术前通过像学和血清学检查确诊的脊柱结核性感染；术前胸片显示没有明显的肺结核感染征象；排除其他肺外结核；术中取标本经病理学检查显示为结核性感染；病原学检查抗酸染色阳性，或渗出液中找出结核分枝杆菌。,The control group comprised healthy Han individuals who underwent physical examinations at our hospital between December 2004 and December 2010 and in whom pulmonary tuberculosis, spinal tuberculosis, and other extrapulmonary tuberculosis were excluded. 对照组包括2004年12月至2010年12月之间在我们医院进行体检的健康汉族人，他们均不患有肺结核、脊柱结核及其他肺外结核。,Cases and controls with any of the following conditions were excluded from the study: cultures of multidrug-resistant M. tuberculosis, diseases affecting the immune system (infection, trauma, tumor); diseases affecting MCP-1expression (coronary artery disease, psoriasis), autoimmune disease, tuberculosis of other organs (or history of this type of disease), genetic disease, undergoing spinal surgery, or free of bacillus Calmette-Guerin (BCG) vaccination. 病例组和对照组中若有下列任何情况都予以排除：培养出耐多药结核分枝杆菌；影响免疫系统疾病（感染，外伤，肿瘤）；影响MCP-1表达水平的疾病（冠心病，银屑病）；自身免疫性疾病；其他器官有结核（或有类似疾病史）；遗传性疾病；有脊椎手术史者；或未接种卡介苗（BCG）疫苗者。,The study was approved by the Ethics Committee of Central South University, and informed consent was obtained from all participants. 这项研究得到了中南大学伦理委员会的批准，并获得了所有参与者的知情同意。 Genomic DNA Extraction Two milliliters of fasting peripheral venous blood was harvested in the morning and placed in acid citrate dextrose tubes. 基因组DNA提取 抽取受试者清晨空腹外周静脉血2mL并加入柠檬酸葡萄糖管中。,White blood cell genomic DNA was extracted using a whole-blood genomic DNA quick extraction kit (Takara, Dalian, China). DNA concentrations were determined using an ultraviolet spectrophotometer. DNA was diluted to 100 ng/uL with double distilled water and cryopreserved at -70. DNA was detected in batches after all samples were collected. 使用全血基因组DNA快速提取试剂盒提取白细胞基因组DNA（Takara，大连，中国），应用紫外线分光光度计测定DNA的浓度。用双蒸水将DNA稀释至100 ng/uL并在- 70下冷冻。所有样品采集完毕后进行DNA分批检测。,Polymerase Chain Reaction (PCR) Primers were synthesized as described previously(5): forward primer: 5-TTCTCTCACGCCAGCAC-3; reverse primer: 5-TGACTTGGCCTTTGCATATATC-3. The amplified fragment was 163 bp. PCR reaction conditions were prenaturation at 95for 5 min, 94 for 1 min, 62 for 30 sec, 72 for 1 min for 30 cycles in total, followed by extension at 72 for 10 min and annealing at 4 . 聚合酶链反应（ PCR ） 引物的合成如前所述：上游引物 ：5TTCTCTCACGCCAGCAC-3;下游引物 ：5-TGACTTGGCCTTTGCATATATC-3。扩增的DNA片段为163 bp，PCR条件为： （1）95 5min （2）94 1min （3）62 30 sec （4）72 1 min 总30个循环 （5）随后72下延伸10分钟，。 （6） 退火4,PCR Product Sequencing If the amount and specificity of the products were as expected, they were treated with shrimp alkaline phosphatase and exonuclease on ice and then incubated at 37 for 90 min and 80 for 15 min to deactivate the enzymes. In the event that the amount and specificity of products were not satisfactory (e.g., if there were confounding bands), the PCR products were retrieved and purified as follows (50uL PCR amplification system as example). PCR产物测序 如果产品的量和特异性正如预期，则在冰块上分别用虾碱性磷酸酶和核酸外切酶处理，然后孵育在3790分钟和8015 min，使酶失活。 如果产品的数量和特异性并不令人满意（例如，如果有混杂带），对PCR产物进行的回收并纯化过程如下（50uL PCR扩增系统为例）。,The target fragment was retrieved under a Vita Light lamp, placed at room temperature for 5 h, ground using a large tip, mixed with 600uL eluting buffer, mixed overnight on a shaking table at 37, and centrifuged at 16,200 g at 4 for 30 min. The supernatant was transferred to a new Eppendorf tube, mixed with 500uL absolute alcohol at -20 for 4 h, and centrifuged at 16,200 g at 4 for 10 min. 在维塔光灯下检索目标片段，在室温下放置5小时，地面使用一个大尖物，用600uL洗脱缓冲液混合后在振动台上37下搅拌整夜，在16200/g 4下离心30 min。