BackgroundTuberculosis (TB) remains a global public health burden, and how immune cell subsets regulate host anti-TB immunity and disease progression remains incompletely understood. While previous studies have focused on single-positive (SP) T cells (CD4+ or CD8+) in TB pathogenesis, the association between CD4+CD8+ double-positive (DP) T cells and TB susceptibility, severity, and treatment outcomes have not been fully elucidated. This study aimed to investigate the relationship between DP T cells and other immune cell subsets with TB, and to explore the potential diagnostic and prognostic value of DP T cells in active TB.MethodsA Genome-Wide Association Study (GWAS) was conducted to analyze 731 immune cell traits and a dataset encompassing 895 patients with TB. Subsequently, a cohort including 647 patients with active TB and 632 healthy controls was used to verify the findings of Mendelian randomization (MR). The correlation between the percentage of DP T cells in lymphocytes and TB severity, treatment efficacy, and Mycobacterium tuberculosis (Mtb)-specific IFN-γ production was evaluated. Finally, a random forest model incorporating the percentage of DP T cells in leukocytes and other peripheral blood parameters was constructed to distinguish severe from mild active TB.ResultsMR analysis suggested potential causal links between the percentage of DP T cells among peripheral leukocytes and TB status. Clinical sample validation showed that the percentage of peripheral DP T cell among leukocytes was significantly lower in patients with active TB than in healthy controls (P < 0.001), and was inversely correlated with disease severity. Additionally, the percentage of DP T cells in leukocytes was positively correlated with Mtb-specific antigen-stimulated IFN-γ production. Flow cytometric analysis demonstrated that DP T cells had a significantly higher frequency of IFN-γ-expressing cells compared to CD8+ SP T cells (P < 0.001). The constructed random forest model effectively distinguished severe from mild TB, with good diagnostic performance (AUC = 0.985).ConclusionsOur findings indicate that DP T cells are closely associated with TB severity, and are positively associated with Mtb-specific IFN-γ response. The percentage of peripheral DP T cells in leukocytes could serve as a potential non-invasive biomarker for TB severity stratification.

Objectives: The mechanism that leads to disseminated tuberculosis in HIV-negative patients is still largely unknown. T cell subsets and signaling pathways that were associated with disseminated tuberculosis were investigated. Methods: Single-cell profiling of whole T cells was performed to identify T cell subsets and enriched signaling pathways that were associated with disseminated tuberculosis. Flow cytometric analysis and blocking experiment were used to investigate the findings obtained by transcriptome sequencing. Results: Patients with disseminated tuberculosis had depleted Th1, Tc1 and Tc17 cell subsets, and IFNG was the most down-regulated gene in both CD4 and CD8 T cells. Gene Ontology analysis showed that non-canonical NF kappa B signaling pathway, including NFKB2 and RELB genes, was significantly down-regulated and was probably associated with disseminated tuberculosis. Expression of several TNF superfamily ligands and receptors, such as LTA and TNF genes, were suppressed in patients with disseminated tuberculosis. Blocking of TNF-alpha and soluble LT alpha showed that TNF-alpha was involved in IFN-gamma production and LT alpha influenced TNF-alpha expression in T cells. Conclusions: Impaired T cell IFN-gamma response mediated by suppression of TNF and non-canonical NF-kappa B signaling pathways might be responsible for disseminated tuberculosis. (c) 2024 The Author(s). Published by Elsevier Ltd on behalf of The British Infection Association. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Purpose: Tuberculosis (TB) is a major global health threat and its diagnosis remains challenging. This study aimed to develop a nomogram that incorporated peripheral blood transcriptional signatures and other blood tests for the diagnosis of tuberculosis. Patients and Methods: Patients with TB, patients with other definite pulmonary diseases (OPD), individuals with latent tuberculosis infection (LTBI), and healthy controls (HC) were retrospectively enrolled between May 2017 and April 2018. The results of the interferon-gamma release assay (IGRA) and blood counts were obtained from medical records, and the transcripts of 10 genes were detected using reverse transcription polymerase chain reaction (RT-PCR). Variable selection was performed using least absolute shrinkage and selection operator regression (LASSO) and multivariate logistic regression was performed for the optimal prediction model with backward direction. The model was displayed as a nomogram, and its performance was evaluated for discrimination ability, calibration ability, and clinical usefulness. Internal validation of the prediction model was conducted using bootstrap resampling. Results: A total of 185 participants were enrolled, including 84 patients with TB and 101 controls. A prediction nomogram composed of IGRA, percentage of neutrophils, and expression levels of CD64, granzyme A (GZMA), and PR/SET domain 1 (PRDM1) was established. The nomogram demonstrated good discrimination, with an unadjusted area under the curve (AUC) of 0.914 (95% CI: 0.875-0.954) and a bootstrap-corrected AUC of 0.914 (95% CI: 0.874-0.947). With a cutoff value of 0.519, the sensitivity and specificity for discriminating PTB from controls were 0.81 and 0.871, respectively. The nomogram also showed good calibration with the Hosmer-Lemeshow test (P=0.58) and good clinical practicality displayed by the decision curve analysis. Conclusion: A nomogram composed of IGRA, percentage of neutrophils, and expression of CD64, GZMA, and PRDM1 was established. The nomogram demonstrated a sensitivity and specificity of 81% and 87%, respectively, for differentiating TB from controls.