弃上清液后转移到新的离心管中，用无水乙醇在20下搅拌4 h，在16200/g 4下离心10 min。,White precipitate was eluted with 70% alcohol, centrifuged at 540 g at 4 for 15 min, dried at room temperature for 5 min, dissolved in 10uL PCR water for 2 h and then sequenced on an ABI3100 sequencer. Sequencing was performed by Shanghai Majorbio Pharm (Shanghai, China). 白色沉淀物用70的酒精进行洗脱，在540g （4）下离心15分钟，在室温下放置5分钟使之干燥，溶解于10ul PCR水洗脱2小时后用ABI3100测序仪开始进行测序。PCR测序由上海Majorbio药业公司进行（上海，中国）。,Statistical Analysis Data were analyzed using SPSS v.13.0 software. The general conditions of the two groups were compared using t tests and tests. The genotype frequency of the control group was calculated to confirm Hardy-Weinberg equilibrium. tests were used to compare differences in genotype frequencies in each group followed by intergroup comparisons of each genotype. Odds ratios (OR) and 95% confidence intervals (CI) were calculated. The results of t tests were expressed as mean SD; p0.05 was considered statistically significant. 采用SPSS v.13.0统计软件对数据进行分析，采用t检验和试验对两组患者的一般情况进行比较。对照组的基因型频率进行了计算，以确认Hardy-Weinberg平衡。接着每个基因型的组间比较，用检验比较各组基因型频率的差异；计算比值比（或）和95%置信区间(CI) ；t 检验的结果均以M SD表示； P 0.05为差异有显著性意义。,Discussion The incidence and progression of tuberculosis is multifactorial, and host predisposing genes play important roles together with pathogen-related factors, host immune status, and socioeconomic and environmental factors(6,7). MCP-1gene polymorphisms can reduce the ability to resist M. tuberculosis infection and thus increase the risk of tuberculosis. 讨论 结核病的发生和发展是多因素的，其中宿主易感基因与病原体相关因素、宿主免疫状态、社会经济和环境因素等共同起到重要作用。MCP-1基因多态性可降低机体抗结核分枝杆菌感染的能力，从而增加患结核病的风险。,Flores-Villanueva et al. reported that the odds of developing tuberculosis were 2.3- and 5.4-fold higher in Mexican carriers of the MCP-1 genotypes GA and GG compared with AA homozygotes, and 2.8- and 6.9-fold higher in Korean carriers of the GA and GG MCP-1 genotypes compared with AA homozygotes. This was identified as the genetic factor with the strongest influence on adult tuberculosis(8). Flores-Villanueva 等人报道，墨西哥MCP-1基因型GA和GG携带者与AA纯合子相比，结核病发生的可能性分别高于2.3和5.4倍；朝鲜人MCP-1基因型GA和GG携带者与AA纯合子相比，则分别高于2.8和6.9倍。此被确定为遗传因素对成人结核病的影响最强。,Another study that focused on Tunisian patients showed that the risk of pulmonary tuberculosis or extrapulmonary tuberculosis was 3.1-fold higher in patients carrying the MCP-1 -2518 GG genotype compared with the MCP-1 -2518GA genotype(9). A meta-analysis showed that the risk of tuberculosis was increased 1.79-fold in an Asian population carrying the MCP-1 -2518 GG genotype (10), whereas another meta-analysis showed that the odds of developing tuberculosis increased 1.51-fold in carriers of the MCP-1 -2518 G allele(4). 另一项以突尼斯患者为对象的研究表明，携带MCP-1 -2518 GG 基因型的患者发生肺结核或肺外结核的风险比携带MCP-1 -2518 GA 基因型的患者高出3.1倍。一项荟萃分析表明，在携带MCP-1-2518 GG基因型的亚洲人群中发生结核病的风险增加1.79倍，而另一项荟萃分析表明，在MCP-1-2518 G同位基因携带者中发生结核病的风险增加1.79倍。,Gene changes in the MCP-1 promoter region can increase MCP-1 gene transcription and serum MCP-1 levels. Feng et al. reported that the MCP-1-2518 G allele was in linkage disequilibrium with the MCP-1+900 C allele, and that MCP-1+900 C could alter MCP-1 protein expression, resulting in susceptibility to tuberculosis (11). 在MCP-1启动子区域的基因变化可以增加MCP-1基因转录和血清MCP-1水平。 Feng等人报道，mcp-1-2518 G等位基因与MCP-1 + 900 C等位基因处于连锁不平衡状态，而MCP-1 + 900 C可以改变MCP-1蛋白的表达，从而引起结核病易感性。,The MCP-1 -2518 G allele increased binding of the TALE protein family members PREP1 and PBX2 to the MCP-1 promoter, resulting in increased transcriptional activation of MCP-1 and increased serum MCP-1 levels(12). High levels of serum MCP-1 can inhibit interleukin-12 expression and selectively inhibit helper T cell immunological reaction type I, leading to reduced resistance to M. tuberculosis (1). 在MCP-1-2518 G等位基因增加TALE蛋白家族成员PREP1和PBX2向MCP-1启动子的结合，从而引起MCP-1的转录激活和使血清MCP-1水平升高。血清高水平的MCP-1可抑制IL-12的表达和选择性地抑制辅助性I型T细胞免疫反应，从而降低机体对结核病的抵抗力。,In addition, overexpression of MCP-1 suppresses interferon- production resulting in reduced granuloma formation, loss of receptor sensitivity to the ligand, and a decreased monocyte chemotactic response (13). Serum MCP-1 can also upregulate interleukin-4 transcription, induce the transformation of Th0 cells into Th2 cells to upregulate the Th2 immunological reaction, and inhibit the Th1 immunological reaction(14). All the above processes can increase an individuals susceptibility to tuberculosis and increase the risk of M. tuberculosis infection dissemination in vivo. 此外，过度表达的MCP-1抑制干扰素-产生并导致肉芽肿形成减少，受体失去对配体的敏感性，降低单核细胞趋化反应。血清MCP-1也可上调白细胞介素-4的转录，诱导Th0细胞转变为Th2细胞上调Th2型免疫反应，并抑制Th1细胞免疫反应。上述的所有过程都会增加个体对结核病的易感性，增加体内的结核杆菌感染播散的可能性。,To date, Chinese studies have focused mainly on patents with pulmonary tuberculosis, and little is known about susceptibility to spinal tuberculosis. Schlesinger et al. found that only 33-50% of spinal tuberculosis patients also had pulmonary tuberculosis, whereas most exhibited no obvious pulmonary focus or history of pulmonary tuberculosis(15). This suggests that the factors influencing the incidence and progression of spinal tuberculosis differ from those associated with pulmonary tuberculosis. 至今，中国人的研究主要集中在肺结核患者，而对脊柱结核的敏感性研究则很少。Schlesinger et al.发现只有33-50 %的脊柱结核患者伴有肺结核，而绝大多数患者没有明显的肺部结核病灶或肺结核史。这表明影响脊柱结核发生和进展的危险因素不同于肺结核的危险因素。,Previous studies have shown that spinal tuberculosis has independent predisposing genotypes(16), and the predisposing genotypes differed between pulmonary tuberculosis and tuberculosis of the bones and joints(17). The internal environment and host immunity are important factors in the pathogenesis of spinal tuberculosis, and host genes may thus play a more important role in spinal tuberculosis compared with pulmonary tuberculosis. 先前的研究已经表明，脊柱结核具有独立的易感基因型，并且这种易感基因型与其他骨关节结核和肺结核的易感基因型不同。内部环境和宿主免疫是脊柱结核发病的重要因素，而与肺结核比较，宿主基因可能在脊柱结核发病中发挥更重要的作用。,The present study detected significant differences in MCP-1 -2518 genotype distributions between the control and case groups. The risk of developing spinal tuberculosis was increased 2.33-fold in carriers of the GG genotype. Carriers of the allele G of MCP-1-2518 also showed an association with an increased risk for spinal tuberculosis. Those indicating that the MCP-1 -2518 GG genotype and G allele may correlate with susceptibility to spinal tuberculosis in the Chinese Han population. 本研究检测到MCP-1- 2518基因型分布在对照组和实验组之间有显著性差异。 GG基因型携带者患有脊柱结核的风险会增加2.33倍。 MCP-1-2518等位基因”G“的携带者也显示其与脊柱结核风险增加有关。这些表明MCP-1 2518 GG基因型和G等位基因可能与中国汉族人群脊柱结核易感性相关。,To the best of our knowledge, no previous study has reported on the correlation between MCP-1 gene polymorphisms and susceptibility to spinal tuberculosis. However, Motsinger et al. reported that MCP-1 -2518 gene polymorphism was not associated with extrapulmonary tuberculosis(18). Several factors may account for this apparent discrepancy. Motsinger et al. recruited black subjects, and recent meta-analys