Diagnosis of tuberculosis remains a challenge when microbiological tests are negative. Immune cell atlas of patients with tuberculosis and healthy controls were established by single-cell transcriptome. Through integrated analysis of scRNA-seq with microarray and bulk RNA sequencing data, a ferroptosis-related gene signature containing ACSL4, CTSB, and TLR4 genes that were associated with tuberculosis disease was identified. Four gene expression datasets from blood samples of patients with tuberculosis, latent tuberculosis infection, and healthy controls were used to assess the diagnostic value of the gene signature. The areas under the ROC curve for the combined gene signature were 1.000, 0.866, 0.912, and 0.786, respectively, in differentiating active tuberculosis from latent infection. During anti-tuberculosis treatment, the expression of the gene signature decreased significantly in cured patients with tuberculosis. In conclusion, the ferroptosis-related gene signature was associated with tuberculosis treatment efficacy and was a promising biomarker for differentiating active tuberculosis from latent infection.

To identify T cell subsets associated with control of tuberculosis, single-cell transcriptome and T cell receptor sequencing were performed on total T cells from patients with tuberculosis and healthy controls. Fourteen distinct subsets of T cells were identified by unbiased UMAP clustering. A GZMK-expressing CD8+ cytotoxic T cell cluster and a SOX4-expressing CD4+ central memory T cell cluster were depleted, while a MKI67-expressing proliferating CD3+ T cell cluster was expanded in patients with tuberculosis compared with healthy controls. The ratio of Granzyme K-expressing CD8+CD161-Ki-67- and CD8+Ki-67+ T cell subsets was significantly reduced and inversely correlated with the extent of TB lesions in patients with TB. In contrast, ratio of Granzyme B-expressing CD8+Ki-67+ and CD4+CD161+Ki-67- T cells and Granzyme A-expressing CD4+CD161+Ki-67- T cells were correlated with the extent of TB lesions. It is concluded that granzyme K-expressing CD8+ T cell subsets might contribute to protection against tuberculosis dissemination.

Tissue-resident MAIT cells in tuberculous pleural effusions, the site of tuberculosis infection, were investigated in the study. Tim-3+CD69+CD103+ and CD39+CD69+CD103+ tissue-resident MAIT cell subsets were identified in tuberculous pleural effusions. Tim-3 expression in MAIT cells was greatly induced and CD39 expression was elevated following ex vivo stimulation with Mycobacterium tubercu-losis antigens. Mycobacterial antigen-stimulated Tim-3+CD69+CD103+ tissue-resident MAIT cells had higher frequency of IFN-g- and granzyme B-producing cells than Tim-3-CD69+CD103+ subset, while CD39+CD69+CD103+ MAIT cells had similar frequency of IFN-g-positive cells but higher ratio of gran-zyme B-producing cells than CD39-CD69+CD103+ subset. Blocking of IL-2, IL-12p70 or IL-18 but not IL -15 led to significantly reduced expression of Tim-3 compared with isotype antibody control. In contrast, CD39 expression was not influenced by any of the cytokines tested. Tim-3+ MAIT cells had higher levels of lipid uptake and lipid content than Tim -3- cells. It is concluded that Tim-3+CD69+CD103+ tissue -resident MAIT cells were elevated in tuberculous pleural effusions and had higher capacity to produce effector molecules of IFN-g and granzyme B.(c) 2022 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

目的 活动性结核病的诊断仍然是结核病防治的重点和难点,差异表达基因有望成为结核病新的诊断靶标.本研究通过检测结核分枝杆菌不同感染状态下人外周血中PR/SET域1(PR/SET domain 1,PRDM1)和GATA结合蛋白2(GATA binding protein 2,GATA2)基因的mRNA表达,分析其作为诊断标志物的能力.方法 收集活动性结核病患者、结核分枝杆菌潜伏感染者和健康人外周血,荧光定量PCR检测外周血单个核细胞中PRDM1和GATA2的mRNA水平,分析各组受试者PRDM1和GATA2基因表达差异及其鉴别活动性结核和潜伏感染的诊断能力.结果 活动性结核病患者外周血单个核细胞中PRDM1和GATA2的表达显著低于潜伏感染者和健康人(H=69.27,P<0.0001;H=37.97,P<0.0001).PRDM1鉴别诊断活动性结核和潜伏感染的曲线下面积为0.8967,灵敏度为80.22％,特异度为87.1％.GATA2鉴别诊断活动性结核和潜伏感染的曲线下面积为0.8061,灵敏度为82.35％,特异度为70.97％.PRDM1和GATA2联合诊断活动性结核和潜伏感染的曲线下面积为0.935,诊断的灵敏度为76.9％,特异度为100％.结论 外周血单个核细胞中PRDM1和GATA2的mRNA表达水平是鉴别活动性结核和潜伏感染者的潜在标志物,有助于活动性结核的辅助诊断.

Objective To investigate the relationship between the CD160 expression and anti-tuberculosis immunity. Methods Fluorescence quantitative real-time PCR was used to detect the expression of CD160 in peripheral blood mononuclear cells (PBMCs). Flow cytometry was used to analyze the expression of CD160 on main subtypes of PBMCs, such as T cells, B cells, NK cells and monocytes. The relationship among CD160 and perforin, granzyme B, granulysin, CD69, CD107 and IFN-γ in NK cells was analyzed by flow cytometry. Results CD160 mRNAs in the PBMCs from patients with active tuberculosis was significantly down-regulated, and the levels of CD160 expression in Mycobacterium tuberculosis (MTB)-positive patients was significantly lower than in MTB-negative patients. The expression of CD160 on B cells and monocytes was lower in patients with active tuberculosis as compared with normal controls, while no significant difference was observed on CD3+ T cells. NK cells from patients with active tuberculosis had significantly lower CD160 expression than those from normal controls. In vitro culture with MTB antigens led to down-regulated expression of CD160 on NK cells. The activation marker CD69 on NK in patients with active tuberculosis was significantly lower than that in normal controls. The expression of perforin, granzyme B, granulysin, CD69 and CD107 in CD160+ NK cells was significantly higher than that of CD160- NK cells. However, the expression of IFN-γ in CD160+ NK cells was significantly lower than that of CD160- NK cells. Conclusion The mRNA and protein expression of CD160 was significantly down-regulated in patients with active tuberculosis. CD160 promotes the activation and degranulation of NK cells associated with tuberculosis antigens, but suppresses the expression of IFN-γ of NK cells. CD160 may become a new target for the diagnosis and treatment of tuberculosis.

Objective:Mycobacterium tuberculosis(Mtb),the causative agent of tuberculosis(TB),causes an estimated 1.6 million human deaths annually,but the pathogenesis of TB remains unclear.Immunity plays a critical role in the onset and outcome of TB.This study aimed to uncover the roles of innate and adaptive immunity in TB.Methods:The gene expression profiles generated by RNA sequencing from human peripheral blood mononuclear cells(PBMCs)stimulated with or without Mtb strain H37Rv antigens were analyzed.A total of 973 differentially expressed mRNAs were identified.Results:The differentially expressed genes were enriched in innate immunity signaling functions.The mesenchymal-epithelial transition factor(MET)gene was significantly upregulated in CD14+monocytes.A MET inhibitor improved the uptake of the BCG strain by monocytes and macrophages as well as inhibited the expression of indoleamine 2,3-dioxygenase(IDO).The expression of IDO was increased in PBMCs stimulated with Mtb antigens,and the IDO inhibitor promoted the expression of CD40,CD83,and CD86.Conclusion:Our results might provide clues regarding the immunomodulatory mechanisms used by Mtb to evade the host defense system.

Objective: Current commercially available immunological tests cannot be used for discriminating active tuberculosis (TB) from latent TB infection. To evaluate the value of biomarker candidates in the diagnosis of active TB, this study aimed to identify differentially expressed genes in peripheral blood mononuclear cells (PBMCs) between patients with active TB and individuals with latent TB infection by transcriptome sequencing. Methods: The differentially expressed genes in unstimulated PBMCs and in Mycobacterium tuberculosis (Mtb) antigen-stimulated PBMCs from patients with active TB and individuals with latent TB infection were identified by transcriptome sequencing. Selected candidate genes were evaluated in cohorts consisting of 110 patients with TB, 30 individuals with latent TB infections, and 50 healthy controls by quantitative real-time RT-PCR. Receiver operating characteristic (ROC) curve analysis was performed to calculate the diagnostic value of the biomarker candidates. Results: Among the differentially expressed genes in PBMCs without Mtb antigen stimulation, interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) had the highest area under curve (AUC) value (0.918, 95% CI: 0.852-0.984, P<0.0001) in discriminating patients with active TB from individuals with latent TB infection, with a sensitivity of 91.86% and a specificity of 84.00%. In Mtb antigen-stimulated PBMCs, orosomucoid 1 (ORM1) had a high AUC value (0.833, 95% CI: 0.752-0.915, P<0.0001), with a sensitivity of 81.94% and a specificity of 70.00%. Conclusion: IFIT3 and ORM1 might be potential biomarkers for discriminating active TB from latent TB infection.

目的 利用原核表达系统表达、纯化结核分枝杆菌潜伏感染相关蛋白Rv2660c,评价其在血清学方面用于结核病诊断的价值.方法 合成Rv2660c全基因核酸序列,构建pET28a-Rv2660c融合表达载体,在原核系统内进行重组表达,亲和层析纯化重组Rv2660c蛋白,采用Western-Blot、ELISA实验分析重组蛋白的免疫反应.结果 pET28a-Rv2660c在大肠杆菌内成功表达重组Rv2660c蛋白,SDS-PAGE电泳鉴定重组蛋白分子量为10.2 KD,与预期一致,Western-Blot结果表明重组蛋白Rv2660c只能与活动性结核患者血清反应,出现一条杂交条带;ELISA结果发现重组Rv2660c蛋白与结核患者血清能产生较强的反应,结核病菌阳患者组、菌阴患者组、潜伏感染组以及健康对照组A450平均值分别为0.52、0.48、0.28和0.25.结论 重组结核分枝杆菌Rv2660c蛋白具有一定的免疫原性,对活动性结核病的诊断具有一定的价值,但对结核分枝杆菌潜伏感染的诊断没有价值.

目的 寻找活动性肺结核患者与潜伏感染者免疫功能差异的重要分子.方法 收集活动性肺结核患者和潜伏感染者PBMCs进行转录组测序,分析数据获得差异表达基因,对差异表达基因使用软件MeV进行聚类分析,用DAVID数据库进行GO分析,用STRING数据库进行蛋白相互作用网络分析,用Cytocapase软件进行KEGG分析和蛋白质复合物分析.结果 共获得差异表达基因98个,其中上调基因67个,下调基因31个;GO分析生物进程富集的词条是"免疫反应"、"天然免疫反应"和"中性粒细胞趋化";KEGG分析结果显示富集词条是"自然杀伤细胞介导的细胞毒作用"、"抗原处理和递呈"和"IL17信号通路"等信号通路,其中IFNG、ITGAM、MMP1和FOS基因连接多个信号通路;构建蛋白相互作用网络,筛选到聚集程度高的分子ELANE、ITGAM、MMP9和ARG1;而蛋白复合物分析显示由上调基因组成的复合物1和复合物2、由下调基因组成的复合物3.结论 连接多个信号通路或者聚集程度高的重要分子和大分子复合物可能在活动性肺结核患者与潜伏感染者免疫功能差异具有重要的作用,有待我们进一步的实验验证及功能研究.

目的 利用原核系统对人乳头瘤病毒(HPV)16型L1蛋白优势抗原表位进行重组表达与纯化,并检测血清学反应.方法 对HPV16型L1蛋白优势抗原表位进行全基因合成,将核酸片段克隆至pET-DsbC原核表达载体,重组质粒转化大肠埃希菌Rosetta(DE3),经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,采用亲和层析对表达产物进行纯化,采用免疫印迹法、酶联免疫吸附试验(ELISA)验证重组蛋白的免疫学活性.结果 成功构建了pET-DsbC-HPV16 L1表达载体,并在大肠埃希菌Rosetta(DE3)中实现了稳定的可溶性表达.重组DsbC-HPV16 L1蛋白经亲和层析进行纯化,相对分子质量为45000,纯度为95％,重组蛋白纯化得率为22％.免疫印迹法结果显示,重组DsbC-HPV16 L1蛋白可与HPV16型阳性血清产生特异的抗原-抗体反应,与重组DsbC蛋白或健康人血清无交叉反应,具有良好的特异性.ELISA结果显示,重组DsbC-HPV16 L1蛋白与HPV16型阳性血清有较强的免疫反应性,A450 nm均值为0.56,高于健康人血清和磷酸盐缓冲液(PBS)阴性对照(A450 nm均值分别为0.24和0.21).结论 采用原核表达系统实现了HPV16型L1蛋白优势抗原表位的可溶性高表达,获得的重组蛋白具有良好的免疫原性和免疫学活性.

目的 研究结核性胸膜炎患者胸腔积液中MAIT细胞IFN-γ免疫应答特征及其与疾病转归的相关性.方法 提取61份结核性胸腔积液PFMCs,用结核分枝杆菌H37Rv裂解液刺激后收集细胞,进行流式抗体染色.另外有17例患者重复收集胸水,10例治疗效果好,胸水吸收,另7例无明显治疗效果,比较其治疗前后IFN-γ+ MAIT细胞比例.结果 结核分枝杆菌抗原刺激后,结核性胸膜炎患者胸腔积液CD8 +T细胞亚群中MAIT细胞对结核抗原应答生成的IFN-γ比例为(30.49 ± 3.23)% .男女性别和不同年龄患者的IFN-γ+ MAIT 细胞比例均无显著差异.痰涂片/培养阳性和阴性患者的IFN-γ+ MAIT 细胞比例分别为(53.44 ± 5.48)%和(33.98 ± 2.45)% ,差异有统计学意义(P=0.0027).治疗效果好的患者治疗后IFN-γ+ MAIT细胞比例明显上升,治疗前后比例分别为(34.3 ±3.69)%和(49.9 ± 3.79)%,差异有统计学意义(P=0.0003).治疗效果不好的患者IFN-γ+ MAIT 细胞比例也有所上升,但差异没有统计学意义.结论 MAIT细胞通过生成细胞因子IFN-γ而在局部抗结核免疫中发挥重要作用.

目的 研究活动性肺结核患者外周血单个核细胞( peripheral blood mononuclear cells, PBMCs)中抑制素βA亚基(inhibin, beta A, INHBA)基因的表达特点.方法 使用密度梯度离心法分离PBMCs,然后用TRIzol提取细胞RNA,逆转成cDNA后,进行荧光定量PCR分析INHBA的表达.结果 结核分枝杆菌H37Rv菌体裂解液和结核分枝杆菌特异性抗原ESAT-6和CFP-10的多肽,显著性提高PBMCs中INHBA基因的表达,配对t检验p值分别为0.019和0.0013;在无抗原刺激的情况下,活动性肺结核与正常对照之间无显著差别(P=0.52);当使用结核分枝杆菌H37Rv菌体裂解液和结核分枝杆菌特异性抗原ESAT-6和CFP-10的多肽刺激PBMCs,INHBA在活动性肺结核患者组的表达显著性的高于正常对照组, t检验P值分别为0.016和0.010.结论 INHBA在结核抗原刺激后在PBMCs表达显著性升高,且在活动性肺结核患者组显著高于正常对照组,可能作为活动性肺结核的诊断标志物.

目的 研究结核性胸膜炎患者胸腔积液中MAIT细胞颗粒酶B(Granzyme B)免疫应答特征及其与疾病转归的相关性.方法 提取25份结核性胸腔积液PFMCs,用结核分枝杆菌H37Rv裂解液刺激后收集细胞,进行流式抗体染色.另外有10例患者重复收集胸水,其中7例治疗效果好,胸水吸收,另3例无明显治疗效果,比较其治疗前后Granzyme B+MAIT细胞比例.结果 结核分枝杆菌抗原刺激后,结核性胸膜炎患者胸腔积液中Granzyme B+MAIT比例为(25.34±5.37)％.男女性和不同年龄患者的Granzyme B+MAIT细胞比例均差异无统计学意义.7例治疗效果好的患者治疗后Granzyme B+MAIT细胞比例明显上升,治疗前后比例分别为(30.41±2.78)％和(40.61±2.61)％,差异有统计学意义(t=9.867,P＜0.000 1).3例治疗效果不好的患者Granzyme B+MAIT细胞比例明显低于治疗效果好的患者,且治疗后比例上升不明显.结论 MAIT细胞生成颗粒酶B通过细胞毒性杀伤作用而在局部抗结核免疫中发挥重要作用.

目的:优化人血小板衍生生长因子BB亚型(PGDF-BB)包涵体复性方法与纯化条件,获得具有较好生物活性的重组PGDF-BB蛋白.方法:对PGDF-BB包涵体以梯度尿素进行变性,选择最佳包涵体变性浓度;比较不同复性条件下的复性率,稳定PGDF-BB包涵体复性方法;参照该蛋白的理化性质,选择适合PGDF-BB重组蛋白的纯化方法.结果:原核系统内实现了PGDF-BB的高表达;通过优化包涵体复性方法,重组蛋白的包涵体复性率可达40％以上;经过多个纯化方法相结合,PGDF-BB的纯度达到95％.结论:通过实验条件的优化,提高了PGDF-BB包涵体复性率,获得高纯度、高生物活性的重组PGDF-BB蛋白.

Germinal centers (GC) are vital to adaptive immunity. BCL6 and miR-155 are implicated in control of GC reaction and lymphomagenesis. FBXO11 causes BCL6 degradation through ubiquitination in B-cell lymphomas. Chronic psychological stress is known to drive immunosuppression. Corticosterone (CORT) is an adrenal hormone expressed in response to stress and can similarly impair immune functions. However, whether GC formation is disrupted by chronic psychological stress and its molecular mechanism remain to be elucidated. To address this issue, we established a GC formation model in vivo, and a GC B cell differentiation model in vitro. Comparing Naive B cells to GC B cells in vivo and in vitro, the differences of BCL6 and FBXO11 mRNA do not match the changes at the protein level and miR-155 levels that were observed. Next we demonstrated that CORT increase, induced by chronic psychological stress, reduced GC response, IgG1 antibody production and miR-155 level in vivo. The effect of chronic psychological stress can be blocked by a glucocorticoid receptor (GR) antagonist. Similarly, impaired GC B cell generation and isotope class switching were observed. Furthermore, we found that miR-155 and BCL6 expression were downregulated, but FBXO11 expression was upregulated in GC B cells treated with CORT in vitro. In addition, we demonstrated that miR-155 directly down-regulated FBXO11 expression by binding to its 3́-untranslated region. The subsequent overexpression of miR-155 significantly blocked the stress-induced impairment of GC response, due to changes in FBXO11 and BCL6 expression, as well as increased apoptosis in B cells both in vivo and in vitro. Our findings suggest perturbation of GC reaction may play a role in chronic psychological stress-induced immunosuppression through a glucocorticoid pathway, and miR-155-mediated post-transcriptional regulation of FBXO11 and BCL6 expression may contribute to the impaired GC response.

目的 筛选适用于活动性肺结核患者PBMCs的内参基因.方法 用密度梯度离心方法分离活动性肺结核患者PBMCs,然后使用结核分枝杆菌 H37Rv裂解液、CFP-10 和 ESAT-6 多肽库、共刺激因子CD28共孵育,或者不添加任何刺激物,培养过夜后收集细胞;使用Trizol提取mRNA,逆转录获得cDNA,然后用6个常用的内参基因ACTB、B2M、DDX5、GAPDH、YWHAZ和18sRNA的特异性引物进行实时定量PCR检测,结果用geNorm 、NormFinder和Bestkeeper软件进行数据分析.结果 18sRNA基因丰度太高,在PBMCs表达不稳定,DDX5、YWHAZ和B2M的稳定最好,适合作为活动性肺结核患者PBMCs的内参基因,而ACTB和GAPDH稳定性略低.结论 筛选出稳定的内参基因DDX5、YWHAZ和B2M,可用于活动性肺结核患者PB-MCs基因表达分析